Toxin Fraction Research Articles

Differential inhibition of Ca 2+ channels in mature rat cerebellar Purkinje cells by sFTX-3.3 and FTX-3.3

Synthetic funnel web spider toxin (sFTX-3.3) is a polyamine amide analogue of FTX, a toxin fraction isolated from the venom of the funnel web spider, Agelenopsis aperta, that blocks P-type Ca 2+ channels. The structures of these polyamine containing compounds are not identical: sFTX-3.3 contains an amide carbonyl oxygen that is absent from the predicted structure of native FTX. Recently, a compound called FTX-3.3 was synthesized with the structure predicted for native FTX. We have compared the effects of polyamine amide sFTX-3.3 and polyamine FTX-3.3, on Ca 2+ channel currents in the soma of mature rat cerebellar Purkinje neurons, in which the predominant Ca 2+ channels are defined as P-type. Differential inhibition by sFTX-3.3 and FTX-3.3 revealed three populations of Ca 2+ channels. One group, mediating ~66% of the current, was blocked by sFTX-3.3 with an IC 50 (concentration producing half maximal inhibition) of 33 nM or by FTX-3.3 with an IC 50 of 55 pM. A second population (5–25% of the total current) was inhibited by sFTX-3.3 with an IC 50 of 33 nM, but was insensitive to FTX-3.3, while a third (10–30%) was blocked by FTX-3.3 with an IC 50 of 125 nM and was resistant to sFTX-3.3. These channels also showed distinctive currentvoltage relationships. Our results suggest that P-type Ca 2+ channels in mature rat cerebellar Purkinje cells may be subdivided according to pharmacological and biophysical properties.

Cytological characterization of Hordeum vulgare L. under the influence of a toxin produced by Drechslera teres (Sacc.) Shoem.

Both application of the crude toxin preparation from Drechslera teres and treatment with fractions of this toxin induced similar cytological defects in the leaf tissue selected from Hordeum vulgare L., like the inoculation of the pathogen: changes in chloroplast- and mitochondria-structure, necrosis of single cells. The toxin fraction 6 with the highest biological activity still triggered cytological detectable changes in the leaf-tissue of the susceptible barley variety ‘Femina’ at a concentration of 1 ppm. Tests of barley genotypes with a defined degree of resistance against the pathogen showed, that the toxin induced cytological damages are useful for a differentiation between resistant and susceptible plants. The host nonspecifity of the D. teres-toxin also could be demonstrated at the ultrastructural level in selected non-host plants.

Specific features of the effect of “murine” toxin and antigen ofYersinia pestis fraction I on cells of plague-sensitive animals

Effects ofYersinia pestis capsular antigen and toxin on the cells of experimental antimals are described. The antigen of bacterial fraction I enhanced the oxidative burst of white mice peritoneal leukocytes but suppressed the activity of guinea pig leukocytes. The effect of “murine” toxin was quite the contrary. Moreover, the effects of the toxin on the phosphorylation of leukocyte membrane and cytosol proteins of various origin differed, this correlating with fluctuations in the activity of tyrosine phosphatase.

Gonyautoxin-3 as a minor toxin in the gastropod Niotha clathrata in Taiwan

Paralytic toxicity was detected in the gastropod mollusc Niotha clathrata collected from South Taiwan in April and November 1993. Each seasonal toxin was partially purified from toxic specimens of N. clathrata by ultrafiltration using a membrane (Diaflo YM-2), followed by chromatography on a column (Bio-Gel P-2). Two toxin fractions (I and II) were then obtained for each seasonal shell toxin. The ratio of fraction I to fraction II for each seasonal shell toxin was about 4:1 according to tetrodotoxin bioassay. Based on analyses by TLC, electrophoresis, and HPLC, fraction I toxin contained tetrodotoxin and its derivative anhydrotetrodotoxin, and fraction II toxin contained gonyautoxin-3 for each seasonal shell toxin.

P-type Ca2+ channels mediate excitatory and inhibitory synaptic transmitter release in crayfish muscle.

The toxin fraction (FTX) and peptide omega-Aga-IVA from the venom of the funnel-web spider Agelenopsis aperta, as well as a synthetic analogue of FTX, specifically block the P-type voltage-dependent Ca2+ channel (VDCC). The effects of these toxins on synaptic transmission were studied in the neuromuscular synapses of the crayfish opener muscle, which has a single excitatory and a single inhibitory motoneuron. FTX selectively and reversibly blocked excitatory and inhibitory postsynaptic currents and potentials in a dose-dependent manner. FTX had no effect on (i) resting and postsynaptic membrane conductance, (ii) postsynaptic L-type VDCC, and (iii) both glutamate- and gamma-aminobutyric acid-induced postsynaptic responses. Mean amplitude and frequency of miniature postsynaptic potentials were unchanged by FTX. The postsynaptic VDCC was inhibited by nifedipine, a selective dihydropyridine antagonist of L-type VDCC, whereas synaptic transmission was unaffected. Transmission was also undisturbed by omega-conotoxin, suggesting that N-type VDCCs are not involved. The peptide omega-Aga-IVA blocked excitatory and inhibitory transmission without affecting postsynaptic VDCC. Synaptic transmission was also blocked by synthetic FTX. We conclude that presynaptic P-type VDCCs are involved in both evoked excitatory and inhibitory transmitter release in crayfish neuromuscular synapses.

A toxin fraction (FTX) from the funnel-web spider poison inhibits dihydropyridine-insensitive Ca2+ channels coupled to catecholamine release in bovine adrenal chromaffin cells.

In adrenal chromaffin cells, depolarization-evoked Ca2+ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca2+ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca2+ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K(+)-depolarization-induced Ca2+ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1-1 microM) and FTX (3:3), a synthetic FTX analogue (1 mM), blocked the [Ca2+]i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca2+]i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by approximately 80 and 70%, respectively, but both substances together abolished the K(+)-evoked catecholamine release, as measured by HPLC. The omega-conotoxin GVIA (0.5 microM) was without effect on K(+)-stimulated 45Ca2+ uptake. Our results indicate that FTX blocks dihydropyridine- and omega-conotoxin-insensitive Ca2+ channels that, together with L-type voltage-sensitive Ca2+ channels, are coupled to catecholamine release.

In Vivo Accumulation of Sodium Pump Inhibitor by Normal and Uremic Erythrocytes

A uremic toxin fraction (fraction 2-3-1) in the middle molecular mass range (300-2000 Da) containing a sodium potassium inhibitor pump was studied. As fraction 2-3-1 was known to be present in minute quantities in urine and plasma, erythrocyte lysates were used as an alternative source. Results show that fraction 2-3-1 is more abundant in erythrocytes than in normal urine. In addition, the fraction is present in a greater quantity in uremic erythrocytes than in normal blood cells. It is concluded that fraction 2-3-1 is concentrated at the erythrocyte level in uremic patients.

Occurrence of a new toxin and tetrodotoxin in two species of the gastropod mollusk Nassariidae

The lethalities of 102 specimens of three species of the gastropod mollusk Nassariidae, collected from fish markets in Taiwan, were examined. The frequency of toxicity in Zeuxis scalaris and Z. castus-like specimens was 94 and 41%, respectively. The range of lethal potency in toxic specimens of Zeuxis scalaris and Z. castus-like was 2–140 and 2–13 mouse units, respectively, while all tissues of Z. castus were non-toxic. The toxins were partially purified from the toxic specimens of Z. scalaris and Z. castus-like. Two toxin fractions were obtained from the extract of each species of Nassariidae by using Bio-Gel P-2 column chromatography. Analyses by thin layer chromatography, electrophoresis, high performance liquid chromatography and ultraviolet spectroscopy, showed that toxin fraction I contained tetrodotoxin while toxin fraction II contained a new neurotoxin.

Ascorbic Acid Derivatives in Two Different Fractions of Uremic Toxins

The middle-molecular-weight uremic toxins which accumulate in uremic plasma seem to be associated with various uremic disorders such as uremic neuropathy and defects in the sodium pump. By a multi-step chromatographic method, two fractions of these toxins were isolated and studied because one inhibits microtubule formation in vitro (fraction 2-5), and the other impairs the sodium pump in living erythrocytes (fraction 2-3). An additional chromatographic method allows the separation of these fractions and isolation of two components: fractions 2-3-V and 2-5-III. Analyses by UV and 1H NMR spectrometry identified these compounds as two different ascorbic acid derivatives. 2-3-V is not yet totally identified and 2-5-III corresponds to ascorbic acid 2-sulfate. These two metabolites exert no toxic effects but they have the same chromatographic behavior as uremic toxins.

Diarrhea in ciguatera fish poisoning: Preliminary evaluation of pathophysiological mechanisms

Ciguatera fish poisoning is a clinical syndrome consisting of a combination of gastrointestinal and neurological symptoms occurring after eating toxincontaining tropical reef fish; it is a major cause of morbidity in Hawaii, the South Pacific, Australia, and the Caribbean. In an effort to define pathophysiological mechanisms responsible for the diarrheal component of the illness, we examined the effect of crude and fractionated toxin preparations on isolated rabbit ileal tissue in a Ussing chamber model. Both the crude toxin preparation (prepared from toxic Ctenochaetus strigosus) and 10% and 50% methanol-chloroform toxin fraction (prepared from a pool of toxic fish samples) gave a striking increase in transepithelial electrical potential difference and short-circuit current. Enterotoxic activity seemed to be mediated by calcium. When examined by light microscopy, the intestinal mucosa was not damaged by the toxin preparations used. Our data demonstrate that toxins involved in ciguatera fish poisoning directly stimulate intestinal fluid secretion without accompanying tissue damage and suggest that calcium is the “second messenger” mediating the process.

Development of an ELISA to assess the potency of horse therapeutic antivenom against Thai cobra venom

An ELISA for the quantitation of antibodies against Naja naja siamensis venom proteins has been developed for use as a possible replacement for the in vivo neutralization assay of antivenom potency. Comparison was made with three preparations of venom proteins as antigens of ELISA; these were the crude venom, a toxin fraction and the purified principle postsynaptic neurotoxin of the Thai cobra. Eight batches of horse monovalent therapeutic anti-cobra antivenom, one of which served as positive reference, were assayed by the ELISAs and also by the in vivo neutrakization assay using mice. When crude venom, the toxin fraction and the pure neurotoxin were used as antigens in the ELISAs, the correlation coefficients between the ELISA antibody titers and in vivo neutralization of the antivenoms were 0.82 ( P < 0.005), 0.94 ( P < 0.001) and 0.95 ( P < 0.001), respectively. Thus, the ELISA which measures only the antibody against the principle toxin of the snake venom should be most suitable for use as an in vitro assay of antivenom potency. The ELISA should also be useful for potency assessment and standardization of antivenoms against other elapid snake venoms whose lethal components are small, poorly immunogenic peptides.

Funnel-web spider venom and a toxin fraction block calcium current expressed from rat brain mRNA in Xenopus oocytes.

Injection of rat brain mRNA into Xenopus oocytes has been shown to induce a calcium current (ICa) that is insensitive to dihydropyridine and omega-conotoxin. We examined the effect of funnel-web spider venom on two aspects of this expressed ICa: (i) the calcium-activated chloride current [ICl(Ca)] and (ii) the currents carried by barium ions through calcium channels (IBa). In the presence of 1.8 mM extracellular calcium, ICl(Ca) tail current became detectable between -30 and -40 mV from a holding potential of -80 mV and reached a maximal amplitude between 0 and +10 mV. Total spider venom partially (83%) and reversibly blocked the calcium-activated chloride current without changing its voltage sensitivity. A chromatographic toxin fraction from the venom also blocked this current (64%). The venom had a minimal effect on INa and IK. Direct investigation of inward current mediated by calcium channels was carried out in high-barium solution. IBa had a higher threshold of activation (-30 to -20 mV) and reached its maximal amplitude at about +20 mV. Total venom or a partly purified chromatographic toxic fraction blocked IBa partially and reversibly without changing its current-voltage characteristics. Furthermore, the extent of the total venom block depended on the concentration of extracellular barium. Only 35% of the IBa was blocked in 60 mM Ba2+, whereas the block increased to 65% and 71%, respectively, for 40 and 20 mM Ba2+. On the basis of these results, we propose that the calcium channels expressed from rat brain mRNA in Xenopus oocytes is similar to the recently discovered P-type channels.

The effect of nortriptyline-specific active immunization on amitriptyline toxicity and disposition in the rabbit

Rabbits were actively immunized by a conjugate of nortriptyline (NT) to study the effect of specific anti-NT antibodies on toxicity and disposition of amitriptyline (AT). Control and immunized rabbits received 115 mg/kg AT intraperitoneally (i.p.). The lethality dose (LD) profile exhibited a gentle slope; LD 100 and LD 0 were separated by 100 mg/kg. Mortality was significantly reduced from LD 67 to LD 43 ( P < 0.05). Total plasma concentrations of the toxin were increased in the immunized group compared to the control group. AUC 0.5−24 h value was 5-fold higher in the immunized group than in the control group. Moreover, a smaller fraction of unbound toxin in plasma was observed in the immunized group than in the control group. These observations indicate that AT was actively sequestred by antibodies. The intensity of this phenomenon was a function of both the antibody affinity constant (10 9 M −1) and the neutralizing capacity (varying from 0.005 to 0.2 mg/kg) of the circulating antibodies in each immunized rabbit. Results clearly show that anti-NT antibodies are able to effectively sequestrate AT.

Rapid purification of the peptide toxins microcystin-LR and nodularin

An isoation procedure was developed to provide within one day microcystin-LR, a cyclic heptapeptide toxin from Microcystis aeruginosa PCC 7806. After ODS (octadecylsilyl) solid phase extraction, the crude toxin fraction was chromatographed using a strong anion exchange column. The toxin was eluted with 0.02 M ammonium bicarbonate. An at least 95% purity was revealed on HPLC separation by monitoring at 214 nm. Application of the procedure to the cyclic pentapeptide toxin nodulation from Nodularia spumigena AV2 was examined.

Rapid purification of the peptide toxins microcystin-LR and nodularin

An isolation procedure was developed to provide within one day microcystin-LR, a cyclic heptapeptide toxin from Microcystis aeruginosa PCC 7806. After ODS (octadecylsilyl) solid phase extraction, the crude toxin fraction was chromatographed using a strong anion exchange column. The toxin was eluted with 0.02 M ammonium bicarbonate. An at least 95% purity was revealed on HPLC separation by monitoring at 214 nm. Application of the procedure to the cyclic pentapeptide toxin nodularin from Nodularia spumigena AV2 was examined.

Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison.

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells.

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and 3H-deoxyglucose-6-phosphate release from human red blood cells

Ming-Shi Jiang, J. E. Fletcher and L. A. Smith. Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and 3H-deoxyglucose-6-phosphate release from human red blood cells. Toxicon 27, 1297–1305, 1989.—At a low concentration of Naja naja kaouthia cardiotoxin (3 μM) Ca 2+, Sr 2+ and Ba 2+ (2 mM), had little to no effect on 3H-deoxyglucose-6-phosphate ( 3H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (≥ 10 μM), Ca 2+ (2 mM), but not Sr 2+ or Ba 2+, significantly enhanced 3H-dGlu-6-p and hemoglobin release. Mn 2+ (2 mM) almost completely inhibited 3H-dGlu-6-p release and hemolysis at both the 3 μM and 10 μM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 μM), Ca 2+ at low concentrations (0.5 mM) enhanced 3H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 μM) induced 3H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. 3H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between 3H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca 2+-dependent. The Ca 2+-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A 2 contamination in the toxin fraction. Mn 2+ is an effective antagonist of cardiotoxin action.

Variations in major toxin composition for six clones of Ptychodiscus brevis

—Extracts from six clones of Ptychodiscus brevis (formerly known as Gymnodinium breve) were analyzed by high performance liquid chromatography for the presence of brevetoxins PbTx-1, PbTx-2, and PbTx-3. Analyses indicated a wide clonal variability of the three toxin fractions in logarithmic phase cultures when normalized on a per cell basis. It appears that a much wider variability exists in toxin content for different P. brevis clones than exists in replicate extraction of multiple cultures of the diploid clone originally isolated by Wilson.

Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli.

A mutant, full-length form of diphtheria toxin was cloned into Escherichia coli K-12 and expressed under BL-1 + EK-1 containment. A gene fragment encoding the catalytic domain of the toxin was subjected to oligonucleotide-directed mutagenesis to produce a three-base mutation of an active site residue; Glu-148 was thereby replaced by Ser. Ser-148 fragment A had less than 1% of the ADP-ribosyltransferase activity of wild-type fragment A. Next, the complementary portion of the toxin structural gene was spliced with the mutated DNA fragment downstream of codon 148 to produce a construct that encoded mutant whole toxin with Ser at position 148. The mutant toxin was indistinguishable from authentic diphtheria toxin by Western blot analysis, but was about 800-fold less cytotoxic than wild-type toxin for BS-C-1 cells. Evidence from subunit exchange experiments indicated that a substantial fraction of the mutant toxin contained a fully functional B moiety, capable of mediating the entry of wild-type fragment A into sensitive mammalian cells. This combination of approaches provides a means of applying recombinant DNA methods in E. coli to study structure-function relationships in whole diphtheria toxin.

Isolation and partial purification of lantana ( Lantana camara L.) toxins

A partially purified toxin fraction and lantadene A were obtained from Lantana camara L. leaves by batch extraction, column chromatography and fractional crystallization. Toxicity was tested in guinea pigs. The total number of chemical entities in the partially purified toxin preparation was 7, the 2 major ones being lantadene A and lantadene B. Lantadene A was nontoxic in itself. Likewise, another fraction containing lantadene A, lantadene B and 3 more components with higher polarity was found to be non-toxic. The toxic component(s) are different from lantadene A/B but appear to resemble them very closely.