UCL University College London Eprints
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Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain. - Poulain, B; Tauc, L; Maisey, EA; Wadsworth, JD; Mohan, PM; Dolly, JO
Botulinum neurotoxins (types A and B), which are microbial proteins consisting of two disulfide-linked chains, inhibit specifically and with high potency the release of acetylcholine from peripheral nerve terminals. As a prerequisite for a long-term development of effective treatments for botulism, the internalization and inhibitory action of the toxin and its constituent chains were examined by electrophysiological methods at identified synapses in Aplysia preparations that allow both intracellular and bath application of the neurotoxins. Intracellular recordings from cholinergic cells of the buccal ganglion demonstrated that extra- or intracellular application of low doses of botulinum neurotoxin results in a specific blockade...
Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release. - Maisey, EA; Wadsworth, JD; Poulain, B; Shone, CC; Melling, J; Gibbs, P; Tauc, L; Dolly, JO
1. The abilities of botulinum neurotoxins, types A and B (single and two-chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic-nerve-diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay. 2. Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly. 3. Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking...
Inhibition of transmitter release by botulinum neurotoxin A. Contribution of various fragments to the intoxication process. - Poulain, B; Wadsworth, JD; Maisey, EA; Shone, CC; Melling, J; Tauc, L; Dolly, JO
1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with...
Multiple domains of botulinum neurotoxin contribute to its inhibition of transmitter release in Aplysia neurons. - Poulain, B; Wadsworth, JD; Shone, CC; Mochida, S; Lande, S; Melling, J; Dolly, JO; Tauc, L
The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably...
Clues to the multi-phasic inhibitory action of botulinum neurotoxins on release of transmitters. - Dolly, JO; Ashton, AC; McInnes, C; Wadsworth, JD; Poulain, B; Tauc, L; Shone, CC; Melling, J
1. With the aim of gaining insight into the mechanism of Ca2(+)-dependent secretion, inhibition of transmitter release by botulinum neurotoxins or their fragments was studied at mammalian motor nerve terminals, cerebrocortical synaptosomes and PC-12 cells. 2. Relative to BoNT type A, the feeble neuromuscular paralytic activity of its two chains and the lack of activity observed with a proteolytic fragment, H2L (lacking H1, the C-terminal half of the heavy chain) highlight a requirement of the intact, disulphide-linked dichain protein for efficient targetting (binding/uptake) to peripheral cholinergic nerve endings. 3. In PC-12 cells, the renatured light chain alone proved equally potent...
Botulinum type F neurotoxin. Large-scale purification and characterization of its binding to rat cerebrocortical synaptosomes. - Wadsworth, JD; Desai, M; Tranter, HS; King, HJ; Hambleton, P; Melling, J; Dolly, JO; Shone, CC
1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of...
Heterologous combinations of heavy and light chains from botulinum neurotoxin A and tetanus toxin inhibit neurotransmitter release in Aplysia. - Poulain, B; Mochida, S; Weller, U; Högy, B; Habermann, E; Wadsworth, JD; Shone, CC; Dolly, JO; Tauc, L
The neuroparalytic activities of botulinum neurotoxin type A (BoNT A), tetanus toxin (TeTx), or homologous and heterologous combinations of their constituent polypeptides were examined at cholinergic and non-cholinergic synapses of Aplysia californica. When applied extracellularly, BoNT A or a mixture of its heavy (HC) and light (LC) chains were far more potent in blocking transmitter release at cholinergic than non-cholinergic synapses. The reverse was true for TeTx or a mixture its constituent chains. Such selectivity was assigned to differences in neuronal targetting and uptake of the neurotoxins since both exhibited similar potencies when injected directly into the cell body of...
Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains. - Poulain, B; Mochida, S; Wadsworth, JD; Weller, U; Habermann, E; Dolly, JO; Tauc, L
1. The effects on the release of transmitter by botulinum neurotoxins (BoNT; types A, B, E), tetanus toxin (TeTx), constituent chains or fragments were studied on identified cholinergic and non-cholinergic synapses in Aplysia. 2. Cholinergic synapses in the buccal ganglion were found to be greater than 100 fold more sensitive to extracellular application of BoNT than to TeTx whereas in non-cholinergic synapses of the cerebral ganglion the potencies of the toxins were reversed. When intracellularly applied TeTx and BoNT were found nearly equipotent. This disparity in the susceptibilities of BoNT and TeTx to inhibit transmission was attributed to differences in...
Comparable 30-kDa apamin binding polypeptides may fulfill equivalent roles within putative subtypes of small conductance Ca(2+)-activated K+ channels. - Wadsworth, JD; Doorty, KB; Strong, PN
Apamin, a peptide neurotoxin from bee venom, blocks small conductance Ca(2+)-activated K+ channels in central synapses and peripheral tissues. Using 125I-apamin, single classes of high affinity binding sites (Kd 1-3 pM) were identified on plasma membranes from rat, rabbit, guinea pig, and bovine brain and from rabbit, guinea pig, and bovine liver. Binding was sensitive to scyllatoxin, dequalinium, gallamine, and d-tubocurarine but not to charybdotoxin, toxin I, or mast cell degranulating peptide. In contrast, saturable binding of 125I-apamin to rat liver plasma membranes was virtually undetectable, thereby providing a correlation with the ability to measure apamin-sensitive Ca(2+)-activated potassium currents in...
Assignment of laminin heavy chains using the lectin Ricinus communis agglutinin-1. - Wadsworth, JD; Okuno, A; Strong, PN
Using high-resolution PAGE and Western-blotting techniques the lectin Ricinus communis agglutinin-1 (RCA-1) was tested for its ability to recognize laminin subunits from the mouse Engelbreth-Holm-Swarm (EHS) tumour and from bovine cardiac and skeletal muscle. Biotinylated RCA-1 recognized both the A and B chains of purified EHS-tumour laminin with a sensitivity comparable to anti-(EHS laminin) antibodies. In cardiac and skeletal muscle RCA-1 also recognized the B chains of laminin, together with a approximately 330 kDa RCA-1-binding glycoprotein that was undetectable in smooth muscle. This glycoprotein was not recognized by antibodies raised to laminin from the EHS tumour. Purification of the 330...
Photolabile derivatives of 125I-apamin: defining the structural criteria required for labeling high and low molecular mass polypeptides associated with small conductance Ca(2+)-activated K+ channels. - Wadsworth, JD; Doorty, KB; Ganellin, CR; Strong, PN
The structure of apamin-sensitive Ca(2+)-activated K+ channels has been investigated using high-affinity, photolabile azidoaryl derivatives of 125I-[alpha-formyl-Cys1]apamin and 125I-[epsilon-formyl-Lys4]-apamin. Labeling patterns suggest that similar structural constraints are required for labeling analogous polypeptides associated with distinct channel subtypes. When photoprobes are coupled at the epsilon-amino-Lys4 position of apamin, comparable low molecular mass (approximately 30 kDa) polypeptides are efficiently labeled on either brain or liver plasma membranes, irrespective of the structure of the photoprobe. However, when photoprobes are coupled at the alpha-amino-Cys1 position of apamin, the pattern of labeling on both brain and liver plasma membranes varies, depending upon the length of...
A novel small conductance Ca2+-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2+ channel probe. - Doorty, KB; Bevan, S; Wadsworth, JD; Strong, PN
Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of 125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (Ki = 1.45 +/- 0.22 nM) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nM) completely blocks apamin-sensitive after-hyperpolarizing slow...
Robust next release problem: Handling uncertainty during optimization - Li, L; Harman, M; Letier, E; Zhang, Y
Uncertainty is inevitable in real world requirement engineering. It has a significant impact on the feasibility of proposed solutions and thus brings risks to the software release plan. This paper proposes a multi-objective optimization technique, augmented with Monte-Carlo Simulation, that optimizes requirement choices for the three objectives of cost, revenue, and uncertainty. The paper reports the results of an empirical study over four data sets derived from a single real world data set. The results show that the robust optimal solutions obtained by our approach are conservative compared to their corresponding optimal solutions produced by traditional Multi-Objective Next Release Problem....
Babel Pidgin: SBSE can grow and graft entirely new functionality into a real world system - Harman, M; Jia, Y; Langdon, WB
Adding new functionality to an existing, large, and perhaps poorly-understood system is a challenge, even for the most competent human programmer. We introduce a 'grow and graft' approach to Genetic Improvement (GI) that transplants new functionality into an existing system. We report on the trade offs between varying degrees of human guidance to the GI transplantation process. Using our approach, we successfully grew and transplanted a new 'Babel Fish' linguistic translation feature into the Pidgin instant messaging system, creating a genetically improved system we call 'Babel Pidgin'. This is the first time that SBSE has been used to evolve and...
Improving 3D medical image registration CUDA software with genetic programming - Langdon, WB; Modat, M; Petke, J; Harman, M
Genetic Improvement (GI) is shown to optimise, in some cases by more than 35%, a critical component of healthcare industry software across a diverse range of six nVidia graphics processing units (GPUs). GP and other search based software engineering techniques can automatically optimise the current rate limiting CUDA parallel function in the Nifty Reg open source C++ project used to align or register high resolution nuclear magnetic resonance NMRI and other diagnostic NIfTI images. Future Neurosurgery techniques will require hardware acceleration, such as GPGPU, to enable real time comparison of three dimensional in theatre images with earlier patient images and...
Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis - Weaver, JMJ; Ross-Innes, CS; Barbera, M; Ong, C-AJ; Lao-Sirieix, P; Smith, L; Grehan, N; Fitzgerald, RC; Knight, O; Nutzinger, B; Abdullahi, Z; Debriram-Beecham, I; Crawte, J; MacRae, S; Noorani, A; Elliott, RF; Li, X; Bornschein, J; Zeki, S; Chettouh, H; De Silva, N; Gregson, E; Yang, T-P; Shannon, N; Lynch, AG; Forshew, T; Murtaza, M; Dunning, MJ; Smith, ML; Anderson, CL; Carvalho, B; Caldas, C; Eldridge, MD; Rosenfeld, N; Tavaré, S; Bower, L; Achilleos, A; Secrier, M; O'donovan, M; Cluroe, A; Miremadi, A; Mahler-Araujo, B; Malhotra, S; Underwood, TJ; May, AP; Hardwick, R; Davies, J; Oloumi, A; Aparicio, S; Edwards, PAW; Hayes, SJ; Yeng, A; Lydon, A-M; Dharmaprasad, S; Greer, S; Preston, S; Oakes, S; Save, V; Paterson-Brown, S; Tucker, O; Alderson, D; Taniere, P; Crichton, C; Kelly, J; Byrne, J; Sharland, D; Holling, N; Boulter, L; Noble, F; Stacey, B; Barr, H; Shepherd, N; Almond, LM; Old, O; Lagergren, J; Gossage, J; Davies, A; Mason, R; Chang, F; Zylstra, J; Peters, C; Sanders, G; Wheatley, T; Berrisford, R; Bracey, T; Harden, C; Bunting, D; Roques, T; Nobes, J; Loo, S; Lewis, M; Cheong, E; Priest, O; Parsons, SL; Soomro, I; Kaye, P; Saunders, J; Pang, V; Welch, NT; Catton, JA; Duffy, JP; Ragunath, K; Lovat, L; Haidry, R; Miah, H; Kerr, S; Eneh, V; Butawan, R; Igali, L; Ford, H; Gilligan, D; Safranek, P; Hindmarsh, A; Sudjendran, V; Metz, A; Carroll, N; Scott, M; O'Neil, JR
Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barrett's esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barrett's esophagus (NDBE; n = 66) and high-grade dysplasia (HGD; n = 43). Unexpectedly, the majority of recurrently mutated genes...
Exploiting prior-phase effort data to estimate the effort for the subsequent phases: A further assessment - Ferrucci, F; Gravino, C; Sarro, F
Context. Development effort estimation is a managerial activity that takes place throughout the life-cycle of the software so that it may benefit from information that becomes available as the project progresses. Researchers investigated the use of prior-phase effort data to estimate the effort in subsequent phases, as well as early phase effort data to estimate the total development effort. Objective. We assessed the usefulness of the effort spent for each phase in order to predict the effort required during the subsequent phase and until the end of the development process. We compared the use of effort data against the use...
A meshless method for the Laplace and Biharmonic equations subjected to noisy boundary data - Jin, B
In this paper, we propose a new numerical scheme for the solution of the Laplace and biharmonic equations subjected to noisy boundary data. The equations are discretized by the method of fundamental solutions. Since the resulting matrix equation is highly ill-conditioned, a regularized solution is obtained using the truncated singular value decomposition, with the regularization parameter given by the L-curve method. Numerical experiments show that the method is stable with respect to the noise in the data, highly accurate and computationally very efficient.
Boundary knot method for some inverse problems associated with the Helmholtz equation - Jin, B; Zheng, Y
The boundary knot method is an inherently meshless, integration-free, boundary-type, radial basis function collocation technique for the solution of partial differential equations. In this paper, the method is applied to the solution of some inverse problems for the Helmholtz equation, including the highly ill-posed Cauchy problem. Since the resulting matrix equation is badly ill-conditioned, a regularized solution is obtained by employing truncated singular value decomposition, while the regularization parameter for the regularization method is provided by the L-curve method. Numerical results are presented for both smooth and piecewise smooth geometry. The stability of the method with respect to the noise...