Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT͞A last from 3 to 12 months, whereas the effects of BoNT͞E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT͞A light chain (BoNT͞ A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25 197. In contrast, the short-duration BoNT͞E serotype is cytoplasmic. Mutations in the BoNT͞A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes.otulinum neurotoxins (BoNTs) are the most potent of all biological substances (1). Although BoNTs are well publicized as a potential biological weapon and as the causative agents in clinical botulism, the potency and myorelaxant actions of BoNTs have been exploited clinically in more than 100 indications, including muscle hyperactivity in cerebral palsy and cervical dystonia, migraines, myofacial pain, and focal hyperhidrosis (2-5). These toxins are specific endoproteases that, collectively, target several distinct proteins in nerve terminals. Motor nerve terminals at neuromuscular junctions are particularly sensitive to these neurotoxins, resulting in a transient and reversible muscle relaxation through inhibition of acetylcholine release. The Clostridium neurotoxin family includes seven serotypes of BoNT (A-G), and a single form of toxin produced by Clostridium tetani (TeNT). These toxins consist of a heavy chain (HC, 100 kDa) and light chain (LC, 50 kDa) linked by a disulfide bond (6, 7). The three-dimensional crystal structures of BoNT͞A (8) and BoNT͞B (9) have been resolved, providing a basis for understanding the structure͞function mechanism of BoNT action. The BoNT-LCs are zinc-dependent endoproteases that specifically cleave one of three soluble Nethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) proteins (10) involved in synaptic vesicle docking and fusion at the nerve terminal (11). The synaptosome-associated protein of 25 kDa (SNAP25) is cleaved at distinct sites near the C terminus by ) and BoNT͞E (R 180 -I 181 ), generating truncated SNAP25 197 (12) and SNAP25 180 (13), respectively.

Summary.-The sera of 73 patients with nasopharyngeal carcinoma (NPC), 28 patients with other carcinomas (OC) and 89 healthy subjects (HS) were tested for IgG and IgA antibodies to Epstein-Barr virus (EBV) viral capsid antigen (VCA). The majority of the NPC sera had IgG titres of 160 or above, whereas the majority of the other sera had titres below 160. For IgA reactivity to EBV-VCA,68 of 73 (93*2%) NPC sera had titres of > 10. In contrast, only 6 of 28 (21-4%) OC sera and none of the HS sera had such titres. The mean serum concentrations of IgG, IgA, IgM and C3' were also determined in 55 NPC and 20 OC patients and 18 HS. They were all significantly higher in the NPC sera than in the HS. Although the concentrations of IgG and C3' were not significantly different in the two carcinoma groups, the concentrations of IgA and IgM were significantly higher in the NPC group than in OC. These findings appear to reflect the intensity of EBV-specific antigenic stimulation in NPC, and the EBV-specific serum IgA reactivity may be a useful aid to the diagnosis of NPC, especially in cases with an occult primary tumour. It may be also of value as a screening test in people at high risk.

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Botulinum neurotoxins (BoNT) are therapeutic proteins that are specific, potent, and effective. They are highly specific in binding to motor neurons but do not bind to other non-neuronal cells. These proteins are zinc-dependent endopeptidases that inhibit exocytosis by specific cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins involved in vesicle docking and fusion. The therapeutic effect of BoNT/A in humans lasts from 3 to 12 months, depending upon the condition treated. Data from animal and cell culture models suggests that the long-lasting duration of inhibition of neurotransmitter release induced by BoNT/A maybe due to the persistence of the endopeptidase activity of the light chain (LC/A) in cells, interactions of the cleaved substrates, and/or the response of the nerve to the temporary disruption of communication with its target tissue. We have analyzed the subcellular localization of the light chains from serotypes A, B, and E and have demonstrated that each light chain displays a distinct distribution within cells. LC/A localizes at the plasma membrane, LC/B is dispersed throughout the cell including the nucleus, and LC/E is mainly cytosolic. Localization is similar in non-neuronal cell lines, suggesting that the signals involved in proper subcellular localization are within the LC sequences and the moiety the light chain interacts with is present in both neuronal and non-neuronal cells.

Summary.-Irrespective of the ethnic origin of the patient, nasopharyngeal carcinoma (NPC), appears to stimulate the production of IgA antibodies against VCA. These antibodies are detected at high frequency and titres in sera from NPC patients but only rarely from control subjects. A majority of relapse-free survivors tested 1-12 years after radiotherapy (RT) sustain a detectable level of IgA anti-VCA. Serum titres of IgA anti-VCA remain relatively unchanged in individual NPC patients after RT, regardless of the disease evolution. These antibodies were detected in serum from one individual 9 months before NPC and the titre rose concomitantly with its clinical onset.