Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin

We attempted to characterize ADP-ribose-amino acid bonds formed by various bacterial toxins. The ADP-ribose-arginine bond formed by botulinum C2 toxin in actin was cleaved with a half-life of about 2 h by treatment with hydroxylamine (0.5 M). In contrast, the ADP-ribose-cysteine bond formed by pertussis toxin in transducin and the ADP-ribose-amino acid linkage formed by botulinum ADP-ribosyltransferase C3 in platelet cytosolic proteins were not affected by hydroxylamine. HgCl2 cleaved the ADP-ribose-amino acid bond formed by pertussis toxin in transducin but not those formed by botulinum C2 toxin or botulinum ADP-ribosyltransferase C3 in actin and platelet cytosolic proteins, respectively. NaOH (0.5 M) cleaved the ADP-ribose-amino acid bonds formed by botulinum C2 toxin and pertussis toxin but not the one formed by botulinum ADP-ribosyltransferase C3. The data indicate that the ADP-ribose bond formed by botulinum ADP-ribosyltransferase C3 differs from those formed by the known bacterial ADP-ribosylating toxins.     

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.     

Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle

Botulinum toxin injection into the pylorus is reported to improve gastric emptying in gastroparesis. Classically, botulinum toxin inhibits ACh release from cholinergic nerves in skeletal muscle. The aim of this study was to determine the effects of botulinum toxin on pyloric smooth muscle. Guinea pig pyloric muscle strips were studied in vitro. Botulinum toxin type A was added; electric field stimulation (EFS) was performed every 30 min for 6 h. ACh (100 microM)-induced contractile responses were determined before and after 6 h. Botulinum toxin caused a concentration-dependent decrease of pyloric contractions to EFS. At a low concentration (2 U/ml), botulinum toxin decreased pyloric contractions to EFS by 43 +/- 9% without affecting ACh-induced contractions. At higher concentrations (10 U/ml), botulinum toxin decreased pyloric contraction to EFS by 75 +/- 7% and decreased ACh-induced contraction by 79 +/- 9%. In conclusion, botulinum toxin inhibits pyloric smooth muscle contractility. At a low concentration, botulinum toxin decreases EFS-induced contractile responses without affecting ACh-induced contractions suggesting inhibition of ACh release from cholinergic nerves. At higher concentrations, botulinum toxin directly inhibits smooth muscle contractility as evidenced by the decreased contractile response to ACh.     

Effect of botulinum D toxin on neutrophils

Activated botulinum D toxin ADP-ribosylates a 22 kDa molecular weight protein in homogenates obtained by sonication of a suspension of rabbit peritoneal neutrophils. The ADP-ribosylation catalyzed by activated botulinum D toxin is inhibited in homogenates obtained from cells pretreated with the toxin, suggesting that it is able to enter into these cells and be activated by them. The rise in intracellular concentration of free calcium in toxin treated cells stimulated by fMet-Leu-Phe is similar to that found in control cells. The basal concentration of intracellular free calcium is significantly elevated in neutrophils treated with the intact but not with the activated form of the botulinum D toxin. Superoxide generation in control and native toxin treated cells stimulated with fMet-leu-Phe, phorbol 12-myristate 13-acetate or opsonized zymosan is the same. The release of beta-glucosaminidase produced by fMet-Leu-Phe or Concanavalin A in botulinum D toxin treated neutrophils was slightly higher than the corresponding release in control cells. Furthermore, the fMet-Leu-Phe-induced increase in the amount of actin associated with the cytoskeleton is not inhibited by botulinum D toxin. These results suggest that the 22 kDa protein which can be ADP-ribosylated by botulinum D toxin is not involved in these stimulated neutrophil responses.     

A型肉毒毒素轻链胞内持留模型的建立

[目的] 建立A型肉毒毒素轻链胞内持留模型来模拟A型肉毒毒素引起的长期中毒。[方法] 设计构建pcDNA3.1-ALC-GFP重组质粒,提取质粒进行PCR验证,并将其转染进Nureo-2a细胞中表达,利用Western Blot与细胞免疫荧光分析验证ALC-GFP的表达情况及其在细胞内的长时间持留,建立A型肉毒毒素轻链的胞内持留模型,并将该模型用于抗肉毒药物的筛选。[结果] 成功构建pcDNA3.1-ALC-GFP重组质粒并将其转染进Nureo-2a细胞中,验证了ALC-GFP在细胞内具有长时间持留性,成功建立了A型肉毒毒素轻链胞内持留模型,并利用该模型筛选出潜在抗肉毒药物CB3。[结论] 成功建立了A型肉毒毒素轻链胞内持留模型,并初步应用于CS1,CE2和CB3药物筛选,为A型肉毒毒素长期中毒的解毒研究奠定了基础。     

用皂土法制造型特异性肉毒诊断血清

用皂土为载体与类毒素结合方法及破伤风类毒素抗原抗体絮状反应方法去除A、B、C、D、E、F型肉毒抗血清原料中的异型和异种抗毒素(破伤风抗毒素)。制备的A、B、C、D、E、F型肉毒诊断血清每1m l均能中和相应型的肉毒毒素10000LD50以上,而中和异型肉毒毒素或破伤风毒素均低于5 LD50;A、B、C、D、E、F各型混合后的混合型血清每1m l能中和各型肉毒毒素亦大于10000 LD50,中和破伤风毒素低于5 LD50,即效价和特异性符合规程要求。     

ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin

The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

The effect of botulinum toxin type D on the triggered and constitutive exocytosis/endocytosis cycles in cultures of bovine adrenal medullary cells.

The extracellular fluid phase marker, horseradish peroxidase, enters chromaffin cells when triggered to secrete catecholamine. This triggered uptake, like secretion, is abolished in cells pre-incubated with botulinum toxin. Endocytosis of horseradish peroxidase into unstimulated cells is unaffected by botulinum toxin but is inhibited when the temperature is reduced. Once internalised by the unstimulated cells, horseradish peroxidase is released back into the extracellular fluid, the rate of release being temperature sensitive but unaffected by carbamylcholine or botulinum toxin. These results suggest that triggered exocytosis is a necessary event to precede triggered endocytosis, and that botulinum toxin may affect only the triggered exocytosis/endocytosis cycle and not the constitutive cycle.     

Double-blind,placebo-controlled study of the safety and efficacy of botulinum toxin type A for patients with glabellar lines

The objective of this study was to evaluate the efficacy and safety of botulinum toxin type A for the treatment of glabellar lines. Patients with moderate or severe glabellar lines at maximal frown received intramuscular injections of placebo or 20 U of botulinum toxin type A (Botox; Allergan, Inc., Irvine, Calif.) distributed among five injection sites (one in the procerus muscle and two in each corrugator supercilii). Follow-up assessments were performed at 7, 30, 60, 90, and 120 days after injections. Efficacy measures were the physician's rating of glabellar line severity at maximal frown and at rest (none, mild, moderate, or severe) and the patient's global assessment of changes in glabellar lines, from +4 (100 percent better) to -4 (100 percent worse). A total of 273 patients were enrolled (botulinum toxin, 202 patients; placebo, 71 patients). All except five patients (botulinum toxin, two patients; placebo, three patients) completed the study. For the physician's rating at maximal frown, the responder rate (percentage of patients with severity ratings of none or mild in follow-up evaluations) for the botulinum toxin group peaked at 77 percent at day 30 and was significantly greater than that for the placebo group at every follow-up visit (p < 0.001). For the patient's assessment, the responder rate (percentage of patients with scores of +2 or more) for the botulinum toxin group peaked at 89 percent at day 30 and was significantly greater than that for the placebo group at every follow-up visit (p < 0.001). Rates of adverse events were similar for the two groups. The only adverse event with an incidence of >/=5 percent was headache (botulinum toxin, 11 percent; placebo, 20 percent). The incidence of blepharoptosis was 1 percent for the botulinum toxin group. Botulinum toxin type A was remarkably safe and effective in reducing glabellar lines.     

Response of type B and E Botulinum toxins to purified sulfhydryl-dependent protease produced by Clostridium botulinum type F.

A sulfhydryl-dependent protease (SHP) was purified from a culture of Clostridium botulinum type F. The enzyme can activate type E progenitor toxin completely but type B progenitor toxin only partially. This may suggest that SHP by itself could completely activate the toxin of proteolytic C. botulinum types A and F in culture. The toxicity of type E progenitor toxin potentiated by the treatment with SHP persisted, whereas that of derivative toxin decreased rapidly by further incubation with SHP. This may indicate that only the progenitor toxin, the complex of the toxic and nontoxic components, activated by SHP withstands the subsequent exposure to the enzyme in cultures of proteolytic C. botulinum.     

Toxicity of botulinum toxin: a stoichiometric model for the locus of its extraordinary potency and persistence at the neuromuscular junction.

The number of botulinum toxin molecules calculated to reach either a diaphragmatic endplate after a minimum lethal dose or a gastrocnemius endplate after a minimal paralyzing dose is in close agreement with both the number of vesicles released per impulse and the finite number of active sites on the endplate for efflux of acetylcholine-containing vesicles. The close agreement in size between botulinum toxin molecules, synaptic vesicles, and the width of neurotubules supports a model in which botulinum toxin physically blocks efflux of acetylcholine. The stoichiometric attachment of botulinum toxin may occur at the critical moment that vesicles fuse with the presynaptic membrane, thereby exposing a receptor. While attachment may be to a ganglioside or neuraminidase-sensitive site, the chemical nature of the receptor is neither defined nor critical for the validity of the model.     

Enzyme linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin.

The enzyme-linked immunosorbent assay using different techniques has been applied to determine botulinum type B toxin. With the so-called "sandwich" technique, about 5,000 mouse ip LD50 of type B toxin can be detected. With the "double-sandwich" technique, about 400 mouse ip LD50 of toxin is detected and different commerical antisera are useful. For accurate quantification of botulinum toxins in culture filtrates, addition of EDTA to samples seems to be necessary. Cross-reactivity of the assay depends on the specificity of the antisera against botulinum type B toxin used and is almost eliminated with antiserum prepared against the toxic component of type B toxin.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Inhibitory effect of ganglioside GTlb on the activities of Clostridium botulinum toxins

Abstract Ganglioside G_Tlb inactivated botulinum toxins. The inactivation of type A, B, E and F toxins was marked but that of type C and D was less. Inactivation of type A toxin by ganglioside was significantly inhibited by one of two toxin fragments. The inactivation of botulinum toxin with ganglioside G_Tlb was affected by the ionic strength of the solvent. The findings indicate that ganglioside G_Tlb may not be implicated in the primary binding site for botulinum toxins on the synaptic membrane.     

Two unlinked cases of foodborne botulism in Italy at the beginning of 2010

The study provides data on two cases of foodborne botulism caused by consumption of commercial vegetable products: artichoke preserve and cream of vegetable soup. By mouse bioassay, Clostridium botulinum toxin in both the suspected food samples was detected and identified as type B toxin. The detection of C. botulinum toxin in the artichoke preserve indicates an inadequate food production technology while the presence of C. botulinum toxin in the vegetable soup appeared to be related to wrong behavior on the part of the consumer and to faulty food preservation. The study confirms that an early identification and reporting of suspected botulism cases is vital in the prevention of accidental widespread outbreaks.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Effect of botulinum D toxin on human neutrophilic leukocytes and localization of its substrates

Botulinum D toxin has been shown to ADP-ribosylate 22-kD proteins in neutrophilic leukocytes, but the function of these GTP-binding proteins remains unknown. In analogy to small GTP-binding proteins like SEC4 to YPT1, it has been suggested that botulinum D toxin substrates might be involved in secretory process of myeloid cells. Three main findings lead to the opposite conclusion. First of all, in human neutrophils, botulinum D toxin does not modify the release of azurophilic and specific granules induced by a chemoattractant (a formylpeptide) or a phorbol ester. Second, botulinum D toxin ADP-ribosylates 24 to 26-kD proteins that are only present in plasma membranes of human neutrophils. The membrane location of these substrates differs largely from that of the GTP-binding proteins involved in exocytosis and located in granules. Finally, since the same quantity of the toxin substrates is present in neutrophils as in their precursors, HL60 cells (which are devoid of specific granules and characterized by immature azurophilic granules and NADPH oxidase), it is unlikely that endogenous botulinum D toxin substrates are directly involved in the secretory responses of neutrophils.     

Clostridium botulinum type C in healthy swine in Japan.

Healthy cattle and swine bred in a district of Japan were examined for the presence of Clostridium botulinum in their liver. Liver specimens were cultivated in chopped meat-glucose medium and the cultures were examined for botulinum toxin. In cattle, none of the cultures of 100 liver specimens yielded the toxin. In swine, however, C1 or C2 toxin was demonstrated in 8 of 100 liver specimens from 36 farms. One of the five farms where the carrier-state swine were present was surveyed for about 2 years to determine whether the carrier-state was transient or resident. C. botulinum type C was found in swine livers and feces, and environmental specimens at extremely high rates during the surveillances, with 76% of specimens yielding botulinum toxin following the culture. These data suggest that it is not uncommon for healthy swine to carry C. botulinum type C in the liver and that there is a close relationship between C. botulinum carrier-state in swine and the presence of this organism in their raising environments. In 20 cattle and 20 swine suffering from parturient paresis of unknown etiology no evidence for involvement of C. botulinum type C was obtained.