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Biomedical subjects

J D Wadsworth

Publications and source records attributed to J D Wadsworth.

17 recordsLinked to original sources
PubMed2001

Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis distinct from other forms of human prion disease: disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and other peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrP(Sc) to investigate the distribution of PrP(Sc) in a range of vCJD tissues. METHODS: We obtained tissues at necropsy from four patients with neuropathologically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrP(Sc) and western blotting using high sensitivity enhanced chemiluminescence. FINDINGS: We could reliably detect PrP(Sc) in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. PrP(Sc) could be detected in tissue homogenates when present at concentrations 10(4)-10(5) fold lower than those reported in brain. Tonsil, spleen, and lymph node were uniformly positive for PrP(Sc) at concentrations in the range of 0.1-15% of those found in brain: the highest concentrations were consistently seen in tonsil. PrP(Sc) was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2.5 and 25%, respectively of those found in brain. Other peripheral tissues studied were negative for PrP(Sc) with the exception of low concentrations in rectum, adrenal gland, and thymus from a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction) were negative for PrP(Sc) at this level of assay sensitivity. INTERPRETATION: We have developed a highly sensitive immunoblot method for detection of PrP(Sc) in vCJD tissues that can be used to provide an upper limit on PrP(Sc) concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should be further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment of the eye, in particular the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for population screening of surgical tissues to assess prevalence of preclinical vCJD infection within the UK and other populations.

Animals↗
PubMed1999

Molecular biology of prion propagation.

The occurrence of new variant Creutzfeldt-Jakob disease and the experimental confirmation that it is caused by the same prion strain as BSE has dramatically highlighted the need for a precise understanding of the molecular basis of prion propagation. The molecular basis of prion-strain diversity, previously a major challenge to the protein-only model, is now becoming clearer. The conformational change thought to be central to prion propagation, from a predominantly alpha-helical fold to one predominantly comprising beta-structure, can now be reproduced in vitro, and the ability of beta-PrP to form fibrillar aggregates provides a plausible molecular mechanism for prion propagation. These and other advances in the fundamental biology of prion propagation are leading to prion diseases becoming arguably the best understood of the neurodegenerative conditions and strategies for the development of rational therapeutics are becoming clearer.

Animals↗
PubMed1999

Strain-specific prion-protein conformation determined by metal ions.

In animals infected with a transmissible spongiform encephalopathy, or prion disease, conformational isomers (known as PrPSc proteins) of the wild-type, host-encoded cellular prion protein (PrPc) accumulate. The infectious agents, prions, are composed mainly of these conformational isomers, with distinct prion isolates or strains being associated with different PrPSc conformations and patterns of glycosylation. Here we show that two different human PrPSc types, seen in clinically distinct subtypes of classical Creutzfeldt-Jakob disease, can be interconverted in vitro by altering their metal-ion occupancy. The dependence of PrPSc conformation on the binding of copper and zinc represents a new mechanism for post-translational modification of PrP and for the generation of multiple prion strains, with widespread implications for both the molecular classification and the pathogenesis of prion diseases in humans and animals.

Binding Sites↗
PubMed1997

Structural diversity among subtypes of small-conductance Ca2+-activated potassium channels.

125I-Apamin and photolabile derivatives of the toxin have been used to investigate the binding properties and subunit composition of small conductance Ca2+-activated potassium channels (SK(Ca) channels) expressed on plasma membranes from rat brain, rabbit liver, or rat pheochromocytoma (PC12) cells. On all preparations, 125I-apamin recognized single classes of acceptor binding sites with similar high affinity (Kd approximately 3-6 pM). Gallamine, however, was found to readily discriminate between 125I-apamin acceptors present in these preparations, showing a maximal approx nine-fold difference in affinity for acceptors expressed by rabbit liver or PC12 cells. Affinity-labeling patterns revealed the expression of different hetero-oligomeric combinations of high (86 or 59 kDa) and low (33 or 30 kDa) molecular mass 125I-apamin-binding polypeptides, consistent with pharmacological differences. Alternative expression of either 86- or 59-kDa polypeptides appeared to be the most important factor influencing gallamine's affinity for SK(Ca) channel subtypes. Both high- and low-molecular-mass polypeptides are integral membrane proteins, the latter being glycosylated in a tissue-specific manner.

Affinity Labels↗
PubMed1997

A novel small conductance Ca2+-activated K+ channel blocker from Oxyuranus scutellatus taipan venom. Re-evaluation of taicatoxin as a selective Ca2+ channel probe.

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 tail K+ currents generated in rat chromaffin cells (mean block 97 +/- 3%, n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 +/- 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.

Animals↗
PubMed1996

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.

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 the spacer arm incorporated into the photoprobe. Spacer arms of approximately 8-9 A efficiently label only high molecular mass polypeptides (86, 59 kDa), accompanied by weak, variable labeling of a 44-kDa component. A shorter spacer arm (5.7 A) results in feeble labeling of 86- and 59-kDa polypeptides and barely detectable labeling of 44- and approximately 30-kDa polypeptides. In contrast, a long spacer arm (12.8 A) efficiently labels only approximately 30-kDa polypeptides. These findings point to close similarities in the topography of the 125I-apamin binding site present on pharmacologically distinct subtypes of apamin-sensitive Ca2+-activated K+ channels and indicates that heterooligomeric association of high and low molecular mass polypeptide subunits may be a general structural feature of members belonging to this family of K+ channels.

Animals↗
PubMed1994

Comparable 30-kDa apamin binding polypeptides may fulfill equivalent roles within putative subtypes of small conductance Ca(2+)-activated K+ channels.

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 rabbit and guinea pig hepatocytes but not in rat hepatocytes. In agreement with membrane binding studies, homobifunctional cross-linkers identified apparently identical 33-kDa 125I-apamin binding polypeptides on brain plasma membranes from all species and analogous but distinct polypeptides on plasma membranes from rabbit, guinea pig, and bovine liver. None of these affinity-labeled polypeptides were detectable on plasma membranes from rat liver. Affinity labeling was abolished on both liver and brain membranes by apamin, scyllatoxin, dequalinium, gallamine, and d-tubocurarine. These results indicate that comparable approximately 30-kDa polypeptides may fulfill equivalent functional roles within putative subtypes of apamin-sensitive small conductance Ca(2+)-activated K+ channels.

Animals↗
PubMed1993

Assignment of laminin heavy chains using the lectin Ricinus communis agglutinin-1.

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 kDa binding glycoprotein from skeletal muscle, using ion-exchange and lectin-affinity chromatography, revealed that in its native form, this glycoprotein is disulphide-bonded to the B chains of laminin. The demonstrated properties of the approximately 330 kDa RCA-1-binding glycoprotein are identical to those reported for the variant M chain of merosin which is known to replace the A chain in laminin from the extrasynaptic regions of skeletal muscle. These results establish that biotinylated RCA-1 can recognize A-, B- and M-chain subunits of laminin isoforms, and that, when used in conjunction with other techniques, they provide a useful method for the assignment of laminin heavy chains.

Animals↗
PubMed1991

Heterologous combinations of heavy and light chains from botulinum neurotoxin A and tetanus toxin inhibit neurotransmitter release in Aplysia.

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 either cell type. When bath-applied, heterologous combinations of the toxins' HC and LC appeared as effective as the parent neurotoxins from whence each HC was derived. Moreover, targetting/internalization was attributable to the analogous N-terminal moieties, H2 and beta 2, of the HC from BoNT A and TeTx. Thus, it may be postulated that the latter regions possess two functional domains, one being distinct and responsible for the divergent neuronal specificity, whereas the other serves a common role in translocating the LC of either toxin. Also, it was shown that the C-terminal portion of the HC of TeTx is unable to play the intracellular role of its counterpart in BoNT A.

Animals↗
PubMed1990

Botulinum type F neurotoxin. Large-scale purification and characterization of its binding to rat cerebrocortical synaptosomes.

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 high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.

Animals↗
PubMed1990

Clues to the multi-phasic inhibitory action of botulinum neurotoxins on release of transmitters.

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 as the whole toxin in reducing Ca2(+)-evoked noradrenaline release, when digitonin-permeabilization was used to overcome the uptake barrier. Treatment of BoNT A with 10 mM dithiothreitol, under non-denaturing conditions, was not very effective in reducing its inter-chain disulphide bond(s) and had little influence on the level of inhibition seen. 4. Altering the intra-synaptosomal concentrations of cyclic nucleotides (c-AMP, c-GMP) or protein kinase C activity failed to affect the reduction of Ca2(+)-dependent K(+)-stimulated noradrenaline release caused by BoNT A or B. On the other hand, raising the cytosolic Ca2+ concentration with the ionophore A23187 reversed the inhibitory effect of BoNT A to a greater extent than that of type B, revealing differences in their actions. 5. Whereas BoNT-induced decrease of Ca2(+)-dependent K(+)-evoked release of noradrenaline was unaffected by destruction of the actin-based cytoskeleton in synaptosomes with cytochalasin D, disassembly of microtubules with colchicine, nocodazole or griseofulvin antagonised the intracellular action of type B but not A. It is speculated that BoNT B blocks transmitter release by interfering with the proposed detachment of synaptic vesicles from microtubules. Establishing the precise involvement of tubulin in the toxin's action may provide a valuable clue to the mechanism of neurotransmitter release or its control.

Animals↗
PubMed1990

Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains.

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 the toxin's acceptors or uptake systems in the two neurone types. 3. Micro-injection into cholinergic neurones of the isolated renatured toxins' chains showed that both light and heavy chains of BoNT are intracellularly required whereas the light chain of TeTx alone is sufficient. 4. The heavy chain of BoNT as well as that of TeTx were found to mediate internalization of active moieties via its amino-terminal half. Furthermore the heavy chain of one toxin could internalize the light chain of the other.

Animals↗
PubMed1989

Multiple domains of botulinum neurotoxin contribute to its inhibition of transmitter release in Aplysia neurons.

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 to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.

Acetylcholine↗
PubMed1989

Inhibition of transmitter release by botulinum neurotoxin A. Contribution of various fragments to the intoxication process.

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 intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.

Acetylcholine↗
PubMed1988

Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release.

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 the evoked release of acetylcholine when bath-applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter. 4. When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide-linked two-chain species. 5. A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively. No blockade was observed when the order of application of the two chains was reversed. 6. These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.

Acetylcholine↗
PubMed1988

Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain.

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 of evoked transmitter release, without changing the quantal size; an intraneuronal site of action has thus been established. In contrast, release from noncholinergic neurons of cerebral ganglion was prevented by the neurotoxin only after injection into the cell. Purified preparations of the individual renatured chains, shown to be nontoxic in a mouse bioassay, failed to affect acetylcholine release when applied extra- or intracellularly. However, inhibition of release was observed after intracellular administration of both chains or when the light chain was injected and the heavy chain was bath-applied. These findings show that both chains are required on the cytosolic side of the neuronal plasma membrane for expression of toxicity and that the cholinergic specificity of the neurotoxin is attributable to its heavy chain, which mediates targeting and subsequent neuronal uptake.

Acetylcholine↗