Botulinum neurotoxins produced by anaerobic bacteria of the genus Clostridium are the most toxic proteins known, with mouse LD50 values in the 1-5 ng/kg range, and are solely responsible for the pathophysiology of botulism. These metalloproteinases enter peripheral cholinergic nerve terminals and cleave proteins of the neuroexocytosis apparatus, causing a persistent, but reversible, inhibition of neurotransmitter release. They are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation, causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals. Keywords: botulinum neurotoxins, dystonia, muscle paralysis, neuroexocytosis, pharmaco-cosmetics, snake neurotoxins. Toxigenic anaerobic spore-forming bacteria of the genus Clostridium produce seven different botulinum neurotoxins (designated BoNT/A to G). Ingestion of BoNT-poisoned food causes an intoxication known as botulism, whose symptoms are the result of a generalized sustained blockade of acetylcholine release (ACh) at somatic and autonomic nerve terminals. They include diplopia, ptosis, dysphagia and paralysis of facial muscles, which progressively descends to the trunk, eventually involving the respiratory and visceral muscles. Dysfunctions of the autonomic nervous system include reduced salivation and lacrimation, nausea, vomiting and abdominal pain. Most patients survive botulism, but complete recovery is slow and may require mechanical ventilation. The recovery time is longer after BoNT/A intoxication than after BoNT/B and BoNT/E intoxications ( Presynaptic snake neurotoxins endowed with PLA2 activity (SPANs) are major components of the venom of four families of venomous snakes (Crotalidae, Elapidae, Hydrophiidae and Viperidae). These neurotoxins play a major role in envenomation of the prey (Harris 1997) by causing a persistent blockade of neurotransmitter release from nerve terminals (Kini 1997;Montecucco and Rossetto 2000;Schiavo et al. 2000). This process is more complicated than botulism, as several venom components are biologically active. However, almost invariably, most of the neurological signs and symptoms are due to the action of the SPANs. In fact, independently on the anatomical site of biting, patients are reported to have ptosis, diplopia, ophthalmoplegia, difficulty in swallowing, respiratory paralysis, abdominal pain and autonomic symptoms (Warrell et al. 1983;Theakston et al. 1990;Connolly et al. 1995;Kularatne 2002;Prasarnpun et al. 2005). The progression of paralysis in isolated nerve-muscle preparations exposed to SPANs is ...
Botulinum neurotoxin type A (BoNT/A) is the most frequent cause of human botulism and, at the same time, is largely used in human therapy. Some evidence indicates that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been directly proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscope, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor synaptic vesicle protein 2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle trans-membrane pH gradient, we show that the neurotoxin translocation into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by no more than two neurotoxin molecules.
Botulinum neurotoxin type A (BoNT/A) is commonly used in human therapy. This treatment may induce immunoresistance and preliminary evaluation of other botulinum neurotoxin serotypes suggested botulinum neurotoxin type C (BoNT/C) to be a good alternative to BoNT/A. Here, we have further characterized the biological activities of BoNT/C using a variety of experimental approaches. Muscle paralysis and time of recovery of mouse hind limb injected with BoNT/A or BoNT/C were assayed with the Digit Abduction Scoring assay. The extent and duration of paralysis were similar with the two toxin serotypes. Extensor digitorum longus or tibialis anterior muscles were dissected at times of complete paralysis and of complete recovery. Muscle weight and force were significantly reduced in mice injected with BoNT/A and BoNT/C, and some atrophy persisted for a long time. In BoNT/C-treated junctions, nerve terminal sprouting was prominent, indicating that the capacity to extend the field of innervation is not hampered by BoNT/C. BoNT/C induced a marked decrease in the frequency of miniature endplate potentials and in the amplitude of endplate potentials. 3,4-diaminopyridine reversed the effect of BoNT/C by increasing the amplitude of synchronized endplate potentials. The present study shows an extensive similarity in the biological activities of BoNT/A and BoNT/C, further supporting the suggestion that BoNT/C is a valid alternative to BoNT/A.
Evidence shows that extracellular ATP signals influence myogenesis, regeneration and physiology of skeletal muscle. Present work was aimed at characterizing the extracellular ATP signaling system of skeletal muscle C2C12 cells during differentiation. We show that mechanical and electrical stimulation produces substantial release of ATP from differentiated myotubes, but not from proliferating myoblasts. Extracellular ATP-hydrolyzing activity is low in myoblasts and high in myotubes, consistent with the increased expression of extracellular enzymes during differentiation. Stimulation of cells with extracellular nucleotides produces substantial Ca(2+) transients, whose amplitude and shape changed during differentiation. Consistently, C2C12 cells express several P2X and P2Y receptors, whose level changes along with maturation stages. Supplementation with either ATP or UTP stimulates proliferation of C2C12 myoblasts, whereas excessive doses were cytotoxic. The data indicate that skeletal muscle development is accompanied by major functional changes in extracellular ATP signaling.
Urease and the cytotoxin VacA are two major virulence factors of the human pathogen Helicobacter pylori, which is responsible for severe gastroduodenal diseases. Diffusion of urea, the substrate of urease, into the stomach is critically required for the survival of infecting H. pylori. We now show that VacA increases the transepithelial flux of urea across model epithelia by inducing an unsaturable permeation pathway. This transcellular pathway is selective, as it conducts thiourea, but not glycerol and mannitol, demonstrating that it is not due to a loosening of intercellular junctions. Experiments performed with different cell lines, grown in a nonpolarized state, confirm that VacA permeabilizes the cell plasma membrane to urea. Inhibition studies indicate that transmembrane pores formed by VacA act as passive urea transporters. Thus, their inhibition by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid significantly decreases toxin-induced urea fluxes in both polarized and nonpolarized cells. Moreover, phloretin, a well-known inhibitor of eukaryotic urea transporters, blocks VacA-mediated urea and ion transport and the toxin’s main biologic effects. These data show that VacA behaves as a low-pH activated, passive urea transporter potentially capable of permeabilizing the gastric epithelium to urea. This opens the novel possibility that in vivo VacA may favor H. pylori infectivity by optimizing urease activity
Peptaibols, by disturbing the permeability of phospholipid membranes, can overcome anticancer drug resistance, but their natural hydrophobicity hampers their administration. By a green peptide synthesis protocol, we produced two water-soluble analogs of the peptaibol trichogin GA IV, termed K6-Lol and K6-NH2. To reduce production costs, we successfully explored the possibility of changing the naturally occurring 1,2-aminoalcohol leucinol to a C-terminal amide. Peptaibol activity was evaluated in ovarian cancer (OvCa) and Hodgkin lymphoma (HL) cell lines. Peptaibols exerted comparable cytotoxic effects in cancer cell lines that were sensitive—and had acquired resistance—to cisplatin and doxorubicin, as well as in the extrinsic-drug-resistant OvCa 3-dimensional spheroids. Peptaibols, rapidly taken up by tumor cells, deeply penetrated and killed OvCa-spheroids. They led to cell membrane permeabilization and phosphatidylserine exposure and were taken up faster by cancer cells than normal cells. They were resistant to proteolysis and maintained a stable helical structure in the presence of cancer cells. In conclusion, these promising results strongly point out the need for further preclinical evaluation of our peptaibols as new anticancer agents.
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