(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin

AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin. METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action. Periodate oxidation and 1H-NMR was used to determine the receptor nature. Affinity chromatography on pustulan-Sepharose column was used to purify D. hansenii killer toxin, probably a 23-kDa protein. The killer toxin character was cureless. CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans. This is a low molecular weight protein, probably encoded by chromosomal genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin. This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.     

Chronic Exposure to Pertussis Toxin Alters Muscarinic Receptor-Mediated Regulation of Cyclic AMP Metabolism in Neuroblastoma × Glioma NG108–15 Hybrid Cells

Chronic pertussis toxin treatment (5 days) of NG108-15 neuroblastoma X glioma hybrid cells had no significant effect on basal cyclic AMP levels whereas it effectively blocked the inhibitory action of acute (10 min) exposure of carbachol (10(-4)M) on intracellular cyclic AMP accumulation, stimulated by prostaglandin E1. This action of pertussis toxin was found to be long lasting: exposure of the cells to pertussis toxin (100 ng/ml) for only 24 h followed by a 5-day withdrawal period still was shown effective on day 7 in abolishing the inhibitory action of carbachol on prostaglandin E1-stimulated cyclic AMP production. Chronic exposure (5 days) of NG108-15 cells to carbachol (10(-5)M) causes an increase in basal cyclic AMP levels by 98%, and a desensitization of the muscarinic inhibition of cyclic AMP accumulation, assessed after a 24-h withdrawal period. When carbachol treatment is carried out in the presence of pertussis toxin (100 ng/ml) both of these effects of carbachol are abolished.     

Effect of Clostridium perfringens Alpha Toxin on Contraction of Isolated Guinea-Pig Diaphragm

The effect of Clostridium perfringens alpha toxin on contraction induced by-electric stimulation of isolated guinea-pig diaphragm was investigated. The toxin inhibited electrically stimulated contraction of the tissue in a dose- and incubation time-dependent manner. Tetrodotoxin resulted in no effect of the action of the toxin. Nifedipine dose-dependently delayed the action of the toxin, but verapamil and diltiazem did not. On the other hand, treatment of the toxin with N-acetylimidazole caused significant reduction of the inhibitory activity of the toxin on contraction, but did not cause significant loss of phospholipase C activity (PN activity) as measured by hydrolysis of p-nitrophenylphosphorylcholine. The data showed that the toxin impairs contraction of isolated guinea-pig diaphragm.     

Effect of local depression of inhibition on electrical activity of the spinal cord

The dorsal cord, dorsal root, and focal potentials in response to peripheral nerve stimulation were investigated in rats with local depression of inhibition in the left or right half of the lumbar segments produced by the action of tetanus toxin. The investigation was carried out at the stage of poisoning when excitation of the neuron population with disturbed inhibition caused generalized excitation of spinal and bulbar motoneurons. Experiments on spinal animals showed that if a cutaneous nerve is stimulated on the side affected by the toxin these responses have a greater amplitude and a much longer duration than those evoked by stimulation of the opposite nerve or responses in healthy rats. The maximal increase in amplitude and duration of the negative component of the focal potential corresponding to the time of the increased P wave of the dorsal cord potential was found in the ventral quadrant on the side affected by the toxin. Besides evoked focal potentials, spontaneous rhythmic negative waves also were recorded in this area. The mechanisms of spread of seizure activity from the focus of depressed inhibition are discussed and the structures generating spreading seizure activity are identified.     

Interaction of snake venom cardiotoxin (a membrane-disruptive polypeptide) with human erythrocytes

The action of 7.2 µM cardiotoxin on 0.25% human erythrocytes in a plasma extender solution was studied by the interaction of toxin with intact red blood cells and subsequent hemolysis of the cells. The binding of toxin to cells was completed within 10 min, whereas the membrane rigidity was weakened in a non-lytic period for about 25 min. The toxin molecules bound almost exclusively to the membrane. The bound toxin could not be liberated with either 0.5% Triton X-100 or 0.1 N NaOH. The degree of binding was slightly reduced in the presence of 10 mM mono- and divalent inorganic salts. The action of toxin might weaken the in situ association of several proteins that are linked with band 3 protein of the membrane, thus making the cells fragile and altering the shape of the cell to a smooth sphere.     

A mechanistic interpretation of the action of toxin II from Anemonia sulcata on the cardiac sodium channel

Cardiac sodium channels, modified by Anemonia sulcata toxin II, have been analyzed by the patch-clamp method. The open state of the modified sodium channels proved to be prolonged highly significantly and reopening from a closed state denoted c*-state frequently occurred, interrupted by silent periods, denoted i*-state. Activation from the c*-state was apparently not affected by toxin action, whereas activation from the i*-state was markedly prolonged. Upon higher depolarizations toxin-induced sodium channels disappeared and this behaviour has been attributed to dissociation of the toxin from the channel by use of a special pulse-protocol. The onset of the toxin effect on the action potential proved to depend on stimulation, and it is concluded that the toxin binds preferentially to the open (o)-state. Taking together the results, a kinetic scheme is suggested for action of the toxin on the cardiac sodium channel.     

Detection of Clostridium novyi type B α toxin by cell culture systems

Ten permanent cell lines were examined for their reaction to the Clostridium novyi alpha toxin. The action of the toxin was determined after 3 days by microscopic examination and the MTT assay. The alpha toxin exhibited the strongest effect on ESH-L cells rather than other cell lines. Vero and SFT-R cells reacted in a comparable way, but less sensitively. We were able to show that the cytopathic effect on the three types of cells was neutralised by the international standard for gas gangrene antitoxin (C. novyi) but in no case by heterologous antisera. Our results have shown that the three cell lines were specific indicators for the detection of the cytopathic effect of alpha toxin. The cytopathic effect can be measured reproducibly by the cell culture assay used. These results are suitable as the starting point for the development of the neutralisation test using cell cultures.     

Changes in plasma membrane fluidity lower the sensitivity of<Emphasis Type="Italic">S. cerevisiae</Emphasis> to killer toxin K1

The possible correlation between plasma membrane fluidity changes induced by modified cultivation conditions and cell sensitivity to the killer toxin K1 of Saccharomyces cerevisiae were investigated. Cells grown under standard conditions exhibited high toxin sensitivity. Both a membrane fluidity drop and fluidity rise brought about markedly reduced sensitivity to the toxin. These results do not fit the hypothesis of physiological relevance of direct toxin-lipid interaction, suggesting that the essential event in killer toxin action is interaction with membrane protein(s) that can be negatively influenced by any changes of membrane fluidity.     

Expression in yeast of a cDNA copy of the K2 killer toxin gene

Summary A cDNA copy of the M2 dsRNA encoding the K2 killer toxin ofSaccharomyces cerevisiae was expressed in yeast using the yeastADH1 promoter. This construct produced K2-specific killing and immunity functions. Efficient K2-specific killing was dependent on the action of the KEX2 endopeptidase and the KEX1 carboxypeptidase, while K2-specific immunity was independent of these proteases. Comparison of the K2 toxin sequence with that of the K1 toxin sequence shows that although they share a common processing pathway and are both encoded by cytoplasmic dsRNAs of similar basic structure, the two toxins are very different at the primary sequence level. Site-specific mutagenesis of the cDNA gene establishes that one of the two potential KEX2 cleavage sites is critical for toxin action but not for immunity. Immunity was reduced by an insertion of two amino acids in the hydrophobic amino-terminal region which left toxin activity intact, indicating an independence of toxin action and immunity.     

Calcium-independent and dependent steps in action of Clostridium perfringens enterotoxin on HeLa and Vero cells.

Purified enterotoxin (20–200 ng/ml) of $\text{Clostridium}$$\text{perfringens}$ rapidly induced bled and balloon formation on HeLa and Vero cells in the presence, but not the absence, of Ca²⁺. The action of the toxin involved two, sequential, temperature-dependent steps: The first was Ca²⁺-independent and included binding of toxin and the bound toxin after 30–60 sec could no longer be removed by washing. The second step was Ca²⁺-dependent and eventually led to bled and balloon formation. On adding Ca²⁺ to cells pretreated with toxin in Ca²⁺-free medium, bled and balloon formation started immediately. The ionophore A23187 mimicked the action of toxin. The effects of sucrose (0.2 M), trypsin-treatment of the cells and various pretreatments of the toxin on the action of enterotoxin were studied.     

The theoretical 3D structure of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry5Ba

Cry5Ba is a δ-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.     

Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.     

Influence of a Ceratocystis ulmi Toxin on Water Relations of Elm (Ulmus americana)

Water-soluble glycopeptides isolated from cultures of Ceratocystis ulmi have been reported to be toxins involved in Dutch elm disease. The influence of the glycopeptides on the water relations of Ulmus americana seedlings was tested by placing cut stems in glycopeptide preparations. After 4 hours in 200 micrograms per milliliter toxin the stem conductance of the seedlings was reduced by 79% and the leaf water potential was reduced by 3 bars to that at which the seedlings wilted, the stomata closed, and transpiration decreased. Decrease in stem conductance as the mode of action of the toxin was further confirmed by forcing toxin through the stem and petiole of elm and measuring the effects on stem conductance. High molecular weight dextrans were found to mimic the action of toxin on stem and petiole conductance, and their ability to do so was found to be correlated with their molecular weight. As low as 4 micrograms of toxin or dextrans were found to measurably decrease the stem and petiole conductance of elms. Disruption of the water-conducting system of elms and other plants by small quantities of high molecular weight compounds may be a factor in diseases with wilting symptoms.     

Factors affecting the susceptibility of sensitive yeast cells to killer toxin K1

Optimum conditions for action of the killer toxin K1 on sensitive strainS. cerevisiae S6 were established. Maximum killing was reached in a very narrow pH range of 4.5–4.6. Maximum susceptibility to toxin was displayed by highly energized fresh cells from the early exponential phase in the presence of an external energy source (at least 200 mmol/L glucose). Further, maintenance of maximum membrane potential was necessary for killer action, as documented by decreasing toxin activity in the presence of increasing concentrations of KCl. The killing was strongly stimulated in the presence of millimolar concentrations of Ca²⁺ and Mg²⁺.     

Actions of β-Bungarotoxin on Amino Acid Transmitter Release

Abstract: The actions of purified β-bungarotoxin (5 or 10 μg/ml) on the metabolic and transmitter-releasing properties of rat cortical synaptosomes was studied. The toxin stimulated control respiratory rates, but prevented the respiratory response to veratrine. Tissue potassium levels were greatly reduced (54%) and endogenous glutamate, aspartate and GABA showed increased levels of release (10- to 25-fold), but other amino acids were unaffected or showed much smaller changes. Tissue levels of these amino acids were reduced in proportion. The toxin inhibited the uptake of [U-¹⁴C]GABA (35%) and [U-¹⁴C]glutamate (53%) over 5-min incubation periods. This uptake-inhibition was Ca²⁺-dependent and was reduced by tetrodotoxin. Miniature-end-plate-potential (m.e.p. p.) frequencies at the locust neuromuscular junction (extensor tibialis) were greatly (four- to sevenfold) accelerated by local application of the toxin (5 μg/ml). This effect was reversible and occupied about 20 min. Amplitudes of m.e.p. p.'s were also increased and muscle membrane depolarization occurred. The results are interpreted as being due to a depolarizing action of the toxin.     

Purification and chemical characterization of the major neurotoxin from the venom of Pelamis plautrus.

A major toxin was isolated from the venom of the sea snake Pelamis platurus (yellow-bellied sea snake) by Sephadex G-50 and carboxymethylcellulose column chromatography. The LD50 of the pure toxin (Pelamis toxin a) was 0.044 mug/g in mice representing a tenfold increase in toxicity after purification. The toxin was homogeneous in acrylamide disc gel electrophoresis and eluted as a single peak after isoelectric focusing in a sucrose density gradient column. The isoelectric point was 9.69; thus it is a highly basic protein. The toxin contained 55 amino acid residues with four disulfide linkages. When all disulfide linkages were reduced and alkylated, the toxic action of the pure toxin disappeared leading to the conclusion that the disulfide bonds of the neurotoxin were essential for toxic action.     

Autonomic effects of scaritoxin in the cat and guinea pig. Cardiac protection by an anti-arrhythmia agent

A purified extract from the muscles of the fish Scarus gibbus (93% scaritoxin and 7% ciguatoxin) was tried on anesthetized cats and isolated guinea-pig atria. Intra-venous injection to cats (10, 20 and 40 mg/kg) induced deep respiratory depression, inhibition of duodenal contractions, increased salivary secretion, mydriasis and lacrymal secretion. Heart rate was slowed by 17 to 44 per cent. Impaired cardiac conduction led to different types of blocks. On the contrary, cardiac excitability was enhanced, as evidenced by the occurence of many auricular and mostly ventricular extra-systoles, often evolving into long periods of ventricular paroxysmal tachycardia. The rate of contraction of isolated guinea-pig atria was equally depressed and this effect was antagonized by atropine. The duration of the refractory period was shortened; an inotropic effect was observed and the action of acetylcholine was potentiated by the toxin. Cholinesterase inhibition by scaritoxin, previously mentioned in other papers, is thus evidenced. Mydriasis and duodenal inhibition may be tentatively explained by a nicotinic effect on the sympatheticoadrenal system. This mechanism may be responsible for cardiac inotropic action and increased excitability. Other mechanisms may be suggested, including a possible digitalis-like effect due to a cyclopentanone ring in the scaritoxin molecule. Interestingly, intra-venous injections of the antiarrythmic drug procainamide (20 to 30 mg/kg) could either prevent or reverse the signs of cardiac hyperexcitability in the anesthetized cat. However, death results usually from respiratory depression, the intimate mechanism of which is not yet known.     

Endogenous protease-activated 66-kDa toxin from Bacillus thuringiensis subsp. kurstaki active against Spodoptera littoralis

The anti-lepidopteran toxin from sporulated Bacillus thuringiensis subsp. kurstaki cells, generated by the proteolytic action of endogenous protease(s) on the protoxin, was purified and studied to identify the effect of such proteolysis on the biochemical nature of the toxin. The active toxin was purified employing anion-exchange chromatography to absolute homogeneity, as indicated by SDS-PAGE and Western blotting. Antisera to the purified toxin (66 kDa) crossreacted with the protoxin (132 kDa) confirming its origin from protoxin. The purified toxin with a pI of 7.95 was derived from the N-terminal region of the protoxin (pI 7.6). Circular dichroism data revealed that the toxin has significant secondary structure and it undergoes pH dependent conformational change. Unlike the toxin generated by exogenous proteases such as trypsin, etc., the endogenous protease(s) activated toxin is highly lethal to a tolerant insect variety of the lepidopteran order, Spodoptera littoralis.     

Morphologic changes in cultures of different tissues exposed to the toxins of C1. perfringens types B, C, E and F]

There were revealed morphological peculiarities of the action of C1. perfringens toxins, types B, C, D, E and F on the cultures of fibroblasts of chick embryo, amniotic cells and intestinal tissue. The toxin type B was characterized by a marked vocuolization of the cell cytoplasm; the action of the toxin of type C was expressed in the swelling of the nuclei and the lysis of the chromatine substance, the toxin of type E casued kariorhexis, and the toxin of type F--hyperchromatosis of the nuclei. All the cultures proved to be insensitive to the toxin of type D. Peculiarity of the morphological affection of the cells permitted to differentiate toxin of type B in the cultures of the fibroblasts of chick embryo, whereas the toxins of types C, E and F--in the cultures of the amniotic cells under control of the reaction of neutralization with the homologous antitoxic sera.