HPLC of trichothecenes — Separation of neosolaniol,NT-1 toxin,and NT-2 toxin

A high pressure liquid chromatographic (HPLC) method is described for the isolation of trichothecenes formed on moldy rice. Extraction of the cultures was followed by purification and fractionation with a C₁₈-Sep Pak cartridge. The polar fraction contained neosolaniol, 4,8-diacetoxy-12,13-epoxytrichothec-9-ene-3,15-diol (NT-1) and 4-acetoxy-12,13-epoxytrichothec-9ene-3,8,15-triol (NT-2), while in another fraction HT-2 toxin, T-2 toxin and acetyl-T-2 toxin were eluted. A high pressure liquid chromatograph equipped with a C₁₈ μ Bondapak column and an R I-detector (differential refractometer) were used for the isolation procedure.     

Ribonucleases in bacterial toxin–antitoxin systems

Toxin–antitoxin (TA) systems are widespread in bacteria and archaea and play important roles in a diverse range of cellular activities. TA systems have been broadly classified into 5 types and the targets of the toxins are diverse, but the most frequently used cellular target is mRNA. Toxins that target mRNA to inhibit translation can be classified as ribosome-dependent or ribosome-independent RNA interferases. These RNA interferases are sequence-specific endoribonucleases that cleave RNA at specific sequences. Despite limited sequence similarity, ribosome-independent RNA interferases belong to a limited number of structural classes. The MazF structural family includes MazF, Kid, ParE and CcdB toxins. MazF members cleave mRNA at 3-, 5- or 7-base recognition sequences in different bacteria and have been implicated in controlling cell death (programmed) and cell growth, and cellular responses to nutrient starvation, antibiotics, heat and oxidative stress. VapC endoribonucleases belong to the PIN-domain family and inhibit translation by either cleaving tRNA^fMet in the anticodon stem loop, cleaving mRNA at -AUA(U/A)-hairpin-G- sequences or by sequence-specific RNA binding. VapC has been implicated in controlling bacterial growth in the intracellular environment and in microbial adaptation to nutrient limitation (nitrogen, carbon) and heat shock. ToxN shows structural homology to MazF and is also a sequence-specific endoribonuclease. ToxN confers phage resistance by causing cell death upon phage infection by cleaving cellular and phage RNAs, thereby interfering with bacterial and phage growth. Notwithstanding our recent progress in understanding ribonuclease action and function in TA systems, the environmental triggers that cause release of the toxin from its cognate antitoxin and the precise cellular function of these systems in many bacteria remain to be discovered. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

SAXS study of the snake toxin α-crotamine

-crotamine is a small toxic protein (42 amino acid residues with three disulphide bridges) present in the venom of Crotallus durissus terrificus. Molecular parameters (R _g=13.7 Å, S=3,000 Ų, V=9,200 ų and D _max=40 Å) were derived from SAXS curves obtained from a solution of this protein at pH=4.5. An excellent agreement between the experimental distance distribution curve and that calculated from a model consisting of two lobes linked by the Cys(18)-Cys(30) disulphide bridge.     

Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH(2), NAD, NADPH(2) and NADP. 2. The binding stoicheiometry is 2moles of NADH(2) and 1mole of NADPH(2)/mole of diphtheria toxin. The binding sites for NADH(2) appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0.01I, pH7 and 25 degrees are 0.7x10(-6)m for NADH(2) and 0.45x10(-6)m for NADPH(2). Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH(2) and NADPH(2) may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH(2) are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin-nucleotide complexes is discussed in this regard.     

Within-host competition selects for plasmid-encoded toxin–antitoxin systems

Toxin–antitoxin (TA) systems are commonly found on bacterial plasmids. The antitoxin inhibits toxin activity unless the system is lost from the cell. Then the shorter lived antitoxin degrades and the cell becomes susceptible to the toxin. Selection for plasmid-encoded TA systems was initially thought to result from their reducing the number of plasmid-free cells arising during growth in monoculture. However, modelling and experiments have shown that this mechanism can only explain the success of plasmid TA systems under a restricted set of conditions. Previously, we have proposed and tested an alternative model explaining the success of plasmid TA systems as a consequence of competition occurring between plasmids during co-infection of bacterial hosts. Here, we test a further prediction of this model, that competition between plasmids will lead to the biased accumulation of TA systems on plasmids relative to chromosomes. Transposon-encoded TA systems were added to populations of plasmid-containing cells, such that TA systems could insert into either plasmids or chromosomes. These populations were enriched for transposon-containing cells and then incubated in environments that did, or did not, allow effective within-host plasmid competition to occur. Changes in the ratio of plasmid- to chromosome-encoded TA systems were monitored. In agreement with our model, we found that plasmid-encoded TA systems had a competitive advantage, but only when host cells were sensitive to the effect of TA systems. This result demonstrates that within-host competition between plasmids can select for TA systems.     

The ζ toxin induces a set of protective responses and dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε(2)) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20-30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1-5×10(-5)). Early after induction ζ toxin alters the expression of ～78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K(+). We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε(2) antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (～10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε(2) antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity.     

Bacteriophages and pathogenicity: more than just providing a toxin?

An increasing number of pathogenicity factors carried by bacteriophages have been discovered. This review considers bacteriophage-bacterium interaction and its relation to disease processes. We discuss the search for new bacteriophage-associated pathogenicity factors, with emphasis on recent advances brought by the use of genomic sequence data and the techniques of genomic epidemiology.     

Pasteurella multocida toxin activates Gβγ dimers of heterotrimeric G proteins

The mitogenic Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida, which causes Pasteurellosis in man and animals. The toxin activates the small GTPase RhoA, the MAP kinase ERK and STAT proteins via the stimulation of members of two G protein families, G_q and G_12/13. PMT action also results in an increase in inositol phosphates, which is due to the stimulation of PLCβ via Gα_q. Recent studies indicate that PMT additionally activates Gα_i to inhibit adenylyl cyclase. Here we show that PMT acts not only via Gα but also through Gβγ signaling. Activation of Gβγ by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) γ and formation of phosphatidylinositol-3,4,5-trisphosphate (PIP₃) as indicated by the recruitment of a PIP₃-binding pleckstrin homology (PH) domain-containing protein to the plasma membrane. Moreover, it is demonstrated that Gβγ is necessary for PMT-induced signaling via Gα. Mutants of Gα_q incapable of binding or releasing Gβγ are not activated by PMT. Similarly, sequestration of Gβγ inhibits PMT-induced Gα-signaling.     

Cross-reactivity of monoclonal antibodies againstClostridium perfringens Θ toxin with streptolysin O

Six monoclonal antibodies to toxin ofClostridium perfringens were characterized. Four antibodies, 1C3, 2D4, 1B9, and 3F11, were nonneutralizing for toxin and were non-cross-reacting with streptolysin O (SLO). The other two antibodies, 3H10 and 2C5, were cross-binding and cross-neutralizing with SLO. Neutralizing activity of 3H10 was higher than that of 2C5 on the basis of the binding activity with toxin and SLO. Both antibodies could inhibit hemolysis even after binding of the toxins to sheep red blood cells and inhibited cardiotoxicity of the toxins in cultured heart cells.     

Do skeletal muscle properties recover following repeat onabotulinum toxin A injections?

Onabotulinum toxin A (BTX-A) is a frequently used treatment modality to relax spastic muscles by preventing acetylcholine release at the motor nerve endings. Although considered safe, previous studies have shown that BTX-A injections cause muscle atrophy and deterioration in target and non-target muscles. Ideally, muscles should fully recover following BTX-A treatments, so that muscle strength and performance are not affected in the long-term. However, systematic, long-term data on the recovery of muscles exposed to BTX-A treatments are not available, thus practice guidelines on the frequency and duration of BTX-A injections, and associated recovery protocols, are based on clinical experience with little evidence-based information. Therefore, the purpose of this study was to investigate muscle recovery following a six months, monthly BTX-A injection (3.5 U/kg) protocol. Twenty seven skeletally mature NZW rabbits were divided into 5 groups: Control (n=5), zero month recovery – BTX-A+0 M (n=5), one month recovery – BTX-A+1 M (n=5), three months recovery – BTX-A+3 M (n=5), and six months recovery – BTX-A+6 M (n=7). Knee extensor strength, muscle mass and percent contractile material in injected and contralateral non-injected muscles was measured at each point of recovery. Strength and muscle mass were partially and completely recovered in injected and contralateral non-injected muscles for BTX-A+6 M group animals, respectively. The percent of contractile material partially recovered in the injected, but did not recover in the contralateral non-injected muscles. We conclude from these results that neither target nor non-target muscles fully recover within six months of a BTX-A treatment protocol and that clinical studies on muscle recovery should be pursued.     

Characterization and comparative analysis of toxin–antitoxin systems in Acetobacter pasteurianus

Journal of Industrial Microbiology & Biotechnology - Bacterial toxin–antitoxin (TA) systems play important roles in diverse cellular regulatory processes. Here, we characterize three...     

Dietary mixing in three generalist herbivores: nutrient complementation or toxin dilution?

We reared larvae of three generalist insect species on plants occurring in their habitats. Individuals of each species were kept either on mixed diets, or on each plant species separately. We measured food plant preference in the mixed-diet group and compared insect performance on single plants to the performance on the mixed diet. For all three insect species, food choice within the mixed-diet groups was non-random and delivered the best overall performance, thus fulfilling the criteria for self-selected diets. When a single diet was as good as the mixed diet for one particular aspect of performance (Adenostyles alliariae and Petasites albus for Miramella alpina; A. alliariae for Callimorpha dominula), it was never the most preferred food plant in the mixed-diet treatment. Whether the benefit achieved by mixing diets is due to nutrient complementation or toxin dilution, we argue that there is no easy way to distinguish between the two hypotheses on the basis of consumption and performance measurements, as has previously been proposed. From the interpretation of utilisation plots, the ANCOVA equivalent of nutritional indices, we were able to gain insight into where in the sequence from ingestion to growth (preingestive, predigestive or postdigestive) single diets caused differences from mixed diets. The elements of this control system which were influenced by single diets varied considerably, both within and between insect species. No food plant was toxic or deterrent to all experimental insect species; a food plant that caused consumption effects (preingestive) for one insect species could be dealt with metabolically (postdigestive) by another; different food plants could cause behavioural effects (preingestive), metabolic effects (postdigestive), or a combination of both effects, all within the same insect species. However, one generality did emerge: once a food was ingested, further growth-relevant effects occurred metabolically (postdigestive) rather than via differential egestion (digestibility). Received: 5 October 1998 / Accepted: 1 March 1999     

A naturally-occurring carboxyl-terminally truncated α-scorpion toxin is a blocker of sodium channels

α-Scorpion toxins constitute a multigene family of evolutionarily conserved venom peptides that inhibit sodium channel inactivation and increase its peak current. Here, we describe the characterization of a new α-scorpion toxin gene expressed in the venom gland of Mesobuthus eupeus that encodes a carboxyl-terminally truncated product of 38 residues (named MeuNaTxα(NT)-1). Synthetic MeuNaTxα(NT)-1 was oxidized to form two disulfide bridges in an alkaline environment and the refolded peptide exhibits different structure and function from the classical α-scorpion toxin. MeuNaTxα(NT)-1 blocks sodium channels on rat dorsal root ganglia (DRG) neurons without impact on the inactivation of the channels. This work provides a clue for evolution-guided design of channel blockers for therapeutic aims.     

β Sheet to α helix transition in the binding subunit of cholera toxin

Circular dichroism spectra have been measured for the binding subunit of cholera toxin in water and in the presence of dodecyl sulfate. In water the protein has an appreciable amount of β structure and almost no α helix. In the presence of dodecyl sulfate the spectrum undergoes drastic change over a time period of approximately four hours, and at equilibrium resembles that expected for a chain with an appreciable amount of α helix but no β structure. The change in helicity is in good agreement with that expected from our formulation of the configuration partition function for the binding subunit. The conformational change may have an important relationship to the means by which the binding subunit permits penetration of the active subunit into the cell.     

ADAM17-overexpressing breast cancer cells selectively targeted by antibody–toxin conjugates

A disintegrin and metalloproteinase 17 (ADAM17) is significantly upregulated not only in malignant cells but also in the pro-inflammatory microenvironment of breast cancer. There, ADAM17 is critically involved in the processing of tumor-promoting proteins. Therefore, ADAM17 appears to be an attractive therapeutic target to address not only tumor cells but also the tumor-promoting environment. In a previous study, we generated a monoclonal anti-ADAM17 antibody (A300E). Although showing no complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity, the antibody was rapidly internalized by ADAM17-expressing cells and was able to transport a conjugated toxin into target cells. As a result, doxorubicin-coupled A300E or Pseudomonas exotoxin A-loaded A300E was able to kill ADAM17-expressing cells. This effect was strictly dependent on the presence of ADAM17 on the surface of target cells. As a proof of principle, both immunotoxins killed MDA-MB-231 breast cancer cells in an ADAM17-dependent manner. These data suggest that the use of anti-ADAM17 monoclonal antibodies as a carrier might be a promising new strategy for selective anti-cancer drug delivery.     

Expression and secretion of β-glucuronidase and Pertussis toxin S1 by Streptomyces lividans

 Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coliβ-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the β-glucuronidase gene was fused with the leader sequence produced up to 30 mg β-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/l. The disappearence of the B. pertussis toxin S1 and β-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans. Received: 19 July 1995/Received revision: 3 November 1995/Accepted: 20 November 1995     

Presence of a dinucleotide fold in cholera toxin: Possible approach to chemoprophylaxis?

The ADP-ribosyltransferase activity of the A₁. subunit of cholera toxin is specifically inhibited by the dye cibacron blue 3GA. The presence of a ‘dinucleotide fold’ in the A₁ subunit is thus established for the first time. This specific inhibition observed in vitro is successfully exploited in vivo for the inhibition of the diarrheal response brought out by the pure toxin in the rabbit ileal-loop test.