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Professor Graham Nicholson

Biography

Graham Nicholson is currently Professor of Neurotoxicology in the Department of Medical and Molecular Bioscience at UTS. He has a BSc(Hons) degree in Pharmacology and a PhD degree, which focused on structure-activity relationships in convulant barbiturates and glutarimides, awarded in 1987. He is now a leading Australian toxinology researcher responsible for isolating and pharmacologically characterising a wide range of ant, spider, snake, scorpion, sea anemone, platypus and dinoflagellate venoms/toxins.

He has over 25 years post-doctoral experience in ion channel and neuromuscular electrophysiology including experience with intracellular, patch clamp, ion flux and radioligand binding techniques and has published over 80 international peer-reviewed publications in the areas of toxins and toxicology.

His laboratory has also presented over 200 papers on toxicology and toxinology research at national and international conferences. His current research is into the isolation and characterisation of neurotoxins acting on neurotransmitter release, receptors or ion channels.

He is particularly interested in charactersing toxins that can be developed as potential therapeutics and biopesticides, and molecular tools in the area of neuroscience. His main interest lies in the isolation and characterisation of various insecticidal neurotoxins from spider and scorpion venoms. Work in this area has led to the identification of five families of insect-selective neurotoxins with novel modes of action on insect ion channels that are currently being investigated as lead compounds for the development of novel environmentally-friendly insecticides. These agents should be useful in controlling vectors and pest insects in animal health, public health and agricultural markets.

This work has been the subject of several ARC Discovery, DEST-ISL and Australian Academy of Science Grants and an International Patent. Graham is currently the Associate Dean (International & External Engagement). He is also a core member of the Centre for Health Technologies and Director of the Neurotoxin Research Group.

Professional

Member of the Editorial Board for:

  • Toxicon
  • Frontiers in Cellular Neuroscience
Image of Graham Nicholson
Associate Dean (International and External Engagement), Faculty of Science
Core Member, Centre for Health Technologies
BSc (Hons) (Syd), GradCertHEd (UTS), PhD (Syd)
 
Phone
+61 2 9514 2230
Room
CB07.07.25

Research Interests

The potency and specificity of many animal, plant or microbial toxins make them attractive drug leads and a number of toxins have proved to be invaluable tools in the study of the structure and function of the nervous system and how nerve cells communicate. We are particularly interested in this area of toxinology since Australia contains a large number of highly toxic animals. This provides us with an, as yet, untapped vast source of highly specific toxins to use as molecular probes, and lead compounds for therapeutic and environmentally-friendly insecticidal agents.

Our laboratory focusses on the biochemical characterisation, target identification and mode-of-action of a number of potential therapeutic or insecticidal neurotoxins using a range of liquid chromatography, mass spectrometry, acute toxicity assays, patch-clamp electrophysiological and organ bath assays.

Can supervise: Yes
  • Samira Aili (PhD student)
  • Slawomir Dziemborowicz (PhD student)
  • Dr Jennifer Koh (Honorary Research Fellow)
  • Dr Francesca Marcon (recent Alumni)
  • Dr Monique Windley (recent Alumni)
  • Dr Youmie Chong (recent Alumni)
  • Dr Benjamin Blacklow (recent Alumni)

Graham coordinates and teaches pharmacology and toxicology to students who are planning to major in biomedical science, biotechnology, or medical science.

In addition, he provides pharmacology lectures in nursing, midwifery and nursing practitioner courses at UTS, and also coordinates first and second year pharmacology teaching for the Postgraduate Medicine degree that UTS's provide service teaching expertise to another university. In 2009 he was awarded the Notre Dame School of Medicine Teaching award for Excellence in Teaching, and was Lecturer of the Year in 2010.

Some areas he has taught at senior levels to pharmacology students include, pharmacokinetics, neuropharmacology, neurotoxicology, and toxinology. He also founded the Medical Science degree at UTS.

Currently he lectures in the following subjects:
UTS, Faculty of Science
Pharmacology 1

Chapters

Nicholson, G.M. 2013, 'Spider Peptides' in Handbook of Biologically Active Peptides, Elsevier Inc., pp. 461-472.
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Bosmans, F., Escoubas, P. & Nicholson, G.M. 2009, 'Spider venom peptides as leads for drug and insecticide design' in de Lima, M.E., Pimenta, A.M.C., Martin-Eauclaire, M.F., Zingali, R.B. & Rochat, H. (eds), Animal Toxins: State of the Art. Perspectives In Health and Biotechnology, Editora UFMG, Belo Horizonte, pp. 269-290.
Spider venoms represent highly complex cocktails of proteins, peptides, neurotransmitters, acylpolyamines and other small molecules employed to subdue and kill their prey or predators. By a process of natural selection, spider venoms have evolved as complex pre-optimized combinatorial peptide libraries, displaying Wide-ranging pharmacological activities.The therapeutic potential of these peptides stems from their highly potent and specific actions to modulate an extensive range of ion channels, receptors, and transporters in vertebrates and invertebrates,as well as their antimicrobial activity. Nevertheless, it is only since the advent of modern analytical technologies that the full potential of this resource is beginning to be exploited for validating novel insecticide targets, and for aiding in the design of novel drugs and bioinsecticides. These techniques have identified that the currently known spider venom pool represents a resource of several million peptides that selectivelytarget specific subtypes ofion channels or receptors. Indeed, some spider toxins are becoming the defining pharmacology for specificsubtypes of ion channels. Furthermore, structure-function studies ofthese molecules are leading not only to the discovery of new molecular tools, but are also providing insight into novel therapeutic approaches for the treatment of cardiovascular diseases, cancer, neuromuscular diseases, pain and to a variety ... of other pathological conditions. Against this background, this review will evaluate peptidic spider toxins as possible lead compounds for new therapeutics and for controlling insect pests. Special attention will be given to their role in the discovery of novel approaches in analgesia and cardiovascular research.
Nicholson, G.M. 2006, 'Spider Venom Peptides' in Handbook of Biologically Active Peptides, Elsevier Inc., pp. 369-379.
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Spider venom peptides and protein toxins are recognized as highly potent and specific molecular tools that modulate neurotransmission via interaction with a variety of ion channels, receptors, and transporters in vertebrates and invertebrates. Spiders have, during their evolution, developed a complex preoptimized combinatorial peptide library of enzymes, neurotoxins, and antimicrobial and cytolytic peptides in their venom glands. The role of spider venom is to paralyze and/or kill prey or predators as rapidly as possible. Therefore, their venoms are particularly rich in neurotoxins that rapidly modify ion conductance (ion channel toxins) and to a lesser extent affect neurotransmitter exocytosis (presynaptic toxins) and interfere with the binding of neurotransmitters (postsynaptic toxins). Many of these peptides are selectively insecticidal or modulate the activity of various targets in vertebrates, including humans. In particular, insect-selective atracotoxins are now being investigated for their possible use as bioinsecticidal agents for the control of phytophagous pests or insect vectors of new or reemerging disease. 2006 Elsevier Inc. All rights reserved.
Gordon, D., Gilles, N., Bertrand, D., Milgo, J., Nicholson, G.M., Sauviat, M., Benoit, E., Shichor, I., Lotan, I., Gurevitz, M., Kallen, R.G. & Heinemann, S.H. 2002, 'Scorpion toxins differentiate among neuronal sodium channel subtypes: Nature's guide for design of selective drugs' in Menez, A. (ed), Prespectives in Molecular Toxicology, John Wiley & Sons, Chichester, UK, pp. 215-238.
Robinson, K., Wilson, M., Brown, D.M., Miller, P.F., Nicholson, G.M., Garman, C. & Van Vorst, S.M. 2000, 'Educational Board Games Promote Social Interaction and Effective Learning in Undergraduate Nursing Students' in Bialon, L. (ed), Social Aspects of Higher Education - Polish and Australian Reflections, Warsaw University of Technology, Warsaw, Poland, pp. 157-168.

Conferences

Pineda, S.S., Jones, A., Nicholson, G.M., Escoubas, P., Mattick, J.S. & King, G.F. 2012, 'Understanding the Chemical Diversity of Spider Venoms Using a Combined Genomic, Transcriptomic and Proteomic Approach', TOXICON, pp. 124-124.
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Windley, M.J., King, G.F. & Nicholson, G.M. 2012, 'An Insecticidal Spider Toxin that Acts as a Positive Allosteric Modulator of Insect Nicotinic Acetylcholine Receptors', TOXICON, pp. 169-170.
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Marcon, F., Leblanc, M., Escoubas, P. & Nicholson, G.M. 2012, 'Isolation and Pharmacological Characterisation of Neurotoxins from the Venom of Three Species of Australian Copperheads (Austrelaps spp.) and the Efficacy of Tiger Snake Antivenom to Prevent or Reverse Neurotoxicity', TOXICON, pp. 170-170.
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de Araujo, A.D.D., Herzig, V., Mobil, M., Windley, M.J., Nicholson, G.M., Alewood, P.F. & King, G.F. 2010, 'Understanding the role of the unusual constrained eight-membered disulfide ring of spider toxins', JOURNAL OF PEPTIDE SCIENCE, pp. 60-61.
Gunning, S.J., Maggio, F., Windley, M.J., Valenzuela, S.M., King, G.F. & Nicholson, G.M. 2009, 'The Janus-faced atracotoxins are specific blockers of invertebrate K-Ca channels', FEBS JOURNAL, pp. 3-3.
Dauly, C., Escoubas, P., Nicholson, G.M., King, G.F. & Hornshaw, M. 2009, 'Nanoscale characterization of spider venom peptides by high resolution LC-MS/MS analysis'.
Nicholson, G.M., Gunning, S.J., Windley, M.J., Maggio, F.J., Valenzuela, S. & King, G.F. 2009, 'Defining the lethal ion channel targets of insecticidal spider toxins'.
Nicholson, G.M., Escoubas, P., Dauly, C. & King, G.F. 2009, 'Spider venoms for novel therapeutics: from Venomics to drug candidates'.
Yamaji, N., Little, M.J., Nishio, H., Billen, B., Villegas, E., Nishiuchi, Y., Tytgat, J., Nicholson, G.M. & Corzo, G. 2009, 'Synthesis, solution structure, and phylum selectivity of a spider ?-toxin that slows inactivation of specific voltage-gated sodium channel subtypes', Journal of Biological Chemistry, pp. 24568-24582.
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Magi 4, now renamed ?-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to ?-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (NaV) channels but, unlike ?-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in NaV channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect NaV channel subtypes showing that ?-hexatoxin-Mg1a selectively slows channel inactivation of mammalian NaV1.1, NaV1.3, and NaV1.6 but more importantly shows higher affinity for insect NaV1 (para) channels. Consequently, ?-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded ?-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion ?-toxins and ?-hexatoxins are distributed in a topologically similar manner in ?-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of ?-hexatoxin-Mg1a for certain mammalian and insect NaV channel subtypes. As such, ?-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity. 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
Sollod, B., Gunning, S.J., Wen, S., Gentz, M.C., Nicholson, G.M. & King, G.K. 2009, 'A dual-target, self-synergizing ion channel toxin from spider venom'.
Marcon, F., Purtell, L.A., Escoubas, P., Graudins, A. & Nicholson, G.M. 2008, 'Isolation and pharmacological characterisation of a presynaptic neurotoxin from the venom of the Australian copperhead (Austrelaps superbus)'.
Blacklow, B.J., Escoubas, P. & Nicholson, G.M. 2008, 'Characterisation of the presynaptic phospholipase A2 neurotoxin complex, -acanthoxin, from the venom of the common death adder (Acanthophis antarcticus)'.
Little, M.J., Ferreira, W.A., Madio, B., Belato, Y., Orts, D.J., Zaharenko, A.J. & Nicholson, G.M. 2008, 'Phyla-selective actions of sea anemone toxins from Bunodosoma caissarum on insect sodium channel gating and kinetics'.
Nicholson, G.M., Yamaji, N., Little, M.J., Nishio, H., Billen, B., Villegas, E., Nishiuichi, Y., Tytgat, J. & Corzo, G. 2008, 'Synthesis, solution structure and pharmacological profile of Magi 4, a spider -toxin that slows inactivation of specific voltage-gated sodium channel subtypes in insects and mammals'.
Windley, M.J., Escoubas, P., Valenzuela, S. & Nicholson, G.M. 2008, 'Characterisation of a family of insect-selective neurotoxins isolated from the African tarantula, Eucratoscelus longiceps'.
Yamaji, N., Little, M.J., Nishio, H., Billen, B., Villegas, E., Nishiuichi, Y., Tytgat, J., Nicholson, G.M. & Corzo, G. 2008, 'Structural and pharmacological profile of Magi 4, a peptide toxin from the venom of the hexathelid spider Macrothele gigas'.
Nicholson, G.M., Gunning, S.J., Maggio, F.J., Windley, M.J., Valenzuela, S. & King, G.F. 2008, 'Identifying novel insecticide targets using insect-specific spider toxins'.
Lossy, B., Graudins, A., Escoubas, P., Hains, P.G. & Nicholson, G.M. 2007, 'Isolation of novel neurotoxins from the venom of the Australian rough-scaled snake (Tropidechis carinatus)'.
Purtell, L.A., Graudins, A., Escoubas, P., Hains, P.G. & Nicholson, G.M. 2007, 'Isolation and characterization of novel neurotoxins from the venom of the Australian copperhead snake (Austrelaps superbus)'.
Escoubas, P., Sollod, B., Nicholson, G.M., Wilson, D. & King, G.K. 2006, 'Venom landscapes: Towards the discovery of novel pharmacological tools via the combined use of MALDI-TOF MS and cDNA'.
Escoubas, P., Lee, M.J., Ross, G., Lazdunski, M. & Nicholson, G.M. 2006, 'Novel insect-selective neurotoxins from the venom of the tarantula Eucratoscelus longiceps target insect Kv channels'.
Escoubas, P., Sollod, B., Nicholson, G.M., Wilson, D., Vinh, J. & King, G.F. 2006, 'Venom landscapes: Towards the discovery of novel pharmacological tools via the combined use of LC-MALDI-TOF and MALDI-TOF/TOF with cDNA analysis'.
Escoubas, P., Quinton, L., Hui, R., Wen, S., Chamot-Rooke, J. & Nicholson, G.M. 2006, 'Spider venom proteome mining and de novo toxin sequencing by LC-MALDI-TOF and FT-ICR mass spectrometry'.
Nicholson, G.M., Chong, Y., Wen, S., Hayes, J.L., Hodgson, W.C., Hains, P.G., Broady, K.W. & King, G.K. 2006, 'Omega-ACTX-Ar1a: a novel insect-selective voltage-gated calcium channel blocker from the venom of the Sydney funnel-web spider'.
Little, M.J., Yamaji, N., Villegas, E., Corzo, G. & Nicholson, G.M. 2006, 'Structure-function relationships of Magi 4, a spider neurotoxin targeting insect and mammalian voltage-gated sodium channels'.
Gunning, S.J., Maggio, F.J., Valenzuela, S., Broady, K.W., King, G.K. & Nicholson, G.M. 2006, 'Pharmacophore mapping of the -atracotoxins: selective insect potassium channel blockers that reveal a novel insecticide target'.
Sollod, B., Gunning, S.J., Wen, S., Nicholson, G.M. & King, G.K. 2006, 'A dual-target, self-synergizing toxin from spider venom'.
Gunning, S.J., Maggio, F.J., Valenzuela, S., Broady, K.W., King, G.K. & Nicholson, G.M. 2005, '-Atracotoxins: Insect potassium channels blockers that reveal a novel insecticide target'.
Sollod, B., Gunning, S.J., Wen, S., Nicholson, G.M. & King, G.K. 2005, 'Evolution of a dual target, self-synergizing toxin: implications for insecticide and pharmaceutical discovery'.
Wen, S., Wilson, D., Kuruppu, S., Korsinczky, M., Hedrick, J., Pang, L., Szeto, T., Hodgson, W.C., Alewood, P.F. & Nicholson, G.M. 2005, 'Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family peptides'.
Gunning, S.J., Maggio, F.J., Valenzuela, S., Broady, K.W., King, G.K. & Nicholson, G.M. 2005, 'Selective actions of -atracotoxins on insect KCa channels: electrophysiological validation of the insect target and pharmacophore'.
Chong, Y., Wen, S., Hayes, J.L., Hains, P.G., King, G.K., Kuhn-Nentwig, L. & Nicholson, G.M. 2005, 'Probing phylogenetically distinct spider venoms for potential biopesticides that target insect voltage-gated calcium channels'.
Nicholson, G.M. 2004, 'Australian spiders: venomous villains or toxic treasures?'.
Gunning, S.J., Maggio, F.J., King, G.K. & Nicholson, G.M. 2004, '-Atracotoxins: Insect potassium channels blockers that reveal a novel insecticide target.'.
Gunning, S.J., Maggio, F.J., King, G.K. & Nicholson, G.M. 2003, 'Do insecticidal J-atracotoxins target insect potassium channels?'.
Chong, Y., Khalife, A., Hains, P.G., Broady, K.W. & Nicholson, G.M. 2003, 'Isolation and characterisation of a novel insect selective neurotoxin from the venom of the female Australian eastern mouse spider (Missulena bradleyi)'.
Gunning, S.J., Chong, Y., Khalife, A., Hains, P.G., Broady, K.W. & Nicholson, G.M. 2003, 'Discovery of a novel sodium channel neurotoxin delta-missulenatoxin Mb1a from the venom of the Eastern mouse spider Missulena bradleyi'.
Hayes, J.L., Wen, S., Yang, Q., Hains, P.G., Broady, K.W. & Nicholson, G.M. 2003, 'Isolation and characterisation of an insect selective neurotoxin omega-atracotoxin-Ar1a from the venom of the female Sydney funnel web spider, Atrax robustus.'.
Wilson, H.I., Hains, P.G. & Nicholson, G.M. 2003, 'The venom of Australian Urodacus scorpions (Arachnidae: Scorpiones: Urodacidae) contains a novel class of insect selective sodium channel blocking toxins'.
Wilson, H.I. & Nicholson, G.M. 2003, 'Characteristics of the venom of Australian urodacid and buthid scorpions: venom production, composition and toxicity'.
Graudins, A., Sung, K., Hains, P.G., Padula, M., Broady, K.W. & Nicholson, G.M. 2002, 'Partial protein and DNA sequences of Latrodectus hasselti, L. hesperus and L. mactans latrotoxins: are they homologous?'.
Gunning, S.J., Khalife, A., Padula, M., Smith, R., Broady, K.W. & Nicholson, G.M. 2002, 'Modulation of sodium channel gating and kinetics by missulenatoxin Mb1a from the Australian eastern mouse spider Missulena bradleyi.'.
Sollod, B., Wilson, D., Wang, X., Reenan, R.A., Nicholson, G.M., Alewood, P.F. & King, G.K. 2002, 'Structure function studies of the insect specific calcium channel blocker omega ACTX Hv2a'.
Graudins, A., Padula, M., Broady, K.W. & Nicholson, G.M. 2000, 'Evidence for red back spider antivenom efficacy in the prevention of envenomation by other widow spiders (genus Latrodectus)', Journal of Toxicology-Clinical Toxicology, Taylor & Francis, London, pp. 205-206.
Nicholson, G.M., Birinyi-Strachan, L.C., Rash, L., Szeto, T., Khalife, A., Hodgson, W.C. & King, G.F. 2000, 'Novel d atracotoxins: spider toxins that target the voltage gated sodium channel'.

Journal articles

Bende, N.S., Dziemborowicz, S., Herzig, V., Ramanujam, V., Brown, G.W., Bosmans, F., Nicholson, G.M., King, G.F. & Mobli, M. 2015, 'The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large ?-hairpin loop', FEBS Journal, vol. 282, no. 5, pp. 904-920.
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Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large ?-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.
Touchard, A., Dauvois, M., Arguel, M.-.J., Petitclerc, F., Leblanc, M., Dejean, A., Orivel, J., Nicholson, G.M. & Escoubas, P. 2014, 'Elucidation of the unexplored biodiversity of ant venom peptidomes via MALDI-TOF mass spectrometry and its application for chemotaxonomy', Journal of Proteomics, vol. 105, pp. 217-231.
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The rise of integrative taxonomy, a multi-criteria approach used in characterizing species, fosters the development of new tools facilitating species delimitation. Mass spectrometric (MS) analysis of venom peptides from venomous animals has previously been demonstrated to be a valid method for identifying species. Here we aimed to develop a rapid chemotaxonomic tool for identifying ants based on venom peptide mass fingerprinting. The study focused on the biodiversity of ponerine ants (Hymenoptera: Formicidae: Ponerinae) in French Guiana. Initial experiments optimized the use of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine variations in the mass profiles of ant venoms using several MALDI matrices and additives. Data were then analyzed via a hierarchical cluster analysis to classify the venoms of 17 ant species. In addition, phylogenetic relationships were assessed and were highly correlated with methods using DNA sequencing of the mitochondrial gene cytochrome c oxidase subunit 1. By combining a molecular genetics approach with this chemotaxonomic approach, we were able to improve the accuracy of the taxonomic findings to reveal cryptic ant species within species complexes. This chemotaxonomic tool can therefore contribute to more rapid species identification and more accurate taxonomies. Biological significance: This is the first extensive study concerning the peptide analysis of the venom of both Pachycondyla and Odontomachus ants. We studied the venoms of 17 ant species from French Guiana that permitted us to fine-tune the venom analysis of ponerine ants via MALDI-TOF mass spectrometry. We explored the peptidomes of crude ant venom and demonstrated that venom peptides can be used in the identification of ant species. In addition, the application of this novel chemotaxonomic method combined with a parallel genetic approach using COI sequencing permitted us to reveal the presence of cryptic ant...
Atakuziev, B.U., Wright, C.E., Graudins, A., Nicholson, G.M. & Winkel, K.D. 2014, 'Efficacy of Australian red-back spider (Latrodectus hasselti) antivenom in the treatment of clinical envenomation by the cupboard spider Steatoda capensis (Theridiidae)', Toxicon, vol. 86, pp. 68-78.
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We describe the first Steatoda capensis envenomation treated with CSL red-back spider antivenom (RBSAV). The patient, a 51-year-old female, developed acute local pain, swelling, redness, and diaphoresis in association with tender lymphadenopathy and hypertension. These features responded, in a dose-dependent manner, to RBSAV. In vitro studies confirmed that RBSAV could neutralize S. capensis venom at equivalent concentrations required to neutralize red-back spider (Latrodectus hasselti) venom. Similar data were obtained using Mexican Latrodectus mactans antivenom (Aracmyn). Although S. capensis yielded similar quantities of venom protein as L. hasselti, pooled S. capensis and Steatoda grossa venom was more rapidly toxic to insects than either L. hasselti or Latrodectus tredecimguttatus venom. By contrast, both Latrodectus venoms were more potent than S. capensis venom in contracting rat isolated mesenteric arteries. Size-exclusion and anion-exchange chromatography was used to purify a 130 kDa fraction from S. capensis venom that induced contracture and loss of twitch tension in chick isolated biventer cervicis nerve-muscle preparations in a manner similar to ?-latrotoxin. This activity was abolished by pre-incubation with RBSAV. We conclude that 'steatodism' may overlap more closely with latrodectism than previously recognized and that this bite should be managed in the same way as for Australian red-back envenomation. 2014 Elsevier Ltd. All rights reserved.
Touchard, A., Labrire, N., Roux, O., Petitclerc, F., Orivel, J., Escoubas, P., Koh, J.M.S., Nicholson, G.M. & Dejean, A. 2014, 'Venom toxicity and composition in three Pseudomyrmex ant species having different nesting modes', Toxicon, vol. 88, pp. 67-76.
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We aimed to determine whether the nesting habits of ants have influenced their venom toxicity and composition. We focused on the genus Pseudomyrmex (Pseudomyrmecinae) comprising terrestrial and arboreal species, and, among the latter, plant-ants that are obligate inhabitants of myrmecophytes (i.e., plants sheltering ants in hollow structures). Contrary to our hypothesis, the venom of the ground-dwelling species, Pseudomyrmex termitarius, was as efficacious in paralyzing prey as the venoms of the arboreal and the plant-ant species, Pseudomyrmexpenetrator and Pseudomyrmexgracilis. The lethal potency of P. termitarius venom was equipotent with that of P. gracilis whereas the venom of P. penetrator was less potent. The MALDI-TOF MS analysis of each HPLC fraction of the venoms showed that P. termitarius venom is composed of 87 linear peptides, while both P. gracilis and P. penetrator venoms (23 and 26 peptides, respectively) possess peptides with disulfide bonds. Furthermore, P. penetrator venom contains three hetero- and homodimeric peptides consisting of two short peptidic chains linked together by two interchain disulfide bonds. The large number of peptides in P. termitarius venom is likely related to the large diversity of potential prey plus the antibacterial peptides required for nesting in the ground. Whereas predation involves only the prey and predator, P. penetrator venom has evolved in an environment where trees, defoliating insects, browsing mammals and ants live in equilibrium, likely explaining the diversity of the peptide structures. 2014 Elsevier Ltd. All rights reserved.
Bende, N.S., Dziemborowicz, S., Mobli, M., Herzig, V., Gilchrist, J., Wagner, J., Nicholson, G.M., King, G.F. & Bosmans, F. 2014, 'A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a', Nature Communications, vol. 5.
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?-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Na v channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv 2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav 1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNa v 1 resistance to Dc1a, we identified two residues within the BgNav 1 domain II S1-S2 loop that when mutated to their PaNav 1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, a concept that will be valuable for the design of insect-selective insecticides. 2014 Macmillan Publishers Limited. All rights reserved.
Aili, S.R., Touchard, A., Escoubas, P., Padula, M.P., Orivel, J., Dejean, A. & Nicholson, G.M. 2014, 'Diversity of peptide toxins from stinging ant venoms', Toxicon, vol. 92, pp. 166-178.
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Ants (Hymenoptera: Formicidae) represent a taxonomically diverse group of arthropods comprising nearly 13,000 extant species. Sixteen ant subfamilies have individuals that possess a stinger and use their venom for purposes such as a defence against predators, competitors and microbial pathogens, for predation, as well as for social communication. They exhibit a range of activities including antimicrobial, haemolytic, cytolytic, paralytic, insecticidal and pain-producing pharmacologies. While ant venoms are known to be rich in alkaloids and hydrocarbons, ant venoms rich in peptides are becoming more common, yet remain understudied. Recent advances in mass spectrometry techniques have begun to reveal the true complexity of ant venom peptide composition. In the few venoms explored thus far, most peptide toxins appear to occur as small polycationic linear toxins, with antibacterial properties and insecticidal activity. Unlike other venomous animals, a number of ant venoms also contain a range of homodimeric and heterodimeric peptides with one or two interchain disulfide bonds possessing pore-forming, allergenic and paralytic actions. However, ant venoms seem to have only a small number of monomeric disulfide-linked peptides. The present review details the structure and pharmacology of known ant venom peptide toxins and their potential as a source of novel bioinsecticides and therapeutic agents.
Marcon, F., Purtell, L., Santos, J., Hains, P.G., Escoubas, P., Graudins, A. & Nicholson, G.M. 2013, 'Characterization of monomeric and multimeric snake neurotoxins and other bioactive proteins from the venom of the lethal Australian common copperhead (Austrelaps superbus)', Biochemical Pharmacology, vol. 85, no. 10, pp. 1555-1573.
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Envenomation by Australian copperheads results mainly in muscle paralysis largely attributed to the presence of postsynaptic ?-neurotoxins. However, poorly reversible neurotoxic effects suggest that these venoms may contain snake presynaptic phospholipase A2 neurotoxins (SPANs) that irreversibly inhibit neurotransmitter release. Using size-exclusion liquid chromatography, the present study isolated the first multimeric SPAN complex from the venom of the Australian common copperhead, Austrelaps superbus. The multimeric SPAN P-elapitoxin-As1a (P-EPTX-As1a) along with two novel monomeric SPANs and a new postsynaptic ?-neurotoxin were then pharmacologically characterized using the chick biventer cervicis nerve-muscle preparation. All SPANs inhibited nerve-evoked twitch contractions at the neuromuscular junction without inhibiting contractile responses to cholinergic agonists or KCl. These actions are consistent with a prejunctional action to inhibit neurotransmitter release, without direct myotoxicity. Furthermore, the multimeric P-EPTX-As1a caused tetanic 'fade' in muscle tension under high frequency nerve stimulation, and produced a triphasic alteration to neurotransmitter release. These actions have been previously noted with other multimeric SPAN complexes such as taipoxin. Moreover, the neurotoxic ?-subunit of P-EPTX-As1a shows high homology to taipoxin ?-chain. Several other coagulopathic and myotoxic high mass proteins including a class PIII snake venom metalloproteinase, C-type lectin, l-amino acid oxidase, acetylcholinesterase and phospholipase B were also identified that may contribute to the overall toxicity of A. superbus venom. In conclusion, clinicians should be aware that early antivenom intervention might be necessary to prevent the onset of irreversible presynaptic neurotoxicity caused by multimeric and monomeric SPANs and that A. superbus venom is potentially capable of producing coagulopathic and myotoxic effects. AbbreviationsAChacetylcholineAChEa...
Bende, N.S., Kang, E., Herzig, V., Bosmans, F., Nicholson, G.M., Mobli, M. & King, G.F. 2013, 'The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels', Biochemical Pharmacology, vol. 85, no. 10, pp. 1542-1554.
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One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large ? hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel. 2013 Elsevier Inc. All rights reserved.
De Araujo, A.D., Herzig, V., Windley, M.J., Dziemborowicz, S., Mobli, M., Nicholson, G.M., Alewood, P.F. & King, G.F. 2013, 'Do vicinal disulfide bridges mediate functionally important redox transformations in proteins?', Antioxidants and Redox Signaling, vol. 19, no. 16, pp. 1976-1980.
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Vicinal disulfide bridges, in which a disulfide bond is formed between adjacent cysteine residues, constitute an unusual but expanding class of potential allosteric disulfides. Although vicinal disulfide rings (VDRs) are relatively uncommon, they have proven to be functionally critical in almost all proteins in which they have been discovered. However, it has proved difficult to test whether these sterically constrained disulfides participate in functionally important redox transformations. We demonstrate that chemical replacement of VDRs with dicarba or diselenide bridges can be used to assess whether VDRs function as allosteric disulfides. Our approach leads to the hypothesis that not all VDRs participate in functionally important redox reactions. Antioxid. Redox Signal. 19, 1976-1980. Mary Ann Liebert, Inc.
Palagi, A., Koh, J.M.S., Leblanc, M., Wilson, D., Dutertre, S., King, G.F., Nicholson, G.M. & Escoubas, P. 2013, 'Unravelling the complex venom landscapes of lethal Australian funnel-web spiders (Hexathelidae: Atracinae) using LC-MALDI-TOF mass spectrometry', Journal of Proteomics, vol. 80, pp. 292-310.
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Spider venoms represent vast sources of bioactive molecules whose diversity remains largely unknown. Indeed, only a small subset of species have been studied out of the ~. 43,000 extant spider species. The present study investigated inter- and intra-species venom complexity in 18 samples collected from a variety of lethal Australian funnel-web spiders (Mygalomorphae: Hexathelidae: Atracinae) using C4 reversed-phase separation coupled to offline MALDI-TOF mass spectrometry (LC-MALDI-TOF MS). An in-depth investigation focusing on four atracine venoms (male Illawarra wisharti, male and female Hadronyche cerberea, and female Hadronyche infensa Toowoomba) revealed, on average, ~. 800 peptides in female venoms while male venoms contained ~. 400 peptides, distributed across most HPLC fractions. This is significantly higher than previous estimates of peptide expression in mygalomorph venoms. These venoms also showed distinct intersexual as well as intra- and inter-species variation in peptide masses. Construction of both 3D and 2D contour plots revealed that peptide mass distributions in all 18 venoms were centered around the 3200-5400. m/. z range and to a lesser extent the 6600-8200. m/. z range, consistent with previously described hexatoxins. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. Biological significance: In the present study we describe the complexity of 18 venoms from lethal Australian funnel-web spiders using LC-MALDI-TOF MS. The study includes an in-depth investigation, focusing on four venoms, that revealed the presence of ~. 800 peptides in female venoms and ~. 400 peptides in male venoms. This is significantly higher than previous estimates of peptide expression in spider venoms. By constructing both 3D and 2D contour plots we were also able to reveal the distinct intersexual as well as intra- and inter-species var...
Graudins, A., Little, M.J., Pineda, S.S., Hains, P.G., King, G.F., Broady, K.W. & Nicholson, G.M. 2012, 'Cloning and activity of a novel ?-latrotoxin from red-back spider venom', Biochemical Pharmacology, vol. 83, no. 1, pp. 170-183.
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The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110-140 kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific ?-latrotoxin (?-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved ?-LTX, ?-LIT and ?-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-?-LTX monoclonal antibody 4C4.1, raised against ?-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve-muscle bioassay. This suggests that there are important structural differences between ?-LTXs in theridiid spider venoms. We therefore cloned and sequenced the ?-LTX from the Australian red-back spider L. hasselti (?-LTX-Lh1a). The deduced amino acid sequence of the mature ?-LTX-Lh1a comprises 1180 residues (?132 kDa) with ?93% sequence identity with ?-LTX-Lt1a. ?-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with ?-LTX-Lt1a, indicates that ?-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that ?-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody. 2011 Elsevier Inc.
Klint, J.K., Senff, S., Rupasinghe, D.B., Er, S.Y., Herzig, V., Nicholson, G.M. & King, G.F. 2012, 'Spider-venom peptides that target voltage-gated sodium channels: Pharmacological tools and potential therapeutic leads', Toxicon, vol. 60, no. 4, pp. 478-491.
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Voltage-gated sodium (Na V) channels play a central role in the propagation of action potentials in excitable cells in both humans and insects. Many venomous animals have therefore evolved toxins that modulate the activity of Na V channels in order to subdue their prey and deter predators. Spider venoms in particular are rich in Na V channel modulators, with one-third of all known ion channel toxins from spider venoms acting on Na V channels. Here we review the landscape of spider-venom peptides that have so far been described to target vertebrate or invertebrate Na V channels. These peptides fall into 12 distinct families based on their primary structure and cysteine scaffold. Some of these peptides have become useful pharmacological tools, while others have potential as therapeutic leads because they target specific Na V channel subtypes that are considered to be important analgesic targets. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides and so far only 0.01% of this diversity been characterised. Thus, it is likely that future research will reveal additional structural classes of spider-venom peptides that target Na V channels. 2012 Elsevier Ltd.
Windley, M.J., Herzig, V., Dziemborowicz, S.A., Hardy, M.C., King, G.F. & Nicholson, G.M. 2012, 'Spider-venom peptides as bioinsecticides', Toxins, vol. 4, no. 3, pp. 191-227.
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Over 10,000 arthropod species are currently considered to be pest organisms. They are estimated to contribute to the destruction of ~14% of the world's annual crop production and transmit many pathogens. Presently, arthropod pests of agricultural and health significance are controlled predominantly through the use of chemical insecticides. Unfortunately, the widespread use of these agrochemicals has resulted in genetic selection pressure that has led to the development of insecticide-resistant arthropods, as well as concerns over human health and the environment. Bioinsecticides represent a new generation of insecticides that utilise organisms or their derivatives (e.g., transgenic plants, recombinant baculoviruses, toxin-fusion proteins and peptidomimetics) and show promise as environmentally-friendly alternatives to conventional agrochemicals. Spider-venom peptides are now being investigated as potential sources of bioinsecticides. With an estimated 100,000 species, spiders are one of the most successful arthropod predators. Their venom has proven to be a rich source of hyperstable insecticidal mini-proteins that cause insect paralysis or lethality through the modulation of ion channels, receptors andenzymes. Many newly characterized insecticidal spider toxins target novel sites in insects. Here we review the structure and pharmacology of these toxins and discuss the potential of this vast peptide library for the discovery of novel bioinsecticides.
Marcon, F., Leblanc, M., Vetter, I., Lewis, R.J., Escoubas, P. & Nicholson, G.M. 2012, 'Pharmacological characterization of ?-elapitoxin-Al2a from the venom of the Australian pygmy copperhead (Austrelaps labialis): An atypical long-chain ?-neurotoxin with only weak affinity for ?7 nicotinic receptors', Biochemical Pharmacology, vol. 84, no. 6, pp. 851-853.
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Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead (Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, ?-EPTX-Al2a, from the venom of A. labialis. ?-EPTX-Al2a (8072.77 Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve-muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. ?-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA 2 value of 7.902 (K B = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal ?7 nAChRs, with an IC 50 of 1.2 ?M, and failed to inhibit ganglionic ?3?2/?3?4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. ?-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain ?-neurotoxins. While ?-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (?1) 2??? nAChRs it lacks the key Ala 28 and Arg 36 residues important for ?7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation. 2012 Elsevier Inc. All rights reserved.
Blacklow, B., Kornhauser, R., Hains, P.G., Loiacono, R., Escoubas, P., Graudins, A. & Nicholson, G.M. 2011, '?-Elapitoxin-Aa2a, a long-chain snake ?-neurotoxin with potent actions on muscle (?1)2??? nicotinic receptors, lacks the classical high affinity for neuronal ?7 nicotinic receptors', Biochemical Pharmacology, vol. 81, no. 2, pp. 314-325.
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In contrast to all classical long-chain ?-neurotoxins possessing the critical fifth disulfide bond, ?-elapitoxin-Aa2a (?-EPTX-Aa2a), a novel long-chain ?-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal ?7-type nicotinic acetylcholine receptors (nAChRs) ?-EPTX-Aa2a (8850 Da; 0.1-1 ?M) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. ?-EPTX-Aa2a (1-10 nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA2 value of 8.311 0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain ?-neurotoxin, ?-bungarotoxin. In contrast, ?-EPTX-Aa2a produced complete, but weak, inhibition of 125I-?-bungarotoxin binding to rat hippocampal ?7 nAChRs (pKI = 3.670), despite high sequence homology and similar mass to a wide range of long-chain ?-neurotoxins. The mostly likely cause for the loss of ?7 binding affinity is a leucine substitution, in loop II of ?-EPTX-Aa2a, for the highly conserved Arg33 in long-chain ?-neurotoxins. Arg 33 has been shown to be critical for both neuronal and muscle activity. Despite this substitution, ?-EPTX-Aa2a retains high affinity for muscle (?1)2??? nAChRs. This is probably as a result of an Arg29 residue, previously shown to be critical for muscle (?1)2??? nAChR affinity, and highly conserved across all short-chain, but not long-chain, ?-neurotoxins. ?-EPTX-Aa2a therefore represents a novel atypical long-chain ?-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes. Copyright 2010 Published by Elsevier Inc. All rights reserved.
Windley, M.J., Escoubas, P., Valenzuela, S.M. & Nicholson, G.M. 2011, 'A novel family of insect-selective peptide neurotoxins targeting insect large-conductance calcium-activated K + channels isolated from the venom of the theraphosid spider Eucratoscelus constrictus', Molecular Pharmacology, vol. 80, no. 1, pp. 1-13.
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Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (?-TRTX-Ec2a, ?-TRTX-Ec2b, and ?-TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that ?-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K + (BK Ca) channel currents with IC 50 values of 3 to 25 nM. In addition, ?-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K + currents, but only at significantly higher concentrations. ?-TRTX-Ec2a and ?-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous ?-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, ?-TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, ?-TRTX-Ec2a and ?-TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BK Ca channels. This study therefore establishes these toxins as tools for studying the role of BK Ca channels in insects and lead compounds for the development of novel insecticides. Co...
Marcon, F. & Nicholson, G.M. 2011, 'Identification of presynaptic neurotoxin complexes in the venoms of three Australian copperheads (Austrelaps spp.) and the efficacy of tiger snake antivenom to prevent or reverse neurotoxicity', Toxicon, vol. 58, no. 5, pp. 439-452.
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The venom of the Australian lowlands copperhead, Austrelaps superbus, produces significant and potentially lethal neurotoxic paralysis in cases of clinical envenomation. However, little is known about the neurotoxic components within this venom or venoms from the related alpine copperhead (Austrelaps ramsayi) or pygmy copperhead (Austrelaps labialis). Using the isolated chick biventer cervicis nerve-muscle preparation, all Austrelaps venoms were found to exhibit potent and rapid inhibition of nerve-evoked twitch contractions and block of contractures to nicotinic agonists, consistent with postsynaptic neurotoxic activity. Following separation by size-exclusion liquid chromatography under non-denaturing conditions, all Austrelaps venoms were found to also contain a high molecular mass fraction with only weak phospholipase A 2 (PLA 2) activity that caused a slow inhibition of twitch contractions, without inhibiting contractures to nicotinic agonists. These actions are consistent with the presence of additional snake presynaptic PLA 2 neurotoxin (SPAN) complexes in all three Austrelaps venoms. However, there was no evidence of direct muscle damage produced by any Austrelaps venom or SPAN complex. Monovalent tiger snake antivenom was effective in neutralising the neurotoxicity of both whole venom and the SPAN complex. However antivenom was unable to effectively reverse whole venom neurotoxicity, or prejunctional SPAN neurotoxicity, once established. Given the strong neurotoxicity of all Austrelaps venoms, particularly A. ramsayi and A. labialis, effective bites from these copperhead species should be considered potentially lethal. Furthermore, clinicians need to be aware of possible irreversible presynaptic neurotoxicity following envenomation from all copperhead species and that early antivenom intervention is important in preventing further development of toxicity. 2011 Elsevier Ltd.
Smith, J.J., Hill, J.M., Little, M.J., Nicholson, G.M., King, G.F. & Alewood, P.F. 2011, 'Unique scorpion toxin with a putative ancestral fold provides insight into evolution of the inhibitor cystine knot motif', Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 26, pp. 10478-10483.
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The three-disulfide inhibitor cystine knot (ICK) motif is a fold common to venom peptides from spiders, scorpions, and aquatic cone snails. Over a decade ago it was proposed that the ICK motif is an elaboration of an ancestral two-disulfide fold coined the disulfide-directed ?-hairpin (DDH). Here we report the isolation, characterization, and structure of a novel toxin [U 1-liotoxin-Lw1a (U 1-LITX-Lw1a)] from the venom of the scorpion Liocheles waigiensis that is the first example of a native peptide that adopts the DDH fold. U 1-LITX-Lw1a not only represents the discovery of a missing link in venom protein evolution, it is the first member of a fourth structural fold to be adopted by scorpion-venom peptides. Additionally, we show that U 1-LITX-Lw1a has potent insecticidal activity across a broad range of insect pest species, thereby providing a unique structural scaffold for bioinsecticide development.
Herzig, V., Wood, D.L.A., Newell, F., Chaumeil, P.-.A., Kaas, Q., Binford, G.J., Nicholson, G.M., Gorse, D. & King, G.F. 2011, 'ArachnoServer 2.0, an updated online resource for spider toxin sequences and structures', Nucleic Acids Research, vol. 39, no. SUPPL. 1.
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ArachnoServer (www.arachnoserver.org) is a manually curated database providing information on the sequence, structure and biological activity of protein toxins from spider venoms. These proteins are of interest to a wide range of biologists due to their diverse applications in medicine, neuroscience, pharmacology, drug discovery and agriculture. ArachnoServer currently manages 1078 protein sequences, 759 nucleic acid sequences and 56 protein structures. Key features of ArachnoServer include a molecular target ontology designed specifically for venom toxins, current and historic taxonomic information and a powerful advanced search interface. The following significant improvements have been implemented in version 2.0: (i) the average and monoisotopic molecular masses of both the reduced and oxidized form of each mature toxin are provided; (ii) the advanced search feature now enables searches on the basis of toxin mass, external database accession numbers and publication date in ArachnoServer; (iii) toxins can now be browsed on the basis of their phyletic specificity; (iv) rapid BLAST searches based on the mature toxin sequence can be performed directly from the toxin card; (v) private silos can be requested from research groups engaged in venoms-based research, enabling them to easily manage and securely store data during the process of toxin discovery; and (vi) a detailed user manual is now available. The Author(s) 2010.
Blacklow, B., Konstantakopoulos, N., Hodgson, W.C. & Nicholson, G.M. 2010, 'Presence of presynaptic neurotoxin complexes in the venoms of Australo-Papuan death adders (Acanthophis spp.)', Toxicon, vol. 55, no. 6, pp. 1171-1180.
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Australo-papuan death adders (Acanthophis spp.) are a cause of serious envenomations in Papua New Guinea and northern Australia often resulting in neurotoxic paralysis. Furthermore, victims occasionally present with delayed-onset neurotoxicity that sometimes responds poorly to antivenom or anticholinesterase treatment. This clinical outcome could be explained by the presence of potent snake presynaptic phospholipase A2 neurotoxin (SPAN) complexes and monomers, in addition to long- and short-chain postsynaptic ?-neurotoxins, that bind irreversibly, block neurotransmitter release and result in degeneration of the nerve terminal. The present study therefore aimed to determine within-genus variations in expression of high molecular mass SPAN complexes in the venoms of six major species of Acanthophis, four geographic variants of Acanthophis antarcticus. Venoms were separated by size-exclusion liquid chromatography under non-denaturing conditions and fractions corresponding to proteins in the range of 22 to >60kDa were subjected to pharmacological characterization using the isolated chick biventer cervicis nerve-muscle (CBCNM) preparation. All venoms, except Acanthophis wellsi and Acanthophis pyrrhus, contained high mass fractions with phospholipase A2 activity that inhibited twitch contractions of the CBCNM preparation. This inhibition was of slow onset, and responses to exogenous nicotinic agonists were not blocked, consistent with the presence of SPAN complexes. The results of the present study indicate that clinicians may need to be aware of possible prejunctional neurotoxicity following envenomations from A.antarcticus (all geographic variants except perhaps South Australia), Acanthophis praelongus, Acanthophis rugosus and Acanthophis. laevis species, and that early antivenom intervention is important in preventing further development of toxicity. 2010 Elsevier Ltd. All rights reserved.
Blacklow, B., Escoubas, P. & Nicholson, G.M. 2010, 'Characterisation of the heterotrimeric presynaptic phospholipase A2 neurotoxin complex from the venom of the common death adder (Acanthophis antarcticus)', Biochemical Pharmacology, vol. 80, no. 2, pp. 277-287.
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While Australo-Papuan death adder neurotoxicity is generally considered to be due to the actions of reversible competitive postsynaptic ?-neurotoxins, the neurotoxic effects are often poorly reversed by antivenom or anticholinesterases. This suggests that the venom may contain a snake presynaptic phospholipase A2 (PLA2) neurotoxin (SPAN) that binds irreversibly to motor nerve terminals to inhibit neurotransmitter release. Using size-exclusion liquid chromatography under non-reducing conditions, we report the isolation and characterisation of a high molecular mass SPAN complex, P-elapitoxin-Aa1a (P-EPTX-Aa1a), from the venom of the common death adder Acanthophis antarcticus. Using the chick biventer-cervicis nerve-muscle preparation, P-EPTX-Aa1a (44,698Da) caused inhibition of nerve-evoked twitch contractions while responses to cholinergic agonists and KCl remained unaffected. P-EPTX-Aa1a also produced significant fade in tetanic contractions and a triphasic timecourse of neuromuscular blockade. These actions are consistent with other SPANs that inhibit acetylcholine release. P-EPTX-Aa1a was found to be a heterotrimeric complex composed of ?, ? and ?-subunits in a 1:1:1 stoichiometry with each subunit showing significant N-terminal sequence homology to the subunits of taipoxin, a SPAN from Oxyuranus s. scutellatus. Like taipoxin, only the ?-chain produced any signs of neurotoxicity or displayed significant PLA2 enzymatic activity. Preincubation with monovalent death adder antivenom or suramin, or inhibition of PLA2 activity by incubation with 4-bromophenacyl bromide, either prevented or significantly delayed the onset of toxicity by P-EPTX-Aa1a. However, antivenom failed to reverse neurotoxicity. Early intervention with antivenom may therefore be important in severe cases of envenomation by A. antarcticus, given the presence of potent irreversible presynaptic neurotoxins. 2010 Elsevier Inc.
Mobli, M., De Arajo, A.D., Lambert, L.K., Pierens, G.K., Windley, M.J., Nicholson, G.M., Alewood, P.F. & King, G.F. 2009, 'Direct visualization of disulfide bonds through diselenide proxies using77Se NMR spectroscopy', Angewandte Chemie - International Edition, vol. 48, no. 49, pp. 9312-9314.
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Chemical Equation Presentation Se-ing is believing: Many proteins are cross-braced by disulfide bonds that frequently play key roles in protein structure, folding, and function. Unfortunately, the methods available for assignment of disulfide-bond connectivities in proteins are technically difficult and prone to misinterpretation. Now disulfide bond connectivities in native proteins can be visualized directly using 77Se NMR spectroscopy. 2009 Wiley-VCH Verlag GmbH & Co. KGaA.
Yamaji, N., Little, M.J., Nishio, H., Billen, B., Villegas, E., Nishiuchi, Y., Tytgat, J., Nicholson, G.M. & Corzo, G. 2009, 'Synthesis, Solution Structure, And Phylum Selectivity Of A Spider Delta-Toxin That Slows Inactivation Of Specific Voltage-Gated Sodium Channel Subtypes', Journal Of Biological Chemistry, vol. 284, no. 36, pp. 24568-24582.
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Magi 4, now renamed ?-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to ?-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (NaV) channels but, unlike ?-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in NaV channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect NaV channel subtypes showing that ?-hexatoxin-Mg1a selectively slows channel inactivation of mammalian NaV1.1, NaV1.3, and NaV1.6 but more importantly shows higher affinity for insect NaV1 (para) channels. Consequently, ?-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded ?-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion ?-toxins and ?-hexatoxins are distributed in a topologically similar manner in ?-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of ?-hexatoxin-Mg1a for certain mammalian and insect NaV channel subtypes. As such, ?-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.
Gunning, S.J., Maggio, F., Windley, M.J., Valenzuela, S.M., King, G.F. & Nicholson, G.M. 2008, 'The Janus-faced atracotoxins are specific blockers of invertebrate K Ca channels', FEBS Journal, vol. 275, no. 16, pp. 4045-4059.
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The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca2+-activated K+ (K Ca) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked KCa channels in cockroach unpaired dorsal median neurons with an IC50 of 2 nm, but it did not significantly affect a wide range of other voltage-activated K +, Ca2+ or Na+ channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca 2+-activated K+ (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC50 ? 10 ?m) and its lack of activity on rat dorsal root ganglion neuron KCa channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate KCa channels and suggests that KCa channels might be potential insecticide targets. 2008 The Authors.
Herzig, V., Khalife, A.A., Chong, Y., Isbister, G.K., Currie, B.J., Churchill, T.B., Horner, S., Escoubas, P., Nicholson, G.M. & Hodgson, W.C. 2008, 'Intersexual variations in Northern (Missulena pruinosa) and Eastern (M. bradleyi) mouse spider venom', Toxicon, vol. 51, no. 7, pp. 1167-1177.
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Venoms of both sexes of Australian Northern (Missulena pruinosa) and Eastern (Missulena bradleyi) mouse spiders were studied in order to determine intersexual variations in venom yield, composition and bioactivity. Females of both species yielded more venom than males. High-performance liquid chromatography (HPLC) and mass spectrometry data further indicate a substantial degree of intersexual variation in the venom composition of both species. In a cricket (Acheta domestica) acute toxicity assay, only small intersexual differences were observed, but M. bradleyi venom was found to be considerably more potent than M. pruinosa venom. In the chick biventer cervicis nerve-muscle preparation, male but not female M. bradleyi venom induced large and sustained muscle contractions with fasciculation and decreased twitch height that could be reversed by CSL funnel-web spider antivenom. In contrast, venoms of both sexes of M. pruinosa did not induce significant effects in the chick biventer cervicis nerve-muscle preparation. We therefore conclude that female M. bradleyi venom and venoms from male and female M. pruinosa appear to contain few, if any, orthologs of ?-missulenatoxin-Mb1a, the toxin responsible for the effects of male M. bradleyi venom in vertebrates. These findings are consistent with clinical reports that mouse spiders, particularly species other than male M. bradleyi, do not appear to be a major medical problem in humans. 2008 Elsevier Ltd. All rights reserved.
Escoubas, P., Quinton, L. & Nicholson, G.M. 2008, 'Venomics: Unravelling the complexity of animal venoms with mass spectrometry', Journal of Mass Spectrometry, vol. 43, no. 3, pp. 279-295.
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Animal venoms and toxins are now recognized as major sources of bioactive molecules that may be tomorrow's new drug leads. Their complexity and their potential as drug sources have been demonstrated by application of modern analytical technologies, which have revealed venoms to be vast peptide combinatorial libraries. Structural as well as pharmacological diversity is immense, and mass spectrometry is now one of the major investigative tools for the structural investigation of venom components. Recent advances in its use in the study of venom and toxins are reviewed. The application of mass spectrometry techniques to peptide toxin sequence determination by de novo sequencing is discussed in detail, in the light of the search for novel analgesic drugs. We also present the combined application of LC-MALDI separation with mass fingerprinting and ISD fragmentation for the determination of structural and pharmacological classes of peptides in complex spider venoms. This approach now serves as the basis for the full investigation of complex spider venom proteomes, in combination with cDNA analysis. Copyright 2008 John Wiley & Sons, Ltd.
King, G.F., Gentz, M.C., Escoubas, P. & Nicholson, G.M. 2008, 'A rational nomenclature for naming peptide toxins from spiders and other venomous animals', Toxicon, vol. 52, no. 2, pp. 264-276.
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Molecular toxinology research was initially driven by an interest in the small subset of animal toxins that are lethal to humans. However, the realization that many venomous creatures possess a complex repertoire of bioactive peptide toxins with potential pharmaceutical and agrochemical applications has led to an explosion in the number of new peptide toxins being discovered and characterized. Unfortunately, this increased awareness of peptide-toxin diversity has not been matched by the development of a generic nomenclature that enables these toxins to be rationally classified, catalogued, and compared. In this article, we introduce a rational nomenclature that can be applied to the naming of peptide toxins from spiders and other venomous animals. Crown Copyright 2008.
King, G.F., Escoubas, P. & Nicholson, G.M. 2008, 'Peptide toxins that selectively target insect NaV and Ca V channels', Channels, vol. 2, no. 2, pp. 100-116.
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Numerous metazoans express venoms for the purpose of defense, competitor deterrence or prey capture. Peptide neurotoxins are particularly well represented in the venoms of arachnids, cnidarians and mollusks and these toxins often possess high affinity and specificity for particular classes of ion channels. Some of these toxins have become the defining pharmacology for certain vertebrate ion channel subtypes. Unfortunately, due to differences in the structure, pharmacology and ion selectivity of insect voltage-gated sodium (NaV) and calcium (CaV) channels compared with their vertebrate counterparts, these peptide toxins have proven less useful for the characterization of insect ion channels. Despite these disparities in channel structure and function, the armament of peptide toxins that specifically modulate the activity of insect ion channels is slowly expanding. This review focuses on insect-selective peptide toxins and their utility for the study of insect NaV and CaV channels. The high affinity and selectivity of some of these neurotoxins means that they have the potential to become the defining pharmacology for specific subtypes of insect ion channels. In addition, it might be possible to exploit the phyletic specificity of these toxins as the basis for rational development of novel classes of ion channel insecticides. 2008 Landes Bioscience.
Nicholson, G.M. 2007, 'Fighting the global pest problem: Preface to the special Toxicon issue on insecticidal toxins and their potential for insect pest control', Toxicon, vol. 49, no. 4, pp. 413-422.
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Arthropod pests are responsible for major crop devastation and are vectors for the transmission of new and re-emerging diseases in humans and livestock. Despite many years of effective control by conventional agrochemical insecticides, a number of factors are threatening the effectiveness and continued use of these agents. These include the development of insecticide resistance and use-cancellation or de-registration of some insecticides due to human health and environmental concerns. Several approaches are being investigated for the design of new (bio)pesticides. These include the development of transgenic plants and recombinant baculoviruses as delivery systems for a variety of insect-selective toxins. Additional approaches for the development of foliar sprays include the rational design of peptidomimetics based on the key residues of these toxins that interact with the insect target. This special issue provides an overview of these phyletically selective animal, plant and microbial toxins and their diverse mechanisms of action to paralyze or kill arthropods. In addition, it reviews their potential for biopesticide discovery and validation of novel insecticide targets and provides an overview of the strengths and weaknesses of biopesticides in the global control of arthropod pests. 2007 Elsevier Ltd. All rights reserved.
Nicholson, G.M. 2007, 'Insect-selective spider toxins targeting voltage-gated sodium channels', Toxicon, vol. 49, no. 4, pp. 490-512.
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The voltage-gated sodium (Nav) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Nav channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Nav channel conductance. Magi 2 and Tx4(6-1) slow Nav channel inactivation via an interaction with site-3. The ?-palutoxins, and most likely ?-agatoxins and curtatoxins, target site-4. However, their action is complex with the ?-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion ?-toxins, but with both ?-palutoxins and ?-agatoxins slowing Nav channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as ?-atracotoxins, are known to target both insect and vertebrate Nav channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped. 2006 Elsevier Ltd. All rights reserved.
Chong, Y., Hayes, J.L., Sollod, B., Wen, S., Wilson, D.T., Hains, P.G., Hodgson, W.C., Broady, K.W., King, G.F. & Nicholson, G.M. 2007, 'The ?-atracotoxins: Selective blockers of insect M-LVA and HVA calcium channels', Biochemical Pharmacology, vol. 74, no. 4, pp. 623-638.
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The ?-atracotoxins (?-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, ?-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the ?-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets. Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1 ?M. To further characterize the target of the ?-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that ?-ACTX-Ar1a, and its homolog ?-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid-low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Cav) currents. This block occurred in the absence of alterations in the voltage-dependence of Cav channel activation, and was voltage-independent, suggesting that ?-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 ?M ?-ACTX-Ar1a failed to significantly affect global Kv channel currents. However, 1 ?M ?-ACTX-Ar1a caused a modest 18% block of insect Nav channel currents, similar to the minor block of Nav channels reported for other insect Cav channel blockers such as ?-agatoxin IVA. These findings validate both M-LVA and HVA Cav channels as potential targets for insecticides. 2007.
Kubista, H., Mafra, R.A., Chong, Y., Nicholson, G.M., Beirao, P.S., Cruz, J.S., Boehm, S., Nentwig, W. & Kuhn-Nentwig, L. 2007, 'CSTX-1, A Toxin From The Venom Of The Hunting Spider Cupiennius Salei, Is A Selective Blocker Of L-type Calcium Channels In Mammalian Neurons', Neuropharmacology, vol. 52, no. 8, pp. 1650-1662.
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The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produ
Nicholson, G.M., Graudins, A., Wilson, H.I., Little, M. & Broady, K.W. 2006, 'Arachnid toxinology in Australia: From clinical toxicology to potential applications', Toxicon, vol. 48, no. 7, pp. 872-898.
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The unique geographic isolation of Australia has resulted in the evolution of a distinctive range of Australian arachnid fauna. Through the pioneering work of a number of Australian arachnologists, toxinologists, and clinicians, the taxonomy and distribution of new species, the effective clinical treatment of envenomation, and the isolation and characterisation of the many distinctive neurotoxins, has been achieved. In particular, work has focussed on several Australian arachnids, including red-back and funnel-web spiders, paralysis ticks, and buthid scorpions that contain neurotoxins capable of causing death or serious systemic envenomation. In the case of spiders, species-specific antivenoms have been developed to treat envenomed patients that show considerable cross-reactivity. Both in vitro and clinical case studies have shown they are particularly efficacious in the treatment of envenomation by spiders even from unrelated families. Despite their notorious reputation, the high selectivity and potency of a unique range of toxins from the venom of Australian arachnids will make them invaluable molecular tools for studies of neurotransmitter release and vesicle exocytosis as well as ion channel structure and function. The venoms of funnel-web spiders, and more recently Australian scorpions, have also provided a previously untapped rich source of insect-selective neurotoxins for the future development of biopesticides and the characterisation of previously unvalidated insecticide targets. This review provides a historical viewpoint of the work of many toxinologists to isolate and characterise just some of the toxins produced by such a unique group of arachnids and examines the potential applications of these novel peptides. 2006 Elsevier Ltd. All rights reserved.
Nicholson, G.M. & Lewis, R.J. 2006, 'Ciguatoxins: Cyclic polyether modulators of voltage-gated ion channel function', Marine Drugs, vol. 4, no. 3, pp. 82-118.
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Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na +- dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera. 2006 by MDPI.
Birinyi-Strachan, L.C., Gunning, S.J., Lewis, R.J. & Nicholson, G.M. 2005, 'Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons', Toxicology and Applied Pharmacology, vol. 204, no. 2, pp. 175-186.
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The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Na v) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Nav channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons, P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Na v currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP, and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Nav channel gating, observed clinically in response to ciguatera poisoning. 2004 Elsevier Inc. All rights reserved.
Birinyi-Strachan, L.C., Davies, M.J., Lewis, R.J. & Nicholson, G.M. 2005, 'Neuroprotectant effects of iso-osmolar D-mannitol to prevent Pacific ciguatoxin-1 induced alterations in neuronal excitability: A comparison with other osmotic agents and free radical scavengers', Neuropharmacology, vol. 49, no. 5, pp. 669-686.
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The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 mM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 mM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 mM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Nav channels, as well as the increased rate of recovery from inactivation of TTX-resistant Na v channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant INa amplitude by P-CTX-1. Additional experiments using hyper- and iso-osmolar d-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na v channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Nav channel subtype, possibly to alter or reduce toxin association. 2005 Elsevier Ltd. All rights reserved.
Wen, S., Wilson, D.T.R., Kuruppu, S., Korsinczky, M.L.J., Hedrick, J., Pang, L., Szeto, T., Hodgson, W.C., Alewood, P.F. & Nicholson, G.M. 2005, 'Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins', Peptides, vol. 26, no. 12, pp. 2412-2426.
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This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 ?M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca 2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed ?-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. 2005 Elsevier Inc. All rights reserved.
Nicholson, G.M. & Little, M.J. 2005, 'Spider neurotoxins targeting voltage-gated sodium channels', Toxin Reviews, vol. 24, no. 3-4, pp. 315-345.
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The voltage-gated sodium (Nav) channel is a target for a number of drugs, insecticides, and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate sodium channel gating and/or kinetics. A number of polypeptide toxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized that interact with several of these sites. Certain huwentoxins and hainantoxins appear to target site 1 to block Nav channel conductance. The ?-atracotoxins and Magi 4 slow Nav-channel inactivation via an interaction with neurotoxin site 3. The ?-palutoxins, and most likely ?-agatoxins and curtatoxins, target site 4. However, their action is complex with the ?-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion ?-toxins, but with both ?-palutoxins and ?-agatoxins slowing Nav channel inactivation, a site 3-like action. Many spider toxins target undefined sites, while others are likely to cross-react with other ion channels due to conserved structures within domains of voltage-gated ion channels. It is already clear, however, that many spider toxins represent highly potent and specific molecular tools to define novel links between sites modulating channel activation and inactivation. Other spider toxins show phyla specificity and are being considered as lead compounds for the development of biopesticides. Others display tissue specificity via interactions with specific Nav channel subtypes and should provide useful tools to delineate the molecular determinants to target ligands to these channel subtypes. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the mentioned areas remains underdeveloped. Copyright 2005 Taylor & Francis Inc.
Tran, Y.H., Craig, A.R., Bartrop, R. & Nicholson, G.M. 2004, 'Time course and regional distribution of cortical changes during acute alcohol ingestion', International Journal Of Neuroscience, vol. 114, pp. 863-878.
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Behavioural effects of alcohol are known to be greater when the blood alcohol is rising, nown as the mellanby effect; however, research investigating the cortical changes during this period is scarce. The objective of tis study was to investigate the efects of consumption of alcohol on cortical activity measured by the electroencephalogram (EEG) during the absorption or rising phase of alcohol. EEG signals were recorded using the entire 10/20 montage system. The experimental design consisted of a repeated measures randomised crossover design in which subjects acted as their ow control. This involved recording two EEG baseline measures, each of which was followed by a placebo or alcohol condition, delivered ovre two days for tech subjects. All subjects has a 50% chance of receiving the alcohol first. All subjects were shown to have mean peak blood alcohol concentration (BAC) levels of around .03%. No significant differences were found between the two baselines. Significant increases inEEG magnitude occurred in thetheta (4-7.75 Hz), alpha 1 98-9.75Hz) and beta 1 (13.25-19.75 Hz) spectrum in the frontal EEG regions, and alpha 1 (8-9.75 Hz) in the central and posterior regions. No significant changes were found in the theta (4-7.75 Hz) or beta (13.5-30Hz) spectrums in the central and psoterior regions. There were also no significant results for alpha 2 (10-13 Hz) in any of the regions. These results suggest that rapid cortical changes occur within the first 35 min after alcohol consumption.
Nicholson, G.M., Little, M.J. & Birinyi-Strachan, L.C. 2004, 'Structure and function of ?-atracotoxins: Lethal neurotoxins targeting the voltage-gated sodium channel', Toxicon, vol. 43, no. 5, pp. 587-599.
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?-Atracotoxins (?-ACTX), isolated from the venom of Australian funnel-web spiders, are responsible for the potentially lethal envenomation syndrome seen following funnel-web spider envenomation. They are 42-residue polypeptides with four disulfides and an 'inhibitor cystine-knot' motif with structural but not sequence homology to a variety of other spider and marine snail toxins. ?-Atracotoxins induce spontaneous repetitive firing and prolongation of action potentials resulting in neurotransmitter release from somatic and autonomic nerve endings. This results from a slowing of voltage-gated sodium channel inactivation and a hyperpolarizing shift of the voltage-dependence of activation. This action is due to voltage-dependent binding to neurotoxin receptor site-3 in a similar, but not identical, fashion to scorpion ?-toxins and sea anemone toxins. Unlike other site-3 neurotoxins, however, ?-ACTX bind with high affinity to both cockroach and mammalian sodium channels but low affinity to locust sodium channels. At present the pharmacophore of ?-ACTX is unknown but is believed to involve a number of basic residues distributed in a topologically similar manner to scorpion ?-toxins and sea anemone toxins despite distinctly different protein scaffolds. As such, ?-ACTX provide us with specific tools with which to study sodium channel structure and function and determinants for phyla- and tissue-specific actions of neurotoxins interacting with site-3. 2004 Elsevier Ltd. All rights reserved.
Alewood, D., Birinyi-Strachan, L.C., Pallaghy, P.K., Norton, R.S., Nicholson, G.M. & Alewood, P.F. 2003, 'Synthesis and Characterization of ?-Atracotoxin-Ar1a, the Lethal Neurotoxin from Venom of the Sydney Funnel-Web Spider (Atrax robustus)', Biochemistry, vol. 42, no. 44, pp. 12933-12940.
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?-Atracotoxin-Ar1a (?-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion ?-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing ?-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. 1H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic ?-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that ?-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic ?-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.
Nicholson, G.M. & Graudins, A. 2003, 'Antivenoms for the treatment of spider envenomation', Journal of Toxicology - Toxin Reviews, vol. 22, no. 1, pp. 35-59.
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There are several groups of medically important araneomorph and mygalomorph spiders responsible for serious systemic envenomation. These include spiders from the genus Latrodectus (family Theridiidae), Phoneutria (family Ctenidae) and the subfamily Atracinae (genera Atrax and Hadronyche). The venom of these spiders contains potent neurotoxins that cause excessive neurotransmitter release via vesicle exocytosis or modulation of voltage-gated sodium channels. In addition, spiders of the genus Loxosceles (family Loxoscelidae) are responsible for significant local reactions resulting in necrotic cutaneous lesions. This results from sphingomyelinase D activity and possibly other compounds. A number of antivenoms are currently available to treat envenomation resulting from the bite of these spiders. Particularly efficacious antivenoms are available for Latrodectus and Atrax/Hadronyche species, with extensive cross-reactivity within each genera. In the case of Latrodectus antivenoms this is of considerable importance in countries where antivenom is unavailable or where certain antivenoms are associated with an unacceptably high risk of adverse reactions. Moreover, Latrodectus and Atrax antivenoms appear to be effective in the treatment of envenomation by closely related Steatoda spiders (family Theridiidae) or the unrelated spider Missulena bradleyi (family Actinopodidae), respectively. The effectiveness of Loxosceles antivenom in the treatment of the necrotic arachnidism resulting from the bite of recluse spiders is less clear mainly due to late presentation of victims. Antivenom is also available for Phoneutria envenomation but is reserved only for severe cases.
Gunning, S.J., Chong, Y., Khalife, A.A., Hains, P.G., Broady, K.W. & Nicholson, G.M. 2003, 'Isolation of ?-missulenatoxin-Mb1a, the major vertebrate-active spider ?-toxin from the venom of Missulena bradleyi (Actinopodidae)', FEBS Letters, vol. 554, no. 1-2, pp. 211-218.
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The present study describes the isolation and pharmacological characterisation of the neurotoxin ?-missulenatoxin-Mb1a (?-MSTX-Mb1a) from the venom of the male Australian eastern mouse spider, Missulena bradleyi. This toxin was isolated using reverse-phase high-performance liquid chromatography and was subsequently shown to cause an increase in resting tension, muscle fasciculation and a decrease in indirect twitch tension in a chick biventer cervicis nerve-muscle bioassay. Interestingly, these effects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax robustus. Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that ?-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium current, a slowing of sodium current inactivation and a hyperpolarising shift in the voltage at half-maximal activation. In addition, ?-MSTX-Mb1a failed to affect TTX-resistant sodium currents. Subsequent Edman degradation revealed a 42-residue peptide with unusual N- and C-terminal cysteines and a cysteine triplet (Cys14-16). This toxin was highly homologous to a family of ?-atracotoxins (?-ACTX) from Australian funnel-web spiders including conservation of all eight cysteine residues. In addition to actions on sodium channel gating and kinetics to ?-ACTX, ?-MSTX-Mb1a caused significant insect toxicity at doses up to 2000 pmol/g. ?-MSTX-Mb1a therefore provides evidence of a highly conserved spider ?-toxin from a phylogenetically distinct spider family that has not undergone significant modification. 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
Nicholson, G.M. & Graudins, A. 2002, 'Spiders of medical importance in the Asia-Pacific: Atracotoxin, latrotoxin and related spider neurotoxins', Clinical and Experimental Pharmacology and Physiology, vol. 29, no. 9, pp. 785-794.
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1. The spiders of medical importance in the Asia-Pacific region include widow (family Theridiidae) and Australian funnel-web spiders (subfamily Atracinae). In addition, cupboard (family Theridiidae) and Australian mouse spiders (family Actinopodidae) may contain neurotoxins responsible for serious systemic envenomation. Fortunately, there appears to be extensive cross-reactivity of species-specific widow spider antivenom within the family Theridiidae. Moreover, Sydney funnel-web antivenom has been shown to be effective in the treatment of mouse spider envenomation. 2. ?-Latrotoxin (?-LTx) appears to be the main neurotoxin responsible for the envenomation syndrome known as 'latrodectism' following bites from widow spiders. This 120 kDa protein binds to distinct receptors (latrophilin 1 and neurexins) to induce neurotransmitter vesicle exocytosis via both Ca2+-dependent and -independent mechanisms, resulting in vesicle depletion. This appears to involve disruption to a process that normally inhibits vesicle fusion in the absence of Ca2+. Precise elucidation of the mechanism of action of ?-LTx will lead to a major advancement in our understanding of vesicle exocytosis. 3. ?-Atracotoxins (?-ACTX) are responsible for the primate-specific envenomation syndrome seen following funnel-web spider envenomation. These peptides induce spontaneous repetitive firing and prolongation of action potentials in excitable cells. This results from a hyperpolarizing shift of the voltage-dependence of activation and a slowing of voltage-gated Na+ channel inactivation. This action is due to voltage-dependent binding to neurotoxin receptor site-3 on insect and mammalian voltage-gated Na+ channels in a manner similar, but not identical, to scorpion ?-toxins and sea anemone toxins. ?-Atracotoxins provide us with highly specific tools to study Na+ channel structure and function. 4. ?- and Janus-faced ACTX, from funnel-web spider venom, are novel neurotoxins that show selective toxicity to in...
Nicholson, G.M., Blanche, T., Mansfield, K. & Tran, Y.H. 2002, 'Differential blockade of neuronal voltage-gated sodium and potassium channels by antidepressant drugs', European Journal of Pharmacology, vol. 452, no. N/A, pp. 35-48.
The effects of a range of antidepressants were investigated on neuronal voltage-gated Na+ and K+ channels. With the exception of phenelzine, all antidepressants inhibited batrachotoxin-stimulated 22Na+ uptake, most likely via negative allosteric inhibition of batrachotoxin binding to neurotoxin receptor site-2 on the Na+ channel. Imipramine also produced a differential action on macroscopic Na+ and K+ channel currents in acutely dissociated rat dorsal root ganglion neurons. Imipramine produced a use-dependent block of Na+ channels. In addition, there was a hyperpolarizing shift in the voltage-dependence of steady-state Na+ channel inactivation and slowed repriming kinetics consistent with imipramine having a higher affinity for the inactivated state of the Na+ channel. At higher concentrations, imipramine also blocked delayed-rectifier and transient outward K+ currents in the absence of alterations to the voltage-dependence of activation or the kinetics of inactivation. These actions on voltage-gated ion channels may underlie the therapeutic and toxic effects of these drugs.
Graudins, A., Gunja, N.Y., Broady, K.W. & Nicholson, G.M. 2002, 'Clinical and in vitro evidence for the efficacy of Australian red-back spider (Latrodectus hasselti) antivenom in the treatment of Brown cupboard spider (Steatoda grossa) envenomation', Toxicon, vol. 40, no. N/A, pp. 767-775.
Graudins, A., Gunja, N., Broady, K.W. & Nicholson, G.M. 2002, 'Clinical and in vitro evidence for the efficacy of Australian red-back spider (Latrodectus hasselti) antivenom in the treatment of envenomation by a Cupboard spider (Steatoda grossa)', Toxicon, vol. 40, no. 6, pp. 767-775.
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We report the case of a 22-year-old female who was bitten on the shoulder by a spider subsequently identified as a female Cupboard spider (Steatoda grossa). She developed nausea, vomiting, and severe local and regional pain, similar to that seen in latrodectism. Symptoms were treated successfully with red-back spider antivenom (RBSAV). We also present in vitro data, which supports this clinical observation, and suggests that S. grossa venom is immunogenically reactive with both RBSAV and latrotoxin (LTx)-specific antibodies by Western blotting. Moreover, the effects of S. grossa venom on the isolated chick biventer cervicis nerve-muscle preparation are dose-dependent and similar to those seen with Latrodectus spp. venoms. S. grossa venom produced a sustained muscle contracture which could be prevented by pre-incubation of venom with RBSAV. Venom effects could also be reversed by the addition of antivenom after application of venom to the preparation. Although severe envenomation is uncommon following the bite of Steatoda spp. it may resemble latrodectism. These results indicate that RBSAV is likely to be effective in reversing symptoms of envenomation and should be considered in the treatment of patients with distressing or persisting symptoms. 2002 Elsevier Science Ltd. All rights reserved.
Graudins, A., Wilson, D., Alewood, P.F., Broady, K.W. & Nicholson, G.M. 2001, 'Cross-reactivity of Sydney funnel-web spider antivenom: Neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms', Toxicon, vol. 40, no. 3, pp. 259-266.
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Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5/10) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders. 2001 Elsevier Science Ltd. All rights reserved.
Grolleau, F., Stankiewicz, M., Birinyi-Strachan, L., Wang, X.-.H., Nicholson, G.M., Pelhate, M. & Lapied, B. 2001, 'Electrophysiological analysis of the neurotoxic action of a funnel-web spider toxin, ?-atracotoxin-Hv1a, on insect voltage-gated Na+ channels', Journal of Experimental Biology, vol. 204, no. 4, pp. 711-721.
The effects of ?-ACTX-Hv1a, purified from the venom of the funnel-web spider Hadronyche versuta, were studied on the isolated giant axon and dorsal unpaired median (DUM) neurones of the cockroach Periplaneta americana under current- and voltage-clamp conditions using the double oil-gap technique for single axons and the patch-clamp technique for neurones. In parallel, the effects of the toxin were investigated on the excitability of rat dorsal root ganglion (DRG) neurones. In both DRG and DUM neurones, ?-ACTX-Hv1a induced spontaneous repetitive firing accompanied by plateau potentials. However, in the case of DUM neurones, plateau action potentials were facilitated when the membrane was artificially hyperpolarized. In cockroach giant axons, ?-ACTX-Hv1a also produced plateau action potentials, but only when the membrane was pre-treated with 3-4 diaminopyridine. Under voltage-clamp conditions, ?-ACTX-Hv1a specifically affected voltage-gated Na+ channels in both axons and DUM neurones. Both the current/voltage and conductance/voltage curves of the ?-ACTX-Hv1a-modified inward current were shifted 10 mV to the left of control curves. In the presence of ?-ACTX-Hv1a, steady-state Na+ channel inactivation became incomplete, causing the appearance of a non-inactivating component at potentials more positive than -40mV. The amplitude of this non-inactivating component was dependent on the holding potential. From this study, it is concluded that, in insect neurones, ?-ACTX-Hv1a mainly affects Na+ channel inactivation by a mechanism that differs slightly from that of scorpion ?-toxins.
Graudins, A., Padula, M., Broady, K. & Nicholson, G.M. 2001, 'Red-back spider (Latrodectus hasselti) antivenom prevents the toxicity of widow spider venoms', Annals of Emergency Medicine, vol. 37, no. 2, pp. 154-160.
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Study objectives: Widow spiders of the genus Latrodectus are found worldwide and produce similar clinical envenomation syndromes. In Australia, red-back spider antivenom (RBS-AV) is effective therapy for Latrodectus hasselti envenomation and it has been reported to reverse envenomation by other widow spiders. This study assessed the efficacy of RBS-AV in preventing in vitro and in vivo toxicity of widow spider venoms of North America and Europe. Methods: The binding of RBS-AV to ?-latrotoxin and Latrodectus venoms (Latrodectus spp mactans, hesperus, lugubris, tredecimguttatus, hasselt) was assayed using Western blotting. Prevention of in vitro toxicity to ?-latrotoxin and the same venoms was tested by pretreating an isolated chick biventer cervicis nerve-muscle preparation with RBS-AV. Prevention of in vivo toxicity was determined by a lethality study in male Balb/c mice to (2.5 to 5 median lethal dose [LD50]) or ?-latrotoxin (10 LD50) preincubated with antivenom or without RBS-AV (control). Results: In Western blots, RBS-AV bound to ?-latrotoxin and similar widow spider proteins in all venoms tested, indicating antigenic similarity with proteins found in RBS venom. Antivenom prevented the typical in vitro muscle contracture and loss of twitch tension seen with ?-latrotoxin and the venoms tested. Control mice rapidly developed signs of envenomation, but mice treated with RBS-AV remained free of signs of envenomation. Conclusion: RBS-AV prevented both in vitro and in vivo toxicity from Latrodectus venoms and ?-latrotoxin in mice. These data suggest that RBS-AV may be clinically effective in the treatment of envenomation resulting from the bite of other widow spiders.
Wang, X., Connor, M., Wilson, D., Wilson, H.I., Nicholson, G.M., Smith, R., Shaw, D., Mackay, J., Alewood, P.F., Christie, M. & King, G.F. 2001, 'Discovery and Structure of a Potent and highly Specific Blocker of Insect Calcium Channels', Journal of Biological Chemistry, vol. 276, no. 43, pp. 40306-40312.
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Gilles, N., Chen, H., Wilson, H.I., Le Gall, F., Montoya, G., Molgo, J., Schoenherr, R. & Nicholson, G.M. 2000, 'Scorpion alpha and alpha-like Toxins Differentially Interact with Sodium Channels in mammalian CNS and Periphery', European Journal of Neuroscience, vol. 12, no. 8, pp. 2823-2832.
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Scorpion alpha-toxins from Leiurus quinquestriatus hebraeus, LqhII and LqhIII, are similarly toxic to mice when administered by a subcutaneous route, but in mouse brain LqhII is 25-fold more toxic. Examination of the two toxins effects in central nervous system (CNS), peripheral preparations and expressed sodium channels revealed the basis for their differential toxicity. In rat brain synaptosomes, LqhII binds with high affinity, whereas LqhIII competes only at high concentration for LqhII-binding sites in a voltage-dependent manner. LqhII strongly inhibits sodium current inactivation of brain rBII subtype expressed in HEK293 cells, whereas LqhIII is weakly active at 2 microM, suggesting that LqhIII affects sodium channel subtypes other than rBII in the brain. In the periphery, both toxins inhibit tetrodotoxin-sensitive sodium current inactivation in dorsal root ganglion neurons, and are strongly active directly on the muscle and on expressed muI channels. Only LqhII, however, induced repetitive end-plate potentials in mouse phrenic nerve-hemidiaphragm muscle preparation by direct effect on the motor nerve. Thus, rBII and sodium channel subtypes expressed in peripheral nervous system (PNS) serve as the main targets for LqhII but are mostly not sensitive to LqhIII. Toxicity of both toxins in periphery may be attributed to the direct effect on muscle. Our data elucidate, for the first time, how different toxins affect mammalian central and peripheral excitable cells, and reveal unexpected subtype specificity of toxins that interact with receptor site 3.
Strachan, L.C., Lewis, R.J. & Nicholson, G.M. 1999, 'Differential actions of pacific ciguatoxin-1 on sodium channel subtypes in mammalian sensory neurons', Journal of Pharmacology and Experimental Therapeutics, vol. 288, no. 1, pp. 379-388.
Pacific ciguatoxin-1 (P-CTX-1), is a highly lipophilic cyclic polyether molecule originating from the marine dinoflagellate Gambierdiscus toxicus. Its effects were investigated on sodium channel subtypes present in acutely dissociated rat dorsal root ganglion neurons, using whole-cell patch clamp techniques. Concentrations of P-CTX-1 ranging from 0.2 to 20 nM had no effect on the kinetics of tetrodotoxin-sensitive (TTX-S) or tetrodotoxin-resistant (TTX-R) sodium channel activation and inactivation, however, a concentration- dependent reduction in peak current amplitude occurred in both channel types. The main actions of 5 nM P-CTX-1 on TTX-S sodium channels were a 13-mV hyperpolarizing shift in the voltage dependence of sodium channel activation and a 22-mV hyperpolarizing shift in steady-state inactivation (h(?)). In addition, P-CTX-1 caused a rapid rise in the membrane leakage current in cells expressing TTX-S sodium channels. This effect was blocked by 200 nM TTX, indicating an action mediated through TTX-S sodium channels. In contrast, the main action of P-CTX-1 (5 nM) on TTX-R sodium channels was a significant increase in the rate of recovery from sodium channel inactivation. These results indicate that P-CTX-1 acts to modify voltage- gated sodium channels present in peripheral sensory neurons consistent with its action to increase nerve excitability. This provides an explanation for the sensory neurological disturbances associated with ciguatera fish poisoning.
Fletcher, J.I., Wang, X., Connor, M., Christie, M.J., King, G.F. & Nicholson, G.M. 1999, 'Spider toxins: A new group of potassium channel modulators', Perspectives in Drug Discovery and Design, vol. 15-16, pp. 61-69.
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Spider toxins that target potassium channels constitute a new class of pharmacological tools that can be used to probe the structure and function of these channels at the molecular level. The limited studies performed to date indicate that these peptide toxins may facilitate the analysis of K+ channels that have proved insensitive to peptide inhibitors isolated from other animal sources. Thus far, two classes of K+ channel-selective spider toxins have been isolated, sequenced, and pharmacologically characterised - the hanatoxins (HaTx) from Grammastola spatulata and heteropodatoxins (HpTx) from Heteropoda venatoria. The hanatoxins block Kv2.1 and Kv4.2 voltage-gated K+ channels. In Kv2.1 K+ channels this occurs as a consequence of a depolarising shift in the voltage dependence of activation and not by occlusion of the channel pore. These toxins show minimal sequence homology with other peptide inhibitors of K+ channels, but they do share some homology with other ion channel toxins from spiders, particularly with regard to the spacing between cysteine residues. We have recently isolated three K+ channel antagonists from the venom of the Australian funnel-web spider Hadronyche versuta; at least two of these toxins are likely to constitute a new class of spider toxins active on K+ channels as they are approximately twice as large as HaTx and HpTx.
Little, M.J., Wilson, H., Zappia, C., Cestle, S., Tyler, M.I., Martin-Eauclaire, M.-.F., Gordon, D. & Nicholson, G.M. 1998, '?-Atracotoxins from Australian funnel-web spiders compete with scorpion ?-toxin binding on both rat brain and insect sodium channels', FEBS Letters, vol. 439, no. 3, pp. 246-252.
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?-Atracotoxins are novel peptide toxins from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion ?-toxins. To analyse their interaction with known sodium channel neurotoxin receptor sites we determined their effect on scorpion toxin, batrachotoxin and saxitoxin binding. Nanomolar concentrations of ?-atracotoxin-Hv1 and ?-atracotoxin-Ar1 completely inhibited the binding of the scorpion ?-toxin AaH II to rat brain synaptosomes as well as the binding of Lqh?IT, a scorpion ?-toxin highly active on insects, to cockroach neuronal membranes. Moreover, ?-atracotoxin-Hv1 cooperatively enhanced batrachotoxin binding to rat brain synaptosomes in an analogous fashion to scorpion ?-toxins. Thus the ?-atracotoxins represent a new class of toxins which bind to both mammalian and insect sodium channels at sites similar to, or partially overlapping with, the receptor binding sites of scorpion ?-toxins. Copyright (C) 1998 Federation of European Biochemical Societies.
Nicholson, G.M., Walsh, R., Little, M.J. & Tyler, M.I. 1998, 'Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activatiom and inactivation', Pflugers Archiv European Journal of Physiology, vol. 436, no. 1, pp. 117-126.
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The present study investigates the actions of robustoxin (atracotoxin-Ar1) purified from the venom of the male Sydney funnel-web spider Atrax robustus on sodium channel gating. Using whole-cell patch-clamp techniques the study assessed the actions of robustoxin on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents in rat dorsal root ganglion cells. Similar to the closely related funnel-web spider toxin versutoxin (?-atracotoxin-Hv1) from Hadronyche versuta, robustoxin had no effect on TTX-R sodium currents but exerted potent effects on TTX-S sodium currents. The main action of robustoxin was a concentratian-dependent slowing or removal of TTX-S sodium current inactivation. This steady-state current was maintained during long-lasting depolarisations at all test potentials. Robustoxin (30 nM) also caused a 13-mV hyperpolarising shift in the voltage midpoint of steady-state sodium channel inactivation (h(?)) leading to a reduced peak current at a holding potential of -80 mV. Moreover there was a steady-state or non-inactivating component present (18% of maximal sodium current) at prepulse potentials that normally inactivate all TTX-S sodium channels (more depolariscd than -40 mV). In addition robustoxin produced a significant increase in the repriming kinetics of the sodium channel when channels returned to the resting state following activation. This increase in the rate of recovery of sodium current appears to explain the use-dependent effects on peak sodium current amplitude at high stimlulation frequencies. Finally 30 nM robustoxin caused an 11-mV hyperpolarising shift in the voltage dependence of the channel but did not markedly modify tail current kinetics. These actions suggest that robustoxin in inhibits conversion of the open state to the inactivated state of TTX-S sodium channels, thus allowing a fraction of the sodium current to remain at membrane potentials at which inactivation is normally complete. Given the recent reclas...
Little, M.J., Zappia, C., Gilles, N., Connor, M., Tyler, M.I., Martin-Eauclaire, M.-.F., Gordon, D. & Nicholson, G.M. 1998, '?-Atracotoxins from Australian funnel-web spiders compete with scorpion ?-toxin binding but differentially modulate alkaloid toxin activation of voltage-gated sodium channels', Journal of Biological Chemistry, vol. 273, no. 42, pp. 27076-27083.
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?-Atracotoxins from the venom of Australian funnel-web spiders are a unique group of peptide toxins that slow sodium current inactivation in a manner similar to scorpion ?-toxins. To analyze their interaction with known sodium channel neurotoxin receptor sites, we studied their effect on [3H]batrachotoxin and 125I-Lqh II (where Lqh is ?-toxin II from the venom of the scorpion Leiurus quinquestriatus hebraeus) binding and on alkaloid toxin-stimulated 22Na+ uptake in rat brain synaptosomes. ?- Atracotoxins significantly increased [3H]batrachotoxin binding yet decreased maximal batrachotoxin-activated 22Na+ uptake by 70-80%, the latter in marked contrast to the effect of scorpion ?-toxins. Unlike the inhibition of batrachotoxin-activated 22Na+ uptake, ?-atracotoxins increased veratridine-stimulated 22Na+ uptake by converting veratridine from a partial to a full agonist, analogous to scorpion ?-toxins. Hence, ?- atracotoxins are able to differentiate between the open state of the sodium channel stabilized by batrachotoxin and veratridine and suggest a distinct sub-conductance state stabilized by ?-atracotoxins. Despite these actions, low concentrations of ?-atracotoxins completely inhibited the binding of the scorpion ?-toxin, 125I-Lqh II, indicating that they bind to similar, or partially overlapping, receptor sites. The apparent uncoupling between the increase in binding but inhibition of the effect of batrachotoxin induced by ?-atracotoxins suggests that the binding and action of certain alkaloid toxins may represent at least two distinguishable steps. These results further contribute to the understanding of the complex dynamic interactions between neurotoxin receptor site areas related to sodium channel gating.
Wilson, H.I. & Nicholson, G.M. 1997, 'Presynaptic snake ?-neurotoxins produce tetanic fade and endplate potential run-down during neuromuscular blockade in mouse diaphragm', Naunyn-Schmiedeberg's Archives of Pharmacology, vol. 356, no. 5, pp. 626-634.
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The present study investigated the ability of a number of presypnatic neurotoxins (snake ?-neurotoxins) to produce nerve-evoked train-of-four fade, tetanic fade and endplate potential run-down during the development of neuromuscular blockade in the isolated mouse phrenic-hemidiaphragm nerve-muscle preparation. All the snake ?-neurotoxins tested, with the exception of notexin, produced train-of-four and tetanic fade of nerve-evoked isometric muscle contractions. Train-of-four fade was not present during the initial depressant or facilitatory phases of muscle tension produced by the snake ?-neurotoxins but developed progressively during the final depressant phase that precedes complete neuro-muscular blockade. The 'non-neurotoxic' bovine pancreatic phospholipase A2 and the 'low-toxicity' phospholipase A2, from Naja naja atra venom failed to elicit train-of-four fade, indicating that the phospholipase activity of the snake ?-neurotoxins is not responsible for the development of fade. Intracellular recording of endplate potentials (EPPs) elicited by nerve-evoked trains of stimuli showed a progressive run-down in EPP amplitude during the train following incubation with all snake ?-neurotoxins except notexin. Again this run-down in EPP amplitude was confined to the final depressant phase of snake ?-neurotoxin action. However when EPP amplitude fell to near uniquantal levels (<3 mV) the extent of toxin induced-fade was reduced. Unlike postjunctional snake ?-neurotoxins, prejunctional snake ?-neurotoxins interfere with acetylcholine release at the neuromuscular junction during the development of neuromuscular blockade. This study provides further support for the hypothesis that fade in twitch and tetanic muscle tension is due to an underlying rundown in EPP amplitude resulting from a prejunctional alteration of transmitter release rather than a use-dependent block of postjunctional nicotinic receptors.
Nicholson, G.M., Little, M.J., Tyler, M. & Narahashi, T. 1996, 'Selective alteration of sodium channel gating by Australian funnel-web spider toxins', Toxicon, vol. 34, no. 11-12, pp. 1443-1453.
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The actions of potent mammalian neurotoxins isolated from the venom of two Australian funnel-web spiders were investigated using both electrophysiological and neurochemical techniques. Whole-cell patch clamp recording of sodium currents in rat dorsal root ganglion neurons revealed that versutoxin (VTX), isolated from the venom of Hadronyche versuta, produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation and a reduction in peak TTX-S sodium current. In contrast, VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. VTX also shifted the voltage dependence of sodium channel activation in the hyperpolarizing direction and increased the rate of recovery from inactivation. Ion flux studies performed in rat brain synaptosomes also revealed that robustoxin (RTX), from the venom of Atrax robustus, and VTX both produced a partial activation of 22Na+ flux and an inhibition of batrachotoxin-activated 22Na+ flux. This inhibition of flux through batrachotoxin-activated channels was not due to an interaction with neurotoxin receptor site 1 since [3H]saxitoxin binding was unaffected. In addition, the partial activation of 22Na+ flux was not enhanced in the presence of ?-scorpion toxin and further experiments suggest that VTX also enhances [3H]batrachotoxin binding. These selective actions of funnel-web spider toxins on sodium channel function are comparable to those of ?-scorpion and sea anemone toxins which bind to neurotoxin receptor site 3 on the channel to slow channel inactivation profoundly. Also, these modifications of sodium channel gating and kinetics are consistent with actions of the spider toxins to produce repetitive firing of action potentials.
Wilson, H.I., Nicholson, G.M., Tyler, M.I. & Howden, M.E.H. 1995, 'Induction of giant miniature end-plate potentials during blockade of neuromuscular transmission by textilotoxin', Naunyn-Schmiedeberg's Archives of Pharmacology, vol. 352, no. 1, pp. 79-87.
The present study investigated the action of textilotoxin, isolated from the venom of the Australian common brown snake Pseudonaja textilis, on neuromuscular transmission in isolated toad nerve-muscle preparations. Initial muscle twitch tension experiments revealed a triphasic pattern of changes in muscle tension and a irreversible binding action of textilotoxin (10 ?g/ml) similar to other snake ?-neurotoxins. This was characterised by an initial depression of twitch tension, followed by a period of enhanced tension, eventually leading to a reduction in tension to complete neuromuscular blockade. These actions on muscle tension were investigated further by assessing the action of textilotoxin on endplate potential amplitude (EPP). This revealed a similar triphasic alteration of the nerve-evoked release of acetylcholine from the motor nerve terminal. These actions on acetylcholine release were confirmed to be of a presynaptic origin since the modal amplitude of miniature end-plate potentials (MEPPs) was not reduced and in twitch tension experiments the muscle still contracted in response to direct muscle stimulation when nerve-evoked release was completely blocked. Interestingly dramatic effects were observed on the spontaneous release of acetylcholine, including an marked increase in MEPP frequency, a skewing of the MEPP amplitude frequency histogram to the right, and a resultant increase in the number of 'giant' MEPPs. These results indicate that textilotoxin causes a presynaptic blockade of neuromuscular transmission involving a disruption of the regulatory mechanism that controls acetylcholine release.
Nicholson, G.M., Willow, M., Howden, M.E.H. & Narahashi, T. 1994, 'Modification of sodium channel gating and kinetics by versutoxin from the Australian funnel-web spider Hadronyche versuta', Pflugers Archiv European Journal of Physiology, vol. 428, no. 3-4, pp. 400-409.
The effects of a neurotoxin (versutoxin VTX), purified from the venom of the Australian Blue Mountains funnel-web spider Hadronyche versuta, on the ionic currents in rat dorsal root ganglion cells were investigated under voltage-clamp conditions using the whole-cell patch-clamp technique. VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. In contrast VTX produced a dose-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation, a reduction in peak TTX-S sodium current but did not markedly slow tail current kinetics of TTX-S sodium currents. This steady-state sodium current was maintained during prolonged depolarizations at all test potentials and the reduction in sodium current amplitude produced by VTX had an apparent K(i) of 37 nM. In the presence of 32 nM VTX the voltage dependence of steady-state sodium channel inactivation (h(?)) also showed a significant 7 mV shift in the voltage midpoint in the hyperpolarizing direction, with no change in the slope factor. In addition there was a steady-state or non-inactivating component present (14 2% of maximal sodium current) at prepulse potentials more depolarized than -40 mV, potentials which normally inactivate all TTX-S sodium channels. Finally, there was an observed increase in the rate of recovery from inactivation in the presence of VTX. These selective actions of VTX on sodium channel gating and kinetics are similar to those of ?-scorpion and sea anemone toxins.
Lloyd, D.R., Nicholson, G.M., Spence, I., Connor, M., Tyler, M.I. & Howden, M.E.H. 1991, 'Frequency-dependent neuromuscular blockade by textilotoxin in vivo', Toxicon, vol. 29, no. 10, pp. 1266-1269.
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D. R. Lloyd, G. M. Nicholson, I. Spence, M. Connor, M. I. Tyler and M. E. H. Howden. Frequency-dependent neuromuscular blockade by textilotoxin in vivo. Toxicon 29, 1266-1269, 1991.-The effect of stimulation frequency on the timecourse of neuromuscular blockade, following the administration of textilotoxin (20 ?g/kg) or ?-bungarotoxin (50 ?/kg), was examined in the interdigital muscles of the hindlimb in anaesthetized mice. While the time of death was variable, neuromuscular blockade of the interdigital muscles occurred at the same time as respiratory failure with both textilotoxin and ?-bungarotoxin only at stimulation rates of 0.5 Hz and above. Textilotoxin (50 ?g/kg) produced an increase in the heart rate prior to death but no change in the shape of the electrocardiogram. 1991.
Nicholson, G.M., Spence, I. & Johnston, G.A.R. 1990, 'Calcium-dependent actions of the convulsant barbiturate, CHEB, on transmitter release at the rat neuromuscular junction', General Pharmacology, vol. 21, no. 5, pp. 741-746.
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1. The effect of convulsant barbiturates on spontaneous and evoked acetylcholine release was studied at the rat neuromuscular junction in vitro. 2. The convulsant barbiturates (+)-5-(1,3-dimethylbutyl)-5-ethyl barbituric acid [(+)-DMBB], 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB), 5-ethyl-5-(3-methylbut-2?-enyl) barbituric acid (3M2B) and 5-ethyl-5-(1,3-dimethylbut-1?-enyl) barbituric acid (1,3M1B) all produced a concentration-dependent increase in miniature end-plate potential (MEPP) frequency. 3. With CHEB (100 ?M) this increase in MEPP frequency was found to be dependent on the [Ca2+]o. CHEB in 0.5 mM [Ca2+]o did not alter MEPP amplitude, but in 1.3 and 2.5 mM [Ca2+]o CHEB significantly reduced the amplitude. 4. At a [Ca2+]o of 0.5 mM, CHEB produced an increase in both EPP amplitude and quantal content, while at 1.3 mM [Ca2+]o CHEB did not alter EPP amplitude or quantal content. 5. The plot of log quantal content vs log [Ca2+]o showed a parallel shift to the left in the presence of 100 ?M CHEB. This change occurred without any alteration in the maximum quantal content. This suggests that the enhancement of transmitter release may be mediated by an effect on calcium fluxes in the pre-junctional nerve terminal. 1990.
Nicholson, G.M., Spence, I. & Johnston, G.A.R. 1988, 'Depolarizing actions of convulsant barbiturates on isolated rat dorsal root ganglion cells', Neuroscience Letters, vol. 93, no. 2-3, pp. 330-335.
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The actions of convulsant barbiturates were studied on dorsal root ganglion (DRG) cells in vitro using intracellular recording techniques. Only the convulsant barbiturates (+)-DMBB and CHEB produced a concentration-dependent depression in the responses to ?-aminobutyric acid (GABA). All convulsant barbiturates were found to produce a direct depolarization of the DRG cell membrane which was accompanied by a decrease in the input resistance of the cell and a reduction in the orthodromic action potential. A sub-population of DRG cells were found to be refractory to these actions but there was no relationship between the cell type (A?, A? and C) and ability to respond to the convulsant barbiturates. 1988.
Nicholson, G.M., Spence, I. & Johnston, G.A.R. 1988, 'Differing actions of convulsant and nonconvulsant barbiturates: An electrophysiological study in the isolated spinal cord of the rat', Neuropharmacology, vol. 27, no. 5, pp. 459-465.
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The effects of various pairs of convulsant and nonconvulsant barbiturates on mono-and polysynaptic activity were studied in the isolated spinal cord of the immature rat, using extracellular recording. The convulsant barbiturates, 5-ethyl-5-(3-methylbut-2'-enyl) barbituric acid (3M2B), 5-ethyl-5-(1,3-dimethylbut-1'-enyl) barbituric acid (1,3M1B) and (+)-5-(1,3-dimethylbutyl)-5-ethyl barbituric acid [(+) DMBB] all increased the monosynaptic reflex at concentrations between 5 and 50 ?M with no change in polysynaptic activity. When the concentration was raised to between 100 and 300 ?M, however, the convulsants all reduced the monosynaptic reflex, thus producing a biphasic dose-response relationship. The nonconvulsant barbiturates phenobarbital, 5-ethyl-5-(3-methylbut-1'-enyl) barbituric acid (3M1B), amylobarbital (3MB) and (-)-5-(1,3-dimethylbutyl)-5-ethyl barbituric acid [(-)DMBB] produced only a decrease in mono- and polysynaptic reflexes. At concentrations which enhanced the monosynaptic reflex, the responses of motoneurones to glycine and eledoisin-related peptide (an analogue of substance P) were reduced by (+)DMBB, while 1,3M1B and 3M2B had no significant effects upon any of the neurotransmitters tested. At concentrations which depressed the monosynaptic reflex, the convulsants all reduced the response to glycine whereas the nonconvulsant barbiturates all increased the response to GABA. With the exception of phenobarbital, both convulsant and nonconvulsant barbiturates produced a direct depolarisation of the presynaptic terminal membrane, with only the convulsants producing a depolarisation of the membrane of the motoneurone. Using another convulsant barbiturate, 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB), this direct depolarising action was found to be calcium-dependent. 1988.
Nicholson, G.M., Spence, I. & Johnston, G.A.R. 1985, 'Effects of a depressant/convulsant pair of glutarimides on neuronal activity in the isolated spinal cord of the immature rat', Neuropharmacology, vol. 24, no. 6, pp. 461-464.
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The effect of a depressant/convulsant pair of glutarimides on the isolated spinal cord of the immature rat was examined using extracellular recording. At concentrations of 300 ?M the depressant ?-butyl-?-methyl glutarimide enhanced the response of motoneurones and dorsal root fibres to ?-aminobutyric acid (GABA) while the convulsant bemegride (?-ethyl-?-methyl glutarimide) decreased both responses to GABA. At this concentration both the convulsant and depressant reduced mono- and polysynaptic reflex activity. Neither the convulsant or depressant had prominant direct actions, with only a small hyperpolarisation being produced by both glutarimides on dorsal root fibres. The overall depressant or convulsant properties of these glutarimides may be due in part therefore to a differential effect on the postsynaptic action of the inhibitory transmitter GABA. Furthermore, the depressant glutarimide reduced the excitatory effects of l-glutamate and the convulsant reduced the inhibitory effects of glycine on spinal neurones; thus, actions on these transmitters may also contribute to the overall effects of these glutarimides. 1985.
Nicholson, G.M., Spence, I. & Johnston, G.A.R. 1985, 'Strychnine-like action of the convulsant barbiturate, CHEB', Neuropharmacology, vol. 24, no. 6, pp. 465-471.
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The effect of 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB) on the isolated spinal cord of the immature rat was examined using extracellular recording. At concentrations less than 20 ?M CHEB increased the monosynaptic reflex (MSR) but depressed the reflex at greater concentrations (30-100 ?M). At concentrations which enhanced the monosynaptic reflex, CHEB reduced the responses of motoneurones to glycine and to a lesser extent to those of l-glutamate. In the presence of strychnine (5 ?M), which enhanced both mono- and polysynaptic reflexes, CHEB produced only slight enhancement of the monosynaptic reflex. At concentrations of 30-100 ?M the responses to ?-aminobutyric acid (GABA), glycine, l-glutamate and eledoisin-related peptide (ERP a substance P and analogue) were all reduced. At these concentrations CHEB directly depolarised the motoneurone membrane. Increases in [Mg2+]0, which reduced spontaneous activity, blocked the enhancement, by CHEB, of the monosynaptic reflex. The actions of CHEB in small doses may be due therefore to its ability to block the action of glycine and thus block tonic inhibition. 1985.
Skerritt, J.H., Johnston, G.A.R., Katsikas, T., Tabar, J., Nicholson, G.M. & Andrews, P.R. 1983, 'Actions of pentobarbitone and derivatives with modified 5-butyl substituents on GABA and diazepam binding to rat brain synaptosomal membranes', Neurochemical Research, vol. 8, no. 10, pp. 1337-1350.
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The effects of a variety of factors known to influence the enhancement of GABA binding by diazepam, were studied upon pentobarbitone stimulation of GABA binding to washed synaptosomal membranes prepared from whole rat brains. The differential kinetics of, and effects of temperature, chloride ions, a benzodiazepine receptor antagonist (Ro15-1788) and picrotoxinin upon pentobarbitone and diazepam enhancement of GABA binding, suggest that these drugs exert their actions upon GABA binding at different loci. The degree of enhancement of diazepam binding and of high affinity GABA binding in chloride-containing media at 25C by members of a series of twelve side chain methyl substituted and/or unsaturated derivatives of 5-butyl-5-ethyl-barbituric acid (pentobarbitone analogs) correlated significantly. For the sedative members of the series, enhancement of high affinity GABA binding correlated with their anaesthetic but not their anticonvulsant activities. It appears likely that the anaesthetic and anticonvulsant activities of barbiturates arise from different molecular actions. 1983 Plenum Publishing Corporation.

National and International Research Collaborators:

  • Glenn King (Institute for Molecular Bioscience, University of Queensland)
  • Richard Lewis (Institute for Molecular Bioscience, University of Queensland)
  • Wayne Hodgson (Department of Pharmacology, Monash University)
  • Pierre Escoubas (Venometech, Valbonne, France)
  • Lucia Kuhn-Nentwig (University of Bern, Switzerland)
  • Claire Dauly (Thermo-Fisher, Paris, France)
  • Alain Dejean (CNRS, UMR Ecologie des Forêts de Guyane, French Guiana)
  • Frank Bosmans (Johns Hopkins University, Baltimore, USA)