Toxicon

Published by Elsevier
Online ISSN: 0041-0101
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Article
Clivorine, a pyrrolizidine alkaloid extracted from Chinese medicinal plant Ligularia hodgsonii Hook significantly inhibited human normal liver L-02 cells proliferation and decreased L-02 cells viability. The results of western blot showed that clivorine strongly evoked phosphorylation of p38 mitogen-activated protein (MAP) Kinase in L-02 cells, but had no effect on extracellular signal-related kinases MAP Kinase phosphorylation. Moreover, another pyrrolizidine alkaloid monocrotaline had no effect on phosphorylation of p38 MAP Kinase in L-02 cells. These studies document the effects of pyrrolizidine alkaloid clivorine on the MAPK cascade and on the growth of human normal liver L-02 cells for the first time, which may be a possible reason for the toxic effects observed in those plants containing pyrrolizidine alkaloids.
 
Article
A strain of a yet unidentified Fusarium sp. produced in addition to enniatins A1, B and B1 a number of minor enniatins. The strain formed a well supported clade with strains identified as Fusarium acuminatum (Gibberella acuminata) in phylogenetic analyses using the TEF-1alpha gene sequences. Two of the minor enniatins were easily recognised as hydroxylated species on the basis of their fragment ion spectra. The hydroxylation could be traced to one of the amino acid moieties using multiple-stage ion trap mass spectrometry. Different approaches for acetylation of the isolated compounds and complete hydrolysis supported the elucidation of the amino acid moiety as 3-hydroxy-2-methylamino-butyric acid, which is equivalent with N-methyl-threonine. The primary structures of the two enniatins were tentatively determined to be cyclo[Hiv-N-Me-Val-Hiv-N-Me-Val-Hiv-N-Me-Thr] and cyclo[Hiv-N-Me-Leu-Hiv-N-Me-Val-Hiv-N-Me-Thr]. The two depsipeptides represent new analogues and were named enniatin P1 and P2, respectively.
 
Article
Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.
 
Article
The 1,3-enone isomer (1) of heptanor-41-oxoyessotoxin (2) was isolated from extracts of Protoceratium reticulatum during large-scale production of yessotoxin (4). We found that 2 readily isomerizes to 1 in the presence of dilute ammonia and present evidence for the existence of 40-epi-2 (3) that also isomerizes to 1. 1-3 were detected by LC-MS methods both in extracts of P. reticulatum cultures and in mussels contaminated with yessotoxins. The isomerization of 2 and 3 into 1 occurs so readily that purification on basic alumina needs to be conducted carefully. No toxic effects were recorded in mice injected intraperitoneally with 1 at a dose of 5,000 microg/kg.
 
Article
Scorpion beta-toxins represent a particular pharmacological group of voltage-gated sodium channel (VGSC) neurotoxins. They typically shift the voltage dependence of activation to more hyperpolarizing potentials and reduce the peak current amplitude by binding to receptor-site 4. Here, we report the purification and functional characterization of the first voltage-gated sodium channel toxins, CeII8 and CeII9, isolated from the scorpion Centruroides elegans (Thorell, 1876), which is responsible for deadly cases of intoxication in Mexico. The soluble venom was fractionated by gel filtration and ion-exchange chromatography, followed by reversed-phase HPLC. The toxins CeII8 and CeII9 were further purified and both their amino acid sequence and molecular weight were determined. Both toxins were electrophysiologically characterized on four mammalian VGSCs (rNa(v)1.2, rNa(v)1.4, hNa(v)1.5 and rNa(v)1.7) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Although CeII8 has the highest sequence similarity with scorpion alpha-toxins, inhibiting the inactivation of VGSCs, 300 nM toxin had a clear beta-toxin effect and was selective towards Na(v)1.7, involved in short-term and inflammatory pain. To the best of our knowledge, CeII8 is the first beta-toxin active on Na(v)1.7. CeII9, a typical anti-mammalian beta-toxin, selectively modulated Na(v)1.4 at a concentration of 700 nM and was, in contrast to CeII8, found to be lethal to mice. Interestingly, both toxins, despite their differences in amino acid sequence, only altered the biophysical properties of a fraction of the expressed sodium channels. Since these effects have also been reported for the beta-toxin CssIV, the bioactive surfaces of the toxins have been compared to each other.
 
Article
Voltage-gated Na(+) channels underlie the action potential upstroke in excitable cells, and both natural and synthetic inactivation inhibitors prolong the Na(+) current (I(Na)). The effects of Na(+) channel mutations on these pharmacological actions are incompletely investigated. Therefore, I compared the effects of inactivation inhibitors on I(Na) in WT or mutant (DeltaKPQ) human cardiac Na(+) channels expressed in HEK-293 cells, by measuring difference currents sensitive to 50muM tetrodotoxin. Veratridine and the pyrethroid tefluthrin prolonged I(Na) in WT and DeltaKPQ without obvious differential effects, while a sea anemone toxin (ATX-II) and a synthetic inotrope (SDZ 201-106) prolonged WT I(Na), but apparently blocked I(Na) in the DeltaKPQ mutant. This block was due, at least in-part, to enhanced steady-state inactivation, with half-inactivation potentials shifted by up to -17mV. Inactivation enhancement by ATX-II also persisted when conditioning depolarizations were abbreviated, and was unaffected by the additional presence of SDZ 201-106 consistent with these agents having unique interactions with DeltaKPQ Na(+) channels. It is concluded that the toxin-binding sites for ATX-II and SDZ 201-106 have allosteric effects converging on a common path affecting steady-state inactivation of DeltaKPQ I(Na). Pharmacological modulation of this path to increase inactivation in mutant Na(+) channels could potentially produce therapeutic benefits.
 
Article
The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na+ and Ca2+ channels. ProTxII is distinct from other known Na+ channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage-dependent activation, and current kinetics in Na(V)1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G(max) appeared at relatively low concentrations (40-100 nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (> or =500 nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba2+, known to screen and/or bind to surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba2+ indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.
 
Article
This study reveals that both cyanobacterial toxicity and turbidity have the potential to reduce the growth and energy storage of young-of-the-year (YOY) perch and thereby influence survival rates. During the 1990's a reduction in recruitment of YOY perch (Perca fluviatilis) occurred along the Swedish East coast. Concurrently, large blooms of filamentous cyanobacteria have increased in the Baltic Proper and in coastal waters. This study examined whether extended exposure to toxic and non-toxic filamentous cyanobacterium Nodularia affect YOY perch growth and feeding behavior under simulated bloom conditions (30 days at 50 μg Chl a L(-1)). Specific growth rate (SGR), the somatic condition index (SCI) and the lipid content of YOY perch (10-12 weeks old) were significantly lower in perch exposed to Nodularia compared to fed controls (no Nodularia). YOY perch exposed to non-toxic Nodularia displayed a higher attack rate than perch living in Nodularia free controls in 2 out of 3 trials. Reductions in growth and energy storage, mediated by cyanobacteria, increase the risk of starvation and predation and could locally influence recruitment of YOY perch.
 
Article
Although the pathology of tetanus toxin poisoning has been linked to an inhibition of neurotransmitter release, the mechanism of this inhibition is unknown. The neuroblastoma x glioma hybrid cell NG-108 is an emerging model in which to study the biochemical effect of tetanus toxin on acetylcholine secretion. In differentiated as well as undifferentiated NG-108 cells, a 4 hr tetanus toxin (10(-8) M) pretreatment had no effect on basal levels of cyclic AMP or cyclic GMP. In addition, toxin pretreatment did not affect agonist induced increases in either cyclic nucleotide. Treatment of NG-108 cells for 4 hr with 10(-10) M tetanus toxin had no effect on the subsequently measured activity of cytosolic protein kinase C. However, a 4 hr pretreatment of undifferentiated or differentiated cells with tetanus toxin (10(-8) or 10(-10) M respectively) significantly attenuated the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C. Direct addition of tetanus toxin (10(-7)-10(-10) M) to isolated protein kinase C did not alter the ability of the enzyme to phosphorylate histone protein. These results suggest that one manifestation of tetanus toxin poisoning may be a disruption in protein kinase C metabolism.
 
Article
There is considerable literature on the pathogenesis of tetanus toxin poisoning; however, the mechanism of action and intracellular substrate of this toxin have not been defined. It was demonstrated that the NG-108 neuroblastoma x glioma cell line is a suitable model in which to study the mechanism of tetanus toxin action, from binding of the toxin to inhibition of transmitter release. Further, it has been shown that tetanus toxin pretreatment attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C (PKC) in this cell line. In the present study a 4-hr tetanus toxin pretreatment (10(-10)-10(-13) M) completely inhibited the mobilization of cytosolic PKC induced by a 30-min exposure to 10 microM neurotensin. Pretreatment with 10(-10) M tetanus toxin for periods as short as 1 hr was sufficient to attenuate the ability of neurotensin to mobilize cytosolic PKC; however, a 30-min pretreatment had no significant effect. At a concentration of 10(-11) M, it was necessary to pretreat the cells for greater than 1 hr to significantly attenuate neurotensin-mobilized PKC activity. The exact role that PKC plays in the secretory process is not yet known; however, these findings suggest that the effect of tetanus toxin on neurotransmitter release is accompanied by an alteration in PKC metabolism in differentiated NG-108 cells.
 
Article
The snake venom proline-rich peptide BPP 10c is an active somatic angiotensin-converting enzyme (sACE) inhibitors. Recently we demonstrated that the anti-hypertensive effect of BPP 10c is not related to the inhibition of sACE alone, thus suggesting that this enzyme is not its only target for blood pressure reduction. In the present work, a biodistribution study in Swiss mice of [(125)I]-BPP 10c in the absence or in the presence of a saturating concentration of captopril, a selective active-site inhibitor of sACE, demonstrated that: (1) [(125)I]-BPP 10c was present in several organs and the renal absorption was significantly high; (2) [(125)I]-BPP 10c showed a clear preference for the kidney, maintaining a high concentration in this organ in the presence of captopril for at least 3h; (3) The residual amount of [(125)I]-BPP 10c in the kidney of animals simultaneously treated with captopril suggest that the peptide can interact with other targets different from sACE in this organ. We also showed that Cy3-labeled BPP 10c was internalized by human embryonic kidney cells (HEK-293T). Taken together, these results suggest that sACE inhibition by captopril affects the tissue distribution of [(125)I]-BPP 10c and that the anti-hypertensive effects of BPP 10c are not only dependent on sACE inhibition.
 
Article
6-Epitetrodotoxin (6-epiTTX) and 11-deoxytetrodotoxin (11-deoxyTTX), isolated from an Okinawan newt, Cynops ensicauda, were tested for sodium-channel blocking effects on the voltage-clamped frog skeletal muscle fiber. In 6-epiTTX, the C-6 -OH is in an epimeric position; in 11-deoxyTTX, C-11 has a methyl in place of a hydroxymethyl group. At pH 7.25, the ED50s for reducing INa are: 4.1 nM (TTX), 96 nM (6-epiTTX), and 445 nM (11-deoxyTTX). In each analogue, the lowered potency can be attributed energetically to the loss of a hydrogen bond. By complementarity, in the sodium-channel receptor for TTX, there must be a hydrogen-acceptor group for the C-6 -OH, and another for the C-11 -OH. Therefore, the TTX molecule is bound to the receptor through an ion-pair (for the guanidinium), and five hydrogen bonds, one each for the -OH on C-9, C-10, C-4, and, as now identified, for C-6 and C-11. Considering the three-dimensional structure of the toxin molecules, these binding sites must be located in a fold or a crevice of the channel protein. If glutamate 387 of rat brain sodium channel II is the ion-pairing site for the guanidinium group, then the carbonyl oxygen of asparagine 388 is the hydrogen acceptor for the C-9 and C-10 -OHs.
 
Article
During November 2005, a dense bloom of Dinophysis spp. dominated (>97%) by Dinophysis acuta in the Galician Rías Baixas (NW Spain), provided a unique opportunity to describe the full toxin profile - including toxins that represent a low percentage and escape detection in analyses of single-cell isolates - in plankton concentrates rich in this species. Detection and identification of toxins were carried out by liquid chromatography coupled to mass spectrometry (LC-MS/MS), a method that is based on their retention times (RT) and the fragmentation patterns of their mass spectra. OA-D8 diol-ester and PTX11 were detected in co-occurrence with okadaic acid analogues (OA, DTX2) and PTX2 in plankton concentrates dominated by D. acuta. The presence of a PTX11-isomer, which was suggested to be PTX13 or a novel PTX11-isomer, released in the sea water, was also confirmed in samples obtained after deployment of passive samplers (SPATT) in situ at the time of the D. acuta bloom maximum. The amount of PTX11 per cell of D. acuta, estimated as PTX2 equivalents, ranged between not detected and 2.2pg cell(-1), and represented a maximum of 2.9% of the total toxin content. The variation in PTX11 content per cell of D. acuta, during a daily cycle, followed the same pattern than that of PTX2, with maxima at 21:00 and 03:00h (dark hours), but the amounts per cell were one order of magnitude lower. This is the first report of PTX11, together with the confirmation of OA-D8 diol-ester in D. acuta populations from Europe.
 
Article
This report describes the preparative scale production of 11-[3H]-tetrodotoxin (TTX) and its evaluation as a substitute for [3H]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [3H]-STX imposed by the international Chemical Weapons Convention served as the primary impetus for this study. We have incorporated on a preparative scale, a nonexchangeable tritium label into the TTX molecule at a specific activity of 12.90 Ci/mmol and recovered material of high radiochemical purity (98%). The resulting 11-[3H]-TTX was found to exhibit site-specific binding characteristics in the receptor assay (dissociation constant(K(d))=4.77+/-1.54nM; maximum binding(B(max))=1. 62+/-0.24pmol/mg of synaptosomal protein). The inhibition constant (K(i)) for the assay was 1.46+/-0.28 nM STX equiv. (n=6), with an estimated detection limit of ca. 2-4 ng STX equiv./ml in a sample extract. Moreover, quantitative comparisons indicated that 11-[3H]-TTX could be used interchangeably with [3H]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[3H]-TTX produced and evaluated herein exhibits physical, chemical and biological characteristics suitable not only for use in the PSP receptor binding assay, but likely for other applications employing [3H]-STX as the radioligand.
 
Article
11-oxoTTX is an analogue 4-5 times more toxic than TTX itself, been rare even in marine animals. Two ions at m/z 320 and 336 corresponding to TTX and 11-oxoTTX (M+H(+)), respectively, were detected in the Brachycephalidae frog Brachycephalus ephippium extracts. The fragment ion pattern of 11-oxoTTX is similar to that TTX, although its possible to verify some specific fragments.
 
Article
Tetrodotoxin was detected in all nine species of newts tested, 6-epitetrodotoxin in six species, and 11-deoxytetrodotoxin in five species. Only one species lacked the analogues, thus suggesting that the analogues were common metabolites among newts. Toxin levels were high in Taricha granulosa and Notophthalmus viridescens followed by Cynops spp. Distinct differences in toxin contents and profiles existed among species and tissues.
 
Article
Tetrodotoxin was oxidized to a hydrated aldehyde, 11-oxo-tetrodotoxin, which shares the specificity of tetrodotoxin for the Na+ channel of the isolated voltage-clamped frog skeletal muscle fiber, but is four to five times more potent. It binds to the solubilized Na+ channel of eel electroplax with a similarly higher potency, because of an equilibrium dissociation constant about 0.25, and a dissociation rate constant 2.4 times slower than those for tetrodotoxin. 11-Oxo-tetrodotoxin can be reduced to regenerate a tetrodotoxin, which is chemically and biologically indistinguishable from the original tetrodotoxin. By reducing with tritiated sodium borohydride, a 3H marker can be inserted regiospecifically to yield 11-[3H]-tetrodotoxin. Because it has a defined specific activity of > 2.5 Ci/mmole, and a 3H marker which does not exchange with solvent proton, 11-[3H]-tetrodotoxin is an ideal tracer for tetrodotoxin. It may enable studies of problems which require higher signals and/or better stability of the marker than those obtainable from currently available tracer Na(+)-channel ligands.
 
Article
An analogue of tetrodotoxin (TTX), 11-oxoTTX, was semi-purified from the red-spotted newt, Notophthalmus viridescens, and identified by ESI-MS and 1H NMR spectra. The levels of TTX, 11-oxoTTX and 6-epiTTX in early stage of development (efts) of this newt were investigated by a post-column fluorescent-HPLC system, and compared with those of adult newts. The level of 11-oxoTTX in both of adults and efts were remarkably high, almost close to those of TTX, while 6-epiTTX was a minor component in both stages. The level of 6-epiTTX in efts (1.8+/-1.3 microg/g, SD, n=10) was significantly larger than that in adults (0.51+/-0.26 microg/g, SD, n=12) (p<0.05), while no significant difference was observed in the levels of TTX and 11-oxoTTX between efts (13+/-7.4 and 9.1+/-5.6 microg/g) and adult newts (16+/-6.3 and 13+/-6.2 microg/g, respectively) (p>0.05).
 
(A) Representative families of skeletal muscle (m1), cardiac muscle (hH1), and N1E-115 mouse neuroblastoma Na currents in the absence and presence of 100 nM BmK 11(2). m1 and hH1 were expressed in HEK 293 cells. Test pulses ranging from 280 to þ 60 mV were applied in 10 mV increments at 5 s intervals. Cells were held at 2120 mV between pulses. Experiments were performed at room temperature; (B) representative examples of current-voltage (I-V ) relations for m1, hH1, and N1E-115 Na channels. The peak Na currents were plotted as a function of test voltage. The I-V relations recorded in the presence of 100 nM BmK 11(2) were superimposed on the I-V relations recorded prior to the toxin application. 
Parameters of the voltage dependence of activation
(A-C) Activation curves for m1, hH1, and N1E-115 Na currents. The activation curves obtained in the presence of 100 nM BmK 11(2) were superimposed on the activation curves generated prior to the toxin application. BmK 11(2) shifted the activation curves for m1 and N1E115 Na currents; (D-F) steady-state availability curves for m1, hH1, and N1E-115 Na currents. The steady-state inactivation was assessed after 500 ms inactivating prepulses followed by a test pulse to 220 mV. The peak amplitudes were normalized and plotted versus the prepulse potentials. The availability curves obtained in the presence of 100 nM BmK 11(2) were superimposed on the availability curves generated prior to the toxin application. BmK 11(2) did not shift the steady-state availability curves of any of the Na channel isoforms. 
Parameters of the voltage dependence of inactivation
(A-C) Representative Na current traces recorded at 220 mV in the presence (thick line) of 100 nM BmK 11(2) were superimposed on Na current traces recorded at 220 mV prior to toxin application (thin line) for m1, hH1, and N1E-115 Na channels. BmK 11(2) slowed current decay of all Na channel isoforms; (D-F) time constants of current decay plotted as a function of test voltage for m1, hH1, and N1E-115 Na currents in the absence and presence of 100 nM BmK 11(2). Note: m1 control data and N1E-115 toxin data were best fitted by a single exponential. 
Article
BmK 11(2) is a 7216Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch. Nanomolar concentrations of the toxin prolong amphibian nerve action potentials without attenuation of the amplitude. The pharmacological action of the toxin and its sequence similarity to other alpha-scorpion toxins suggest that BmK 11(2) selectively alters voltage-gated Na channels. In order to test whether BmK 11(2) preferentially modulates the gating or kinetics of certain channel isoforms, we applied BmK 11(2) to muscle, heart and neuronal Na channels. 100nM BmK 11(2) increased the peak current amplitude of skeletal muscle (micro1) and neuronal (N1E-115) Na currents by 40 and 20%, respectively, and reduced the cardiac Na (hH1) current by 15%. The toxin slowed current decay of all isoforms, most prominently in N1E-115 (tau(BmK)/tau(Control)=12), micro1 (11), and less so for hH1 (1.3). BmK 11(2) shifted the voltage dependence of activation of micro1 and N1E-115 currents. BmK 11(2) had no effect on steady-state inactivation, use-dependent availability, and the kinetics of entry into slowly recovering inactivated states.
 
Article
Pa-11, a phospholipase A2 isolated from the venom of an Australian elapid snake Pseudechis australis, was chemically modified and its enzymic, neuromuscular and lethal activities were studied. Carboxymethylation of Met-8 gave a derivative with 2% of the enzymic activity and less than 3% of the lethal activity of native Pa-11; it had about 5% of the original ability to block directly and indirectly stimulated mouse phrenic nerve-hemidiaphragm preparations. Nitrophenylsulfenylation of tryptophanyl residues at positions 31 and 69 caused loss of all activities. Amidination of all 14 lysyl residues gave a derivative with 41% and 16% of the enzymic and lethal activities, respectively, but with less than 5% of the original neuromuscular blocking activity. Mono-carbamoylation of lysyl residues at positions 58, 63, 81 and 85 was achieved. The most abundant derivative, 58-carbamoyl-lysine Pa-11 was enzymically 130% and lethally 100% as active as native Pa-11, but it had only about 20% of the native's neuromuscular activity in vitro. 63-Carbamoyl-lysine Pa-11 had 10% of the enzymic and 20% of the lethal activities, respectively; however, it retained at least 50% of its ability to block neuromuscular transmission in vitro, while losing most of its activity to block directly stimulated muscle contractions. 81- and 85-Carbamoyl derivatives have the same enzymic and lethal activities as the original protein, but the 85 derivative had less than 10% of the native neuromuscular activity. Hence, modifications of lysine residues at positions 58, 63 and 85 seem to be particularly significant in altering the neuromuscular, but not enzymic, activity of Pa-11, perhaps by altering the ability of the toxin to bind to its target on nerve and muscle membranes. Modification at position 63 appeared to lead to a dissociation of effects on neuromuscular transmission and directly on muscle cells.
 
Article
The actions of the 12 alpha-saxitoxinol, 12 beta-saxitoxinol and a C-12 ethylene thioketal derivative of saxitoxin, as well as those of 11 alpha-(OSO3)-saxitoxin, 11 beta-(OSO3)-saxitoxin and 11 alpha-(OH)-saxitoxin, have been examined on the isolated squid giant axon. Each of these analogues acted similarly to saxitoxin in blocking specifically the sodium channel. The relative potencies are: STX (1); 11 beta-(OSO3)-STX (gonyautoxin III) (0.42); 11 alpha-(OSO3)-STX (gonyautoxin II) (0.20); 11 alpha-(OH)-STX (0.10); 12 alpha-saxitoxinol (0.0021); 12 beta-saxitoxinol (0.0005). Thus, the presence of a bulky and negatively charged sulphate group on C-11 does not materially affect the biological activity of STX. Hydrogen bonding at the C-12 position is probably an important means of binding of STX to the membrane receptor site. The difference between the epimers of saxitoxinol suggests that the H in one of them may be geometrically better aligned than that in the other, with the hydrogen acceptor group in the receptor.
 
Article
L. Martínez de Villarreal, R. Velazco-Campos, A. Piñeyro López and R. González Alanís. Effects of toxin T-544 from the Karwinskia humboldtiana (buckthorn) plant upon mouse embryos explanted at 11 days. Toxicon 28, 449-452, 1990.-Eleven-day mouse embryos were exposed to the K. humboldtiana toxin T-544 for 24 hr. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered protein levels. There was a significant difference in malformations in those embryos exposed to 0.05 and 0.2 μg/ml of toxin compared with controls. Embryo protein content was significantly lower in those embryos exposed to 0.1 μg/ml of T-544 compared with control group.
 
Article
Lys49 phospholipase A2 homologues constitute a group of catalytically-inactive proteins, present in the venoms of many crotalid snakes, which induce myonecrosis. Current evidence supports the mapping of their toxic site to the C-terminal region, where amino acids comprised within the sequence 115-129 appear to play a central role in toxicity. This study evaluated the possible toxic effects of several synthetic peptides corresponding to the sequence 115-129 of different Lys49 myotoxins, using in vitro cytotoxicity and in vivo myotoxicity assays. Peptides varied widely in their activities, ranging from fully toxic to harmless. Thus, the toxic actions of Lys49 myotoxins cannot always be reproduced by their free peptides 115-129. Peptides from Agkistrodon p. piscivorus (AppK) and A. contortrix laticinctus Lys49 myotoxins exerted both cytotoxicity and myotoxicity. Random scrambling of peptide AppK resulted in complete loss of toxicity, demonstrating that its specific sequence of residues, rather than their simple presence or frequency, confers its ability to damage muscle. Peptide AppK synthesized with D-amino acids retained both activities of the natural L-enantiomer, suggesting that its mechanism of action does not involve the recognition of a proteic receptor/acceptor site on muscle cells, but possibly the binding to other structures, such as negatively-charged membrane phospholipids.
 
Article
The region comprising amino acid residues 115-129 of myotoxin II, a Lys49 phospholipase A2 from the venom of Bothrops asper, was previously shown to constitute a heparin binding site, and to be associated with its toxic activities. The corresponding synthetic peptide, KKYRYYLKPLCKK, was coupled to diphtheria toxoid as a carrier, and utilized as an immunogen in mice, to explore the possible protection from the myotoxic activity induced by myotoxin II in vivo. Mice receiving peptide-carrier injections produced antibodies to peptide 115-129, which cross-reacted to myotoxin II, as determined by enzyme-immunoassay. In contrast, no antibodies against peptide 115-129 were detected in mice immunized with myotoxin II, despite the strong antibody response to the whole antigen. Thus, region 115-129 of myotoxin II is not an immunodominant B-cell epitope in the mouse. After immunization with conjugated peptide or myotoxin II, mice were challenged with myotoxin II, and the extent of myonecrosis was estimated by determining their plasma creatine kinase activity, in comparison to non-immunized mice. After the challenge, both the group immunized with myotoxin II, and the group immunized with peptide 115-129, had a significant reduction of myonecrosis. These results demonstrate that region 115-129 of myotoxin II constitutes a neutralizing epitope, and provide further evidence for the relevance of this region in its myotoxic effect in vivo.
 
Article
Nineteen 12,13-epoxytrichothecene mycotoxins were tested for their relative capabilities to inhibit protein synthesis in Vero cells and rat spleen lymphocytes. Although the lymphocytes were generally more sensitive to the mycotoxins, good correlation existed between the relative potencies of the various trichothecenes in the two cell systems. The most potent mycotoxins (T-2, verrucarin A and roridin A) have acetyl side groups on, or a hydrocarbon chain between, carbons 4 and 15 of the basic ring structure. Loss of side groups from either of these positions or an isovaleryl group at carbon 8 resulted in reduced protein synthesis inhibition (T-2 to HT-2, neosolaniol or diacetoxyscirpenol). Any combination of loss from all three positions (T-2 triol, T-2 tetraol, 15-monoacetyl DAS, scirpentriol, fusarenon X and deoxynivalenol) further weakens their effect. Reduction of the hydroxyl groups to hydroxides, forming verrucarol and deoxyverrucarol, reduced their effectiveness by over a thousand-fold compared to the most potent mycotoxins. Addition of side groups resulted in reduced effectiveness only when an acetyl group was added to the carbon 3 position of T-2 (acetyl T-2) and deoxynivalenol (3-acetyl deoxynivalenol) or on substitution of an epoxide across the 9,10 carbons of diacetoxyscirpenol (beta-epoxide DAS). Effects of combining these and other mycotoxins were additive and showed no synergism or competition for binding to the active site. When in vitro effects of the mycotoxins were compared with results from whole animal lethality tests, several of the trichothecenes were weak inhibitors of protein synthesis in vitro but had in vivo toxicities similar to that of T-2 toxin. Thus, the in vitro cell response of a given trichothecene is not always an accurate predictor of toxicity in whole animals.
 
Article
Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with (125)I using chloramine T method, and separated by gel filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6)cpm/mouse) of (125)I native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain.
 
Article
The immunoglobulin fractions IgG, F(ab')2 and Fab of scorpion and snake antivenoms possess pharmacokinetic characteristics that are significantly different from their respective venoms. The venoms (and their toxins) are several fold faster in their distribution into the tissues than any of the immunoglobulin fraction. In rabbits, F(ab')2 possessed the fastest disposition rate constants and the longest distribution half lives. In the physiologically based pharmacokinetic experiments carried out in mice F(ab')2 possessed the highest Cp(max), smallest AUC and the shortest t1/2beta in the different tissues while Fab had values in between IgG and F(ab')2. Rescue experiments in anaesthetized rats challenged with lethal doses of venoms or toxins and infused with border-line neutralizing doses of antivenoms, showed that rats infused with F(ab')2 completely recovered, those infused with IgG partially rescued and none of the rats infused with Fab survived. It is concluded that F(ab')2 of scorpion and snake antivenoms possess pharmacokinetic characteristics that render it the most suitable for use in serotherapy of scorpion and snake envenoming.
 
Article
A novel approach for the production of [125I]amatoxin and anti-amanitin is described. The antigen and the starting material for Bolton Hunter iodination is prepared by periodic acid oxidation of the gamma-delta-dihydroxy-isoleucine side chain of alpha-amanitin followed by reductive amination. The antigen seems to be of low apparent toxicity. Chemical, spectroscopic and immunological evidence suggests that the hapten has a secondary structure similar to alpha-amanitin, but a modified tryptophanyl ring system. The establishment of a clinically useful iodinated radioimmunoassay system is possible, because the ring system seems to be of limited immunological importance.
 
Article
Botulinum neurotoxin, purified to homogeneity from Clostridium botulinum (Type A), was found to be highly neurotoxic (greater than 8 X 10(7) mouse LD50/mg protein). Labelling of this pure neurotoxin with 125I-iodine to high specific radioactivity was achieved without appreciable loss of biological activity. This was used to demonstrate saturable binding sites for this toxin at the neuromuscular junction, following in vivo administration into mice. A demonstrable inhibitory effect of the neurotoxin on release of acetylcholine from rat cerebrocortical synaptosomes indicates that it affects synapses in the central nervous system. Kinetic studies on the binding of 125I-labelled neurotoxin to brain synaptosomes yielded an association rate constant of 2.3 x 10(5)M-1s-1; dissociation plots were biphasic and the predominant species showed a rate constant of 1.2 X 10(-4)s-1. The saturable binding component is heat-sensitive and inactivated by trypsin. Preliminary studies showed that botulinum neurotoxin associates with plasma membrane fractions of synaptosomes and that binding does not result in any gross structural changes, at least in the majority of the toxin molecules.
 
Article
Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.
 
Article
The diffusion from the site of intramuscular injection of 900 kDa botulinum neurotoxin-hemagglutinin complex (BoNT/A-complex) and 150 kDa free-botulinum neurotoxin (free-BoNT/A) was compared. Radioiodinated compounds were injected into the gastrocnemius muscle of rats (70Units (U) 125I-BoNT/A-complex, 67 or 344 U free-125I-BoNT/A, or free-125I-iodide) and the eyelids of rabbits (24 U 125I-BoNT/A-complex or 108 U free-125I-BoNT/A), and measured in various tissues at different time points. There were no detectable systemic effects or generalized botulinum neurotoxin toxicity in either rats or rabbits, indicating that most of the toxin, whether as 125I-BoNT/A-complex or free-125I-BoNT/A, remained at the injection site. In rats, 125I-BoNT/A-complex and free-125I-BoNT/A diffused in a pattern that was grossly similar. Almost no radioactivity was recovered from the brain. Radioactivity recovered from distant tissues (thyroid, skin, and contralateral muscle) was primarily attributable to either low molecular weight 125I-containing peptides or 125I-iodide. After injection into rabbit eyelids, neither 125I-BoNT/A-complex nor free-125I-BoNT/A spread to distant structures, including the eye. The results indicate that most of the neurotoxin does not diffuse from the injection site, whether in free or complexed form, and this may reduce the potential for systemic effects.
 
Article
A three-compartment open pharmacokinetic model best fitted the data obtained following the i.v. injection of the venom, toxin and the immunoglobulin fractions into either rabbits or mice. The venom and toxin, however, possessed pharmacokinetic characteristics that were significantly different from the immunoglobulin fractions. The venom and toxin had very highly significantly greater disposition rate constants to the shallow and deep tissue compartments and overall elimination rate constant from the central compartment than any of the immunoglobulin fractions. This was reflected in other pharmacokinetic parameters, including highly significantly smaller areas under the curve (AUC) and highly significantly greater volumes of the central compartment (Vc), shallow tissue compartment (Vt shallow), deep tissue compartment (Vt deep) and total body clearance (TBC). In rabbits, F(ab')2 possessed the fastest disposition rate constants and the shortest distribution half-lives, while Fab showed the slowest disposition rate constants and the longest distribution half-lives. The same picture occurred in mice except that the values for Fab were between those of F(ab')2 and IgG. The time needed by the venom and toxin to reach maximum tissue concentration (tmax) ranged between 7 and 15 min and 60 and 180 min for the shallow and deep tissue compartments, respectively. The immunoglobulin fractions required 8-26-fold these times to attain tmax; F(ab')2 was the fastest to achieve its maximal concentration. Following i.m. injection, very fast absorption of venom and toxin took place, with the toxin reaching tmax within 5-20 min and 90% of the injected dose absorbed within 60 min. The bioavailability factor (F) was 0.82 and 0.88 for the venom and toxin, respectively. Fab had an F-value of 0.36 and required 4.3 and 47.4-fold the time taken by the venom and toxin to achieve tmax. The calculated values of F for F(ab')2 and IgG were 0.25 and 0.26, respectively. In the physiologically based pharmacokinetics (PBPK), the venom and toxin reached tmax in the different organs studied very rapidly while the immunoglobulin fractions required several-fold this time to attain tmax. F(ab')2 possessed the highest CPmax, the smallest AUC and the shortest t1/2 beta in the different tissues; Fab had values between F(ab)2 and IgG. It is concluded that F(ab')2 possesses pharmacokinetic characteristics that render it most suitable for use in serotherapy of snake and scorpion envenoming. It should be injected i.v. in doses higher than calculated neutralizing doses to compensate for the slow rate of distribution. Because of slow and incomplete absorption, the i.m. injection of the immunoglobulin fractions would be of little value in serotherapy.
 
Article
Toxic heptapeptides from a water bloom of the cyanobacterium Microcystis aeruginosa were purified by HPLC. The unoxidised fraction was iodinated with 125I plus 127I by the lactoperoxidase/H2O2 method, further purified by HPLC, and the non-iodinated and three iodinated fractions administered i.p. to male mice. All iodinated fractions were toxic, with symptoms and pathological lesions of the liver identical with those caused by non-iodinated peptide. Radioactivity was concentrated in the liver of mice at death.
 
Article
Sea anemones produce a series of toxic polypeptides and proteins with molecular weights in the range 3000-5000 that act by binding to specific receptor sites on the voltage-gated sodium channel of excitable tissue. This article reviews our current knowledge of the molecular basis for activity of these molecules, with particular emphasis on recent results on their receptor binding properties, the role of individual residues in activity and receptor binding, and their three-dimensional structures as determined by nuclear magnetic resonance spectroscopy. A region of these molecules that constitutes at least part of the receptor binding domain is proposed.
 
Poster
We present a retrospective study of 129 medical files concerning seafood poisonings (SFPs) registered at the central hospital of Tahiti (French Polynesia) between 1999 and 2005. Even if during that period most of the described cases (96%) concerned the ichtyosarcotoxism ciguatera, it is interesting to note that we also registered three other SFPs: tetrodotoxism, carchatoxism and lyngbyatoxism due to the consumption of tetraodon/diodon species, sharks or sea turtles, respectively. In ciguatera, cardiovascular symptoms were the primary criteria of severity with bradycardia and hypotension observed at 75% and 43%, respectively. Neurological manifestations (such as cerebellar syndrome, language troubles, diplopia or polyradiculoneuritis), trouble and/or loss of consciousness and dyspnoea were secondary criteria of severity. Body temperature was reported under 36.5 degrees C in 48 of 80 documented files. This observation, which has not previously been described in humans, may be related to possible central effects of the ingested toxin. The last remark concerns two extremely severe cases of ciguatera fish poisoning in which physicians had suspected an inflammatory neuropathy called the Guillain-Barré syndrome (GBS). Even if it is premature to conclude any correlation between the intoxication and the appearance of GBS, it is interesting to note that in both pathologies, morphological disturbances of nerve fibres have been reported.
 
Article
Toxicity studies of riddelliine, a member of a class of pyrrolizidine alkaloids, were conducted because riddelliine has been found to contaminate human food sources. Groups of male and female Fischer rats were administered riddelliine by gavage in phosphate buffer at doses up to 10 mg/kg, and B6C3F1 mice at doses up to 25 mg/kg, five times a week. The animals were necropsied after 13 weeks of treatment or after a 7 or 14 week recovery period. Body weight gains were inversely related to dose in both rats and mice. Body weight of the 1.0 and 3.3 mg/kg female rats and 10.0 and 25.0 mg/kg mice remained depressed during the 14 week recovery period. At 13 weeks, significant findings included dose-related hepatopathy and intravascular macrophage accumulation in rats and hepatocytomegaly in mice. During the 14 week recovery period these lesions persisted and hepatic foci of cellular alteration in male rats and bile duct proliferation in female rats and male and female mice increased in severity. In the 10 mg/kg group of female rats adenomas of the liver occurred in two of ten at 13 weeks and in one of five at the 14 week recovery period. In separate studies, the frequency of micronucleated erythrocytes in peripheral blood was increased in male mice administered a single dose (150 mg/kg) of riddelliine. Increases in unscheduled DNA and S-phase syntheses were detected in primary hepatocytes from rats and mice treated with riddelliine at doses up to 25.0 mg/kg for 5 or 30 days. In mating trials in rats and mice, pup weights from treated dams at birth and during suckling were lower than controls. Thus, riddelliine is genotoxic and carcinogenic and may cross the placenta and/or be found in milk, causing developmental toxicity in rodents.
 
Article
Gymnodimine-A and 13-desmethyl spirolide C are marine toxins belonging to the cyclic imine group produced by Karenia selliformis and Alexandrium ostenfeldii/peruvianum dinoflagellates, respectively. The aim of this work was to analyze the pharmacological properties of both toxins (at sub-lethal doses) on neuromuscular excitability, when injected locally to isoflurane-anesthetized mice, using a multimodal minimally-invasive in vivo electrophysiological approach. The main effect of both toxins was a marked reversible time- and dose-dependent decrease of the compound muscle action potential recorded from the tail muscle in response to caudal motor nerve stimulation. The dose-response curves of gymnodimine-A and 13-desmethyl spirolide C effect on the maximal amplitude of compound muscle action potential revealed 50% inhibitory doses of 51 ng/mouse (i.e. 1.6 μg/kg or 3.3 nmol/kg mouse) and 0.18 ng/mouse (i.e. 6 ng/kg or 0.01 nmol/kg mouse), respectively. The blocking effect occurred without significant modification of motor nerve excitability parameters. It is concluded that the inhibition of the mouse compound muscle action potential, induced by gymnodimine-A and 13-desmethyl spirolide C, results from an action of these toxins at the level of the skeletal neuromuscular junction, since both cyclic imine toxins are known to interact and block muscle-type nicotinic acetylcholine receptors. In the present in vivo study, 13-desmethyl spirolide C was about 300 fold more active than gymnodimine-A on equimolar basis. The present in vivo approach, associated to recent progress in chemical synthesis of cyclic imine toxins, paves the way for more detailed structure-activity studies to obtain new and more potent synthetic analogs.
 
Article
Alexandrium ostenfeldii is a widespread toxic dinoflagellate that has recently bloomed across the Adriatic Sea, seriously threatening both shellfish consumers and aquacultures. In 2007 we reported on preliminary studies carried out on field samples and cultures of A. ostenfeldii. At the time, along with three major spirolides - among which 27-hydroxy-13,19-didesmethyl spirolide C (3) proved to be a novel compound - a number of new minor spirolides were detected. Unfortunately, for all of them only Mass Spectrometry-based structural hypotheses could be ventured due to their very small amount. In the present paper we report on isolation and High Resolution Mass Spectrometry- and NMR-based structural elucidation of two of those minor spirolides detected in our previous study.
 
Article
Marine sponges have been shown to produce metabolites with cell growth- and endocrine-altering activities. We tested extracts from two species: the 'brown variable sponge' (Anthosigmella varians) and the 'West Indian bath sponge' (Spongia barbara), for effects on the cell cycle regulatory protein, cyclin B1; cell cycle growth-phase (sub-G1/apoptosis, G1, S, and G2/M); and cell survival in SW-13 human adrenal carcinoma cultures. Polyacrylamide gel electrophoresis studies indicated a 70-90% reduction in cyclin B1 levels by treatment with these agents. Microscopic examination of cultures with DAPI staining showed dense and fragmented DNA fluorescence, characteristic of apoptosis, in both sponge extract-treated cultures but not in controls. Flow cytometry analysis showed a 16-fold increase in the percentage of cells entering apoptosis (sub-G1 phase of cell cycle) by treatment with Anthosigmella varians extract (p <0.01) and a 10-fold increase using Spongia barbara extract (p <0.01) During this same time, the percentage of cells in G2/M was increased 1.6-fold by Anthosigmella varians extract (p <0.01) and 2.0-fold by Spongia barbara extract (p <0.01) Cell growth/survival studies also indicated a time-dependent decline in the percentage confluence of cell cultures exposed to Anthosigmella varians or Spongia barbara extracts. These experiments demonstrate that some species of marine sponges have metabolites which are capable of interfering with the mammalian cell cycle and with the survival of human adrenal carcinoma cells in culture.
 
Article
A combination of several equilibrium chromatographic steps permitted us to purify thirteen proteins from the venom of Buthus occitanus tunetanus using the mouse as the test animal for lethality measurement. These proteins have been characterized by their amino acid composition and, for seven of them, by their N-terminal or complete amino acid sequence. The structural data obtained correlate well with the pharmacological and antigenic properties of the neurotoxins.
 
Article
This study presents the effects of two synthetic disulfide bond variants of cal16b, a 13-mer Ca(2+) channel blocker conotoxin, on pro- and anti-inflammatory cytokine gene transcription in the murine macrophage-like cell line J774A.1 stimulated with LPS. The globular form (cal16b_1) acted as an anti-inflammatory agent; in contrast, the ribbon disulfide variant (cal16b_2) had a pro-inflammatory effect. Our results suggest that the pro- and anti-inflammatory effects are mediated by the three-dimensional structure.
 
Article
Some species of marine sponge have been shown to produce metabolites with endocrine-altering and cell growth regulatory properties. Since cell division and differentiation are controlled, in part, by the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK/ERK) cascade, we tested extracts (1.0mg/ml) from six shallow water marine species obtained in the Florida Keys for effects on MAPK/ERK(l,2) (sub-variant of EC 2.7.1.37) activity in incubations with SW-13 human adrenal carcinoma cells in culture. In these short-term incubations, extracts from two species, the purple bleeding sponge (Iotrochota birotulata) and the West Indian bath sponge (Spongia barbara), significantly inhibited MAPK/ERK(1,2) activity (to 51 and 44% of control levels, respectively) without altering cell survival. Western blots for phosphorylated and total ERK showed that ERK(2) predominated over ERK(1) by a factor of about 4:1 and that the phosphorylated forms of these isozymes were strongly suppressed by active extracts from both sponges. Another species, the green sponge (Haliclona veridis), whose extract has been shown previously to activate guanylate cyclase and to inhibit adenylate cyclase in a variety of mammalian tissues, was found not to affect MAPK/ERK(1,2) in human adrenal carcinoma cultures but did lyse and kill most of these cultured cells. Extracts from the sheepswool sponge (Hippospongia lachne) and the bleeding sponge (Oligoceras hemorrhages) did not significantly affect either MAPK/ERK(1,2) activity or the survival of attached cells. An extract from the fire sponge (Tedania ignis) did not alter MAPK/ERK(1,2) activity but did modestly decrease cell viability. These studies document for the first time species-specifc effects of marine sponge extracts on the MAPK/ERK(1,2) cascade and on the growth and survival of human adrenal carcinoma cells in culture.
 
Article
Bm-TFF2 is an amphibian trefoil factor purified from the Bombina maxima skin secretion that is highly toxic to mammals. We previously reported that Bm-TFF2 activates human platelets via protease-activated receptor 1. In this study, for a better understanding of platelet activation induced by Bm-TFF2, we used affinity chromatography and pharmacological inhibitors to investigate the downstream signaling pathway. Using Bm-TFF2-affinity chromatography, Gq was specifically eluted from the Bm-TFF2-coulped column. Pharmacological inhibitors such as U73122, Xestospongin C, BAPTA-AM and Gö6976 can significantly inhibit Bm-TFF2-induced platelet aggregation. These results suggested that Gq activation and the downstream PLCβ-IP3 receptor-cytoplasmic Ca(2+)-PKC signaling pathway is crucial for Bm-TFF2 to stimulate platelet aggregation. Furthermore, Bm-TFF2 induced strong platelet shape change at the concentrations of 5nM, in which the Ca(2+) mobilization of the platelets stimulated was not detectable. The p160(ROCK) inhibitorY27632 totally inhibited the shape change, indicating that Bm-TFF2 may activate the G12/13 pathway which leads to the activation of RhoA-p160(ROCK). In conclusion, Bm-TFF2 induced platelet activation mainly via the Gq and G12/13 signaling pathway. This study on the signaling pathway of Bm-TFF2 stimulation may help us understand the toxicity of B. maxima skin secretion to the human platelets.
 
Article
Bothrops erythromelas venom (BeV) has been responsible for many snake accidents in Brazil. We investigated the plasmatic pharmacokinetic of BeV labeled with (131)I in the absence and the presence of anti-Bothrops serum (BAS). A higher percentage of BeV plasmatic radioactivity and longer elimination were found in the presence of BAS. Our results showed a redistribution of venom from the tissue to vascular compartment associated with the treatment of envenomed mice with anti-venom 15 min after venom injection.
 
Article
The production of secondary metabolites by cyanobacteria is extensively varied. Anabaena spp. have been shown to produce the toxins saxitoxin, anatoxin-a, anatoxin-a(s), cylindrospermopsin and microcystin. In this study we show cytotoxicity associated with Anabaena circinalis 131C that could not be attributed to its high saxitoxin content. Analytical, ELISA and enzymatic methods excluded the presence of other known cyanobacterial toxins leading to the suggestion of novel toxicity.
 
Article
K. Harada, K. Ogawa, K. Matsuura, H. Nagai, H. Murata, M. Suzuki, Y. Itezono, N. Nakayama, M. Shirai and M. Nakano. Isolation of two toxic heptapeptide microcystins from an axenic strain of Microcystis aeruginosa, K-139. Toxicon 29, 479-489, 1991.-Two toxic heptapeptides were isolated from an axenic Microcystis aeruginosa strain, K-139. Using mainly a non-destructive NMR method, we determined the structure of the major toxin to be 7-desmethylmicrocystin LR which lacks an N-methyl group of the dehydroalanine moiety of microcystin LR. The minor toxin was deduced to be 3,7-didesmethylmicrocystin LR. The chromatographic and NMR analyses of 7-desmethylmicrocystin LR were compared with those of 3-desmethylmicrocystin LR.
 
Top-cited authors
José María Gutiérrez
  • University of Costa Rica
Bruno Lomonte
  • University of Costa Rica
Wayne Carmichael
  • Wright State University
Ramachandra Kini
  • University of Mysore
Lourival D Possani
  • Universidad Nacional Autónoma de México