The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in humans and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Clostridium botulinum strains produce large BoNTs toxin complexes, which include auxiliary non-toxic proteins that appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist BoNT translocation across the intestinal mucosal layer. In this study, we visualize for the first time a series of botulinum serotype D toxin complexes using negative stain transmission electron microscopy (TEM). The complexes consist of the 150-kDa BoNT, 130-kDa nontoxic non-hemagglutinin (NTNHA), and three kinds of hemagglutinin (HA) subcomponents: 70-kDa HA-70, 33-kDa HA-33, and 17-kDa HA-17. These components assemble sequentially to form the complex. A novel TEM image of the mature L-TC revealed an ellipsoidal-shaped structure with "three arms" attached. The "body" section was comprised of a single BoNT, a single NTNHA and three HA-70 molecules. The arm section consisted of a complex of HA-33 and HA-17 molecules. We determined the x-ray crystal structure of the complex formed by two HA-33 plus one HA-17. On the basis of the TEM image and biochemical results, we propose a novel 14-mer subunit model for the botulinum toxin complex. This unique model suggests how non-toxic components make up a "delivery vehicle" for BoNT.Different strains of Clostridium botulinum produce seven distinct serotypes of neurotoxins (BoNTs), 2 classified A through G. BoNT has attracted much interest in recent years due to extensive research on its biochemistry, determination of its crystal structure, and investigations into the pharmacology and applications of BoNTs as therapeutic agents for the treatment of human disease (1-3). After ingestion of BoNT, the BoNT is absorbed from intestinal epithelial cells into the bloodstream, after which it consequently reaches the neuromuscular junctions. BoNT enters nerve cells via receptor-mediated endocytosis, where it cleaves specific sites on target proteins, inhibiting release of neurotransmitters from peripheral cholinergic synapses through its zinc protease activity (4 -6). This process causes muscular paralysis in humans and animals, leading to the disease botulism.Toxins with serotypes A-D and G are encoded by two gene clusters in close proximity to each other; cluster 1 contains the bont and ntnha genes, and cluster 2 contains three genes : ha-70, ha-33, and ha-17 (7, 8). Therefore, botulinum TC consists of five components: BoNT, non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, HA-33, and HA-17). All serotypes of BoNT associate non-covalently with auxiliary non-toxic proteins, thereby forming large toxin complexes (TCs). Serotype A-D strains produce the M-TC (BoNT⅐NTNHA complex) and L-TC (BoNT⅐NTNHA⅐HAs complex) in the culture medium, while serotype E and F strains produce only M-TC. The major biological function of t...
We examined the frequency of occurrence of cell membrane damage and repair in sonoporation using pulsed ultrasound and microbubbles. Our results should prove useful for improving pulsed-ultrasound sonoporation.
To elucidate the mechanisms of localized genesis and development of atherosclerosis and anastomotic intimal hyperplasia in man, a coculture of bovine aortic endothelial cells (ECs) and smooth muscle cells (SMCs) was prepared, and the effects of a shear flow on the uptake of lipoproteins by the cells was studied by incubating the EC-SMC coculture as well as an EC monoculture with a culture medium containing either DiI-LDL or DiI-Ac-LDL and subjecting to a laminar shear flow. It was found that in both the presence and absence of a shear flow that imposed the ECs an area mean shear stress of 13.3 dynes/cm2, the uptake of LDL by an EC-SMC coculture was much greater than that by an EC monoculture, whereas that of Ac-LDL was almost the same. The uptake of LDL by an EC monoculture increased slightly by being exposed to a shear flow, whereas that by an EC-SMC coculture did not. In contrast to this, the uptake of Ac-LDL by both an EC monoculture and an EC-SMC coculture decreased drastically by a shear flow, suggesting that the action of a shear flow on the uptake of Ac-LDL by vascular cells is very different from that of LDL.
To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H2O2 and investigated the roles of protein kinase C delta (PKC delta) and Ca2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H2O2 caused the accumulation of p53, which was inhibited by rottlerin (a PKC delta inhibitor) but not by BAPTA-AM (an intracellular Ca2+ chelator). PKC delta itself was activated through the phosphorylation at tyrosine residues. H2O2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKC delta contributes to the accumulation of p53 and that Ca2+ plays a role in downstream signals of p53 leading to apoptosis in H2O2-treated BAEC.
A large size botulinum toxin complex (L-TC) is composed of a single neurotoxin (BoNT), a single nontoxic nonhaemagglutinin (NTNHA) and a haemagglutinin (HA) complex. The HA complex is comprised of three HA-70 molecules and three arm structures of HA-33/HA-17 that consist of two HA-33 and a single HA-17. In addition to the mature L-TC, smaller TCs are present in cultures: M-TC (BoNT/NTNHA), M-TC/HA-70 and immature L-TCs with fewer HA-33/HA-17 arms than mature L-TC. Because L-TC displays higher oral toxicity than pure BoNT, it was presumed that nontoxic proteins are critical for food poisoning. In this study, the absorption of TCs across intestinal epithelial cells was assessed by examining the cell binding and monolayer transport of serotype D toxins in the rat intestinal epithelial cell line IEC-6. All TCs, including pure BoNT, displayed binding and transport, with mature L-TC showing the greatest potency. Inhibition experiments using antibodies revealed that BoNT, HA-70 and HA-33 could be responsible for the binding and transport. The findings here indicate that all TCs can transport across the cell layer via a sialic acid-dependent process. Nonetheless, binding and transport markedly increased with number of HA-33/HA-17 arms in the TC. We therefore conclude that the HA-33/HA-17 arm is not necessarily required for, but facilitates, transport of botulinum toxin complexes.
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