Wheat germ in?vitro translation to produce one of the most toxic sodium channel specific toxins

Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Aah (Androctonus australis hector)II protein that requires the proper formation of four disulphide bonds. Soluble, recombinant N-terminal GST (glutathione S-transferase)-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified rAahII (recombinant AahII) toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice. An LD₅₀ (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD₅₀ of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active rAahII. In addition, NbAahII10 (Androctonus australis hector nanobody 10), a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified rAahII upon injection in mice.     

Persistence of specific antigenic protein in the serum of chickens given intravenously botulinum toxin type B, C, D, E or F

A sublethal dose of Clostridium botulinum progenitor toxin of each of types B, C, D, E, and F was injected once intravenously into chickens. Blood samples were withdrawn periodically from the chickens to determine the toxin remaining in the serum by the mouse injection test and by enzyme-linked immunosorbent assay (ELISA) for both toxic and nontoxic components composing the progenitor toxin. Both components were detected by ELISA for at least a few days after the serum had became innocuous to mice, indicating a higher stability of the antigenicities of both components than the lethal toxicity in the chicken serum. For the diagnosis of botulism, it seems justified to recommend detection of the antigen (toxic component or nontoxic component or both) by ELISA even if no toxin is detected by the mouse test. Such immunological tests would no doubt contribute to an increase in the rate of diagnosis of human and animal botulism cases, particularly when blood sampling is delayed.     

Biochemical,functional, structural and phylogenetic studies on Intercro,a new isoform phospholipase A2 from Crotalus durissus terrificus snake venom

Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA₂ (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin.     

A Novel Insecticidal Toxin from Photorhabdus luminescens, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of Manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.     

Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal binary toxin during sporulation. The binary toxin is composed of toxic BinA (41.9 kDa) and receptor binding BinB (51.4 kDa) polypeptides and is active against vectors of filariasis, encephalitis and malaria. The toxin has been tested with limited use for the control of vector mosquitoes for more than two decades. The binA gene from a local ISPC-8 strain of B. sphaericus that is highly toxic to Culex and Anopheles mosquito species was cloned into pET16b and expressed in Escherichia coli. The purified BinA protein differs by one amino acid (R197 M) from BinA of the highest toxicity strains 1593/2362/C3-41. Majority of the expressed protein was observed in inclusion bodies. BinA inclusions alone from E. coli did not show toxic activity, like reported previously. However, the active form of BinA could be purified to homogeneity from the soluble fraction of E. coli cell lysate, grown at reduced temperature after isopropyl β-d-thiogalactopyranoside induction. The purified BinA protein with and without poly-histidine tag showed LC₅₀ dose of 82.3 and 66.9 ng ml⁻¹, respectively, at 48 h against Culex quinquefasciatus larvae. The secondary structure of BinA is expected to be mainly β strands as estimated using far-UV circular dichroism. The estimates matched well with the secondary structure predictions using amino acid sequence. This is the first report of large-scale purification and accurate toxicity estimation of soluble B. sphaericus BinA. This can help in design and synthesis of improved bacterial insecticide.     

On the nucleotide binding domain of fructose-1,6-bisphosphatase.

Native chicken liver fructose-1,6-bisphosphatase (Fru-P₂ase) can bind to blue dextranSepharose affinity column and is not displaced by its sugar-phosphate substrate; however; it is readily eluted by the inhibitor 5′-AMP. Treatment of Fru-P₂ase with pyridoxal 5′-phosphate (pyridoxal-P) in the presence of the substrate, fructose 1,6-bisphosphate, followed by reduction with NaBH₄ leads to the formation of active pyridoxal-P derivatives of the enzyme showing diminished sensitivity to AMP inhibitor. The modified enzyme does not bind to the affinity column. On the other hand, in the presence of AMP modification of Fru-P₂ase with pyridoxal-P occurs at the catalytic site; this modification does not alter its binding behavior toward the dye ligand. Blue dextran can also protect Fru-P₂ase against AMP inhibition, and it is a competitive desensitizer for the nucleotide ligand. The results establish that blue dextran binds specifically to the allosteric site of the enzyme, and that the structure of this site may resemble that of the dinucleotide fold in other enzymes. Like native Fru-P₂ase, digestion of pyridoxal-P-Fru-P₂ase (with regulatory properties altered) with subtilisin causes a severalfold increase in the catalytic activity measured at pH 9.2, without significant change in the activity at pH 7.5, and produces a peptide with 56 amino acids. The residual subunit, M_r ~ 30,000, was found to contain all of the incorporated pyridoxal-P.     

Histidine as an essential residue in the active site of the copper enzyme galactose oxidase.

The pH dependence of the oxidation of β-methyl-d-galactopyranoside by galactose oxidase at 1.33 mm O₂ has been determined. The k_cat exhibits a bell-shaped dependence on the ionization of at least two groups in the enzyme-substrate complex, pK_b' = 6.3 and pK_a' = 7.1, respectively. The pH-independent value for k_cat at 1.33 mm O₂ (nonsaturating) and saturating glycoside is 1435 s^?; the pH optimum is 6.7. Galactose oxidase is inactivated rapidly by iodoacetamide. Although the reaction is much slower, iodoacetate also inactivates the enzyme. The inactivation by iodoacetamide obeys saturation kinetics; at pH 7.0 k₃ = 2.19 min^?1 and K_i = 5.1 mM; k₃ but not K_i exhibits a bell-shaped pH dependence, with pK_a values of 6.3 and 7.6, respectively. Labeling with [¹⁴C]iodoacetamide establishes that one carboxamidomethyl group is incorporated per enzyme molecule. This incorporation parallels the loss of enzymatic activity. Only N-3-carboxymethylhistidine is detected in chromatograms following hydrolysis of the labeled protein. The protein-bound copper is not lost as a consequence of alkylation. Apogalactose oxidase does not react with iodoacetamide. The alkylation is inhibited by the oxidation of an active center tryptophan residue (s) by N-bromosuccinimide. The fraction of residual enzyme activity remaining after tryptophan oxidation corresponds to the extent of labeling by [¹⁴C]iodoacetamide. Although alkylation causes little change in the spin Hamiltonian parameters of the Cu(II) atom, it nearly abolishes both the optical activity and optical absorbance of the metal. The native tryptophan fluorescence of the enzyme, which is a sensitive probe of its active site, is also markedly affected. Since binding of a substrate, β-methyl-d-galactopyranoside, reduces fluorescence as it does in the active enzyme and binding of CN^? at the Cu(II) site as detected by electron spin resonance appears unaffected by the alkylation, the effect of alkylation is on catalysis, per se. Both a catalytic and a subtle conformational role for the active site histidine are inferred from the results.     

Tightly Bound Binary Toxin in the Cell Wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.     

环亚胺毒素gymnodimine的研究进展

Gymnodimine(GYM)是1994年从新西兰牡蛎中被鉴定出的藻毒素.其由凯伦藻（Karenia selliformis）产生,结构中含有一个位于螺环上的亚胺氮,属于环亚胺毒素.亚胺是GYM的毒理功能基团,具有很高的小鼠腹腔注射急性致死毒性,口服毒性很小,但详细的毒理作用机制尚不清楚.本文基于有限的研究资料,系统综述了GYM的结构、来源生物、毒性机理、携带生物、地理分布、降解代谢、剂量响应关系及风险评估等研究现状,并对今后藻毒素的重点研究方向进行了展望.     

Alanine-162 of Bacillus thuringiensis Cyt2Aa2 toxin is essential for membrane binding and oligomerisation

Cyt2Aa2 is a cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. It is specifically toxic to dipteran larvae in vivo and is also active against several cell types, such as erythrocytes. The active toxin is proposed to bind to the cell membrane, and membrane pore formation by toxin oligomerisation leads to cell lysis. This study aimed to characterise the role of residues (I139, S159, L160, S161, A162, D209 and V215) potentially involved in the membrane binding of Cyt2Aa2. All mutants, except I139A and V215A, showed similar characteristics to the wild-type toxin after proteinase K cleavage. Three mutants, S159A, L160A and S161A, showed high haemolytic activity but low toxicity against Aedes aegypti. Membrane interaction assays showed that these mutants could bind to rat red blood cells (rRBCs) and oligomerise. The mutant D209N had no haemolytic activity but was still mildly toxic to A. aegypti. The mutant A162V could not lyse rRBCs, even at high concentrations, and showed no toxicity against A. aegypti. Our data suggest that alanine 162 of the Cyt2Aa2 toxin is involved in membrane binding and oligomerisation. Substitution of this amino acid altered the conformation of the toxin and affected its biological activity.     

Target cell specificity of the Pasteurella haemolytica leukotoxin is unaffected by the nature of the fatty-acyl group used to activate the toxin in vitro

The leukotoxin (LktA) of Pasteurella haemolytica is active only against cells of ruminant origin. It is synthesised as an inactive protoxin encoded by the lktA gene and post-translationally modified to the active toxin by the product of the lktC gene. The LktA and LktC proteins were expressed separately in Escherichia coli and partially purified. Active LktA was produced in vitro in the presence of LktC and acyl-acyl carrier protein (ACP) charged separately in vitro with a fatty-acyl group. The toxic activity and target cell specificity of LktA and adenylate cyclase toxin (CyaA), a toxin active against a wide variety of mammalian cells, were investigated after activation with ACP charged with different fatty acids. Palmitoyl-ACP produced the most active toxin in both cases and, although other fatty acids were also effective, the fatty acid preference was the same for the in vitro activation of both toxins. Activated LktA remained ruminant cell-specific whichever acyl group was used to acylate the A protoxin.     

Properties and use of botulinum toxin and other microbial neurotoxins in medicine.

Crystalline botulinum toxin type A was licensed in December 1989 by the Food and Drug Administration for treatment of certain spasmodic muscle disorders following 10 or more years of experimental treatment on human volunteers. Botulinum toxin exerts its action on a muscle indirectly by blocking the release of the neurotransmitter acetylcholine at the nerve ending, resulting in reduced muscle activity or paralysis. The injection of only nanogram quantities (1 ng = 30 mouse 50% lethal doses [U]) of the toxin into a spastic muscle is required to bring about the desired muscle control. The type A toxin produced in anaerobic culture and purified in crystalline form has a specific toxicity in mice of 3 x 10(7) U/mg. The crystalline toxin is a high-molecular-weight protein of 900,000 Mr and is composed of two molecules of neurotoxin (ca. 150,000 Mr) noncovalently bound to nontoxic proteins that play an important role in the stability of the toxic unit and its effective toxicity. Because the toxin is administered by injection directly into neuromuscular tissue, the methods of culturing and purification are vital. Its chemical, physical, and biological properties as applied to its use in medicine are described. Dilution and drying of the toxin for dispensing causes some detoxification, and the mouse assay is the only means of evaluation for human treatment. Other microbial neurotoxins may have uses in medicine; these include serotypes of botulinum toxins and tetanus toxin. Certain neurotoxins produced by dinoflagellates, including saxitoxin and tetrodotoxin, cause muscle paralysis through their effect on the action potential at the voltage-gated sodium channel. Saxitoxin used with anaesthetics lengthens the effect of the anaesthetic and may enhance the effectiveness of other medical drugs. Combining toxins with drugs could increase their effectiveness in treatment of human disease.     

Detection of neutral sugars in purified type G botulinum progenitor toxin and the effects of some glycolytic enzymes on its molecular dissociation and oral toxicity

Arabinose and galactose were detected in purified type G botulinum toxin (Mr about 500,000) of Clostridium argentinense. The i.p. LD50/mg N of type G progenitor toxin was one-tenth, but the oral LD50/mg N twice that of type A-L toxin. The lysozyme-, endo-beta-galactosidase-, and N-glucanase-treated toxins each had a molecular mass of about 300,000. The oral toxicity of the endo-beta-galactosidase or N-glucanase-treated toxin was one-fifth that of untreated progenitor toxin. On DEAE-Sephadex chromatography, the N-glucanase-treated toxin dissociated into two fractions, nontoxic and toxic. SDS-PAGE of the toxic fraction showed a single band with a Mr of about 150,000, and after dithiothreitol treatment, two bands with Mr of 100,000 and 50,000.     

A Study on the Safety and Effects of Amorpha fruticosa Fruit Extract on Spontaneously Hypertensive Rats with Induced Type 2 Diabetes

Metabolic syndrome is characterized by a variety of diagnostic criteria: obesity, dyslipidemia, type 2 diabetes, and arterial hypertension. They contribute to the elevated risk of cardiovascular morbidity and mortality. The potential for Amorpha fruticosa L. (Fabaceae) to improve diabetes and metabolic disease is promising, based on in vitro tests. This is why a further investigation of the species is needed. Additionally, a toxicity review in relation to safety revealed that to date, there are no published data regarding the toxicity of A. fruticosa towards humans. This species could provide abundant and cheap resources because it is an aggressive invasive plant that grows almost unrestrictedly. The objective of this study was to evaluate the acute toxicity of a purified extract of A. fruticosa (EAF), and to assess its antioxidant, antihypertensive, and antihyperglycemic activity in streptozotocin-induced diabetic spontaneously hypertensive rats (SHRs). The EAF was slightly toxic (LD₅₀ = 2121 mg/kg, b.w.) when administered orally, and moderately toxic (LD₅₀ = 316 mg/kg, b.w.) at intraperitoneal administration, both in mice. The oral administration of EAF (100 mg/kg) for 35 days to SHRs caused significant decreases in the systolic pressure, blood glucose levels, and MDA quantity. It also increased the hepatic level of the endogenous antioxidant GSH, not only in diabetic SHRs, but also in the control group. An additional potential benefit to human health might be conferred through the environmental management of A. fruticosa based on its large-scale use for medicinal purposes, as this aggressive invasive species brings problems to natural habitats in many European countries.     

The X-ray structure of a snake venom Gln48 phospholipase A2 at 1.9A resolution reveals anion-binding sites

Phospholipase A2 is an "interfacial" enzyme and its binding to negatively charged surfaces is an important step during catalysis. The Gln48 phospholipase A2 from the venom of Vipera ammodytes meridionalis plays the role of chaperone and directs a toxic His48 PLA2 onto its acceptor. In the venom the two phospholipases A2 exist as a postsynaptic neurotoxic complex, Vipoxin. The X-ray structure of Gln48 PLA2, complexed to sulphate ions, which mimic the negatively charged groups of anionic membranes, has been determined by the molecular replacement method and refined to 1.9A resolution. The protein forms a homodimer stabilized by ionic, hydrophobic, and hydrogen-bond interactions. The structure reveals two anion-binding sites per subunit. These sites are probably involved in interactions with the negatively charged membrane surface and, in this way, in the "targeting" of the toxic component to the receptors of the postsynaptic membranes. In the absence of the chaperone subunit the toxin changes the target of the physiological attack. A comparison of the homodimeric Gln48 PLA2 structure with that of the heterodimeric Vipoxin reveals differences in regions involved in the pharmacological activity of the toxin. This fact, except the active site histidine substitution, can explain the absence of toxicity in the Gln48 protein in comparison to the His48 phospholipase A2.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Purification and chemical and biological characterizations of seven toxins from the Mexican scorpion, Centruroides suffusus suffusus

Seven polypeptides highly toxic to mice were isolated from the venom of the scorpion, Centruroides suffusus suffusus (Css), and their chemical and toxic properties were characterized. It was shown that the most active toxins by intracerebroventricular injection are less active when injected subcutaneously. The complete amino acid sequence (66 residues) of toxin II (Css II) has been determined. The C-terminal end is amidated as found for most other scorpion toxins. Css II is a beta-type toxin, previously used to define the binding site for activation of the sodium channel. Using rat brain synaptosomes, we demonstrated that all Css toxins compete with 125I-Css II to bind to site 4 and should be considered as beta-scorpion toxins. Specific binding parameters for Css VI, one of the most active toxins, were determined: KD = 100 pM; capacity in binding sites, 2.2 pmol of toxin/mg of synaptosomal protein. Css VI was shown to inhibit gamma-aminobutyric acid uptake by synaptosomes: K 0.5 = 100 pM, which agrees with its KD. Competition experiments between the seven Css toxins and 125I-Css II for antiserum raised against Css II demonstrated that all these toxins have common antigenic properties.     

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.     

Acute oral toxicity of Yersinia pseudotuberculosis to fleas: implications for the evolution of vector-borne transmission of plague

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