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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
CB04.04.48K

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

Book Chapters

Nicholson, G.M. 2013, 'Spider peptides' in Kastin, A (eds), The Handbook of Biologically Active Peptides, Elsevier, San Diego, USA, pp. 461-472.
Spider peptide and protein toxins are recognized as highly potent and specific molecular tools that modulate signaling in excitable cells via interaction with a variety of ion channels, receptors, and transporters in vertebrates and invertebrates. By a process of natu- ral selection, spider venoms have evolved as complex preoptimized combinatorial peptide libraries, displaying extremely diverse pharmacology with a wide range of molecular targets, designed to subdue or kill their prey or predators. The discovery that these disulfide-rich proteins and peptides bind to their cognate receptors with high affinity, and in many cases with outstanding selectivity, means that spider venoms are now being investigated as sources of molecular tools for probing ion channel and synaptic neuro- transmission, for validating novel insecticide targets, and as lead compounds in therapeutic and biopesticide discovery pipelines.
Simpson, A.M., Swan, M.A., Liu, G.J., Tao, C.Z., O'Brien, B., Ch'ng, E., Castro, L.M., Ting, H.J., Elgundi, Z., An, T., Lutherborrow, M., Torpy, F.R., Martin, D.K., Tuch, B.E. & Nicholson, G.M. 2013, 'Insulin trafficking in a glucose responsive engineered human liver cell line is regulated by the interaction of ATP-sensitive potassium channels and voltage- gated calcium channels' in Molina, Francisco Martin (eds), Gene Therapy - Tools and Potential Applications, InTech, Rijeka, Croatia, pp. 703-726.
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Type I diabetes is caused by the autoimmune destruction of pancreatic beta () cells [1]. Current treatment requires multiple daily injections of insulin to control blood glucose levels. Tight glucose control lowers, but does not eliminate, the onset of diabetic complications, which greatly reduce the quality and longevity of life for patients. Transplantation of pancreatic tissue as a treatment is restricted by the scarcity of donors and the requirement for lifelong immunosuppression to preserve the graft, which carries adverse side-effects. This is of particular concern as Type 1 diabetes predominantly affects children. Lack of glucose control could be overcome by genetically engineering "an artificial -cell" that is capable of synthesising, storing and secreting insulin in response to metabolic signals. The donor cell type must be readily accessible and capable of being engineered to synthesise, process, store and secrete insulin under physiological conditions.
Bosmans, F., Escoubas, P. & Nicholson, G.M. 2009, 'Spider venom peptides as leads for drug and insecticide design' in de Lima ME, Pimenta AMC, Martin-Eauclaire M-F, Zingali RB, Rochat H (eds), Animal Toxins: State of the Art. Perspectives In Health and Biotechnology, Editora UFMG, Belo Horizonte, pp. 269-290.
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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 Kastin, A (eds), The Handbook of Biologically Active Peptides, Elsevier, San Diego, USA, pp. 369-379.
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Spider peptide and protein toxins are recognised as highly potent and spefici molecular tools that modulate neurotransmission via interaction with a variety of ion channels, receptors and transporters in vertebrates and invertebrates. The recent discovery of the diversity of thesepeptides means that spider venoms are only now being considered as combinatorial peptide libraries useful for probing synaptic neurotransmission, for validating novel insecticide targets and for aiding in the design of therapeutics and biopesticides. These studies are being greatly assistaed bu the determination of the pharmacophore of these toxins. This review details the discovery, structure and function of these spider venom peptide toxins.
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.

Conference Papers

de Araujo, A.D., Herzig, V., Mobli, 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', 31st European Peptide Symposium, Copenhagen, Sweden, September 2010 in Journal of Peptide Science, John Wiley & Sons Inc, Malden MA 02148 USA, pp. 60-61.
Gunning, S.J., Maggio, F.J., Windley, M.J., Valenzuela, S., King, G.F. & Nicholson, G.M. 2009, 'Janus-faced atracotoxins are specific blockers of invertebrate KCa channels', 34th Congress of the Federation of European Biochemical Societies, Prague, Czech Republic, July 2009.
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', 16th World Congress on Animal, Plant and Microbial Toxins, March 2009.
Yamaji, N., Little, M.J., Nishio, H., Villegas, E., Billen, B., Tytgat, J., Nicholson, G.M. & Corzo, G. 2009, 'Synthesis, solution structure and phyla-specificity of a spider +-toxin that slows inactivation of specific voltage-gated sodium channel subtypes', 16th World Congress on Animal, Plant and Microbial Toxins, Recife Brazil, March 2009.
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', 16th World Congress on Animal, Plant and Microbial Toxins, Canberra, ACT, April 2009.
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)', 8th Asia-Pacific Congress on Animal, Plant & Microbial Toxins, Hanoi and Halong Bay, Vietnam, December 2008.
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)', 8th Asia-Pacific Congress on Animal, Plant & Microbial Toxins, Hanoi and Halong Bay, Vietnam, December 2008.
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', 8th Asia-Pacific Congress on Animal, Plant & Microbial Toxins, Hanoi and Halong Bay, Vietnam, December 2008.
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', 3rd International Congress on Natural Peptides to Drugs, Zermatt, Switzerland, April 2009.
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)', 27th International Congress of the European Association of Poisons Centres and Clinical Toxicologists, Athens, Greece, May 2007.
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', 54th American Society of Mass Spectrometry, Seattle, WA, USA, May 2006.
Kubista, H., Mafra, R.A., Chong, Y., Nicholson, G.M., Beirao, P.S., Cruz, J.S., Boehm, S., Nentwig, W. & Kuhn-Nentwig, L. 2006, 'CSTX-1, a neurotoxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in rat neurons', 5th Forum of European Neuroscience Societies, Vienna, Austria, July 2006.
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', 15th World Congress on Animal, Plant and Microbial Toxins, Glasgow, Scotland, July 2006.
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', 15th World Congress on Animal, Plant and Microbial Toxins, Glasgow, Scotland, July 2006.
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', 15th World Congress on Animal, Plant and Microbial Toxins, Glasgow, Scotland, July 2006.
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', 15th World Congress on Animal, Plant and Microbial Toxins, Glasgow, Scotland, July 2006.
Sollod, B., Gunning, S.J., Wen, S., Nicholson, G.M. & King, G.K. 2006, 'A dual-target, self-synergizing toxin from spider venom', 15th World Congress on Animal, Plant and Microbial Toxins, Glasgow, Scotland, July 2006.
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', Venoms to Drugs 3, Heron Island, QLD, August 2005.
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', Venoms to Drugs 3, Heron Island, QLD, August 2005.
Wen, S., Wilson, D., Kuruppu, S., Korsinczky, M.L., 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', 7th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cebu City, Philippines, October 2005.
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', 7th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cebu City, Philippines, October 2005.
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', 7th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cebu City, Philippines, October 2005.
Nicholson, G.M. 2004, 'Australian spiders: venomous villains or toxic treasures?', 22nd International Congress of Entomology, Brisbane, August 2004.
Gunning, S.J., Maggio, F.J., King, G.K. & Nicholson, G.M. 2004, '+-Atracotoxins: Insect potassium channels blockers that reveal a novel insecticide target.', 8th Symposium of the Pan-American Section of the International Society of Toxinology, Angra dos Reis, Brazil, September 2004.
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)', 14th World Congress on Animal, Plant and Microbial Toxins, Adelaide, September 2003.
Gunning, S.J., Maggio, F.J., King, G.K. & Nicholson, G.M. 2003, 'Do insecticidal J-atracotoxins target insect potassium channels?', 14th World Congress on Animal, Plant and Microbial Toxins, Adelaide, September 2003.
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', 14th World Congress on Animal, Plant and Microbial Toxins, Adelaide, September 2003.
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.', 14th World Congress on Animal, Plant and Microbial Toxins, Adelaide, September 2003.
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', 14th World Congress on Animal, Plant and Microbial Toxins, Adelaide, September 2003.
Wilson, H.I. & Nicholson, G.M. 2003, 'Characteristics of the venom of Australian urodacid and buthid scorpions: venom production, composition and toxicity', 14th World Congress on Animal, Plant and Microbial Toxins, Adelaide, September 2003.
Graudins, A., Sung, K., Hains, P.G., Padula, M.P., Broady, K.W. & Nicholson, G.M. 2002, 'Partial protein and DNA sequences of Latrodectus hasselti, L. hesperus and L. mactans latrotoxins: are they homologous?', 6th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cairns, July 2001.
Gunning, S.J., Khalife, A., Padula, M.P., 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.', 6th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cairns, July 2001.
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', 6th Asia Pacific Congress on Animal, Plant and Microbial Toxins, Cairns, July 2002.
Wang, X., Connor, M., Wilson, D., Sollod, B., Nicholson, G.M., Alewood, P.F., Christie, M. & King, G.K. 2001, 'Discovery and structure of a potent and highly specific blocker of insect calcium channels', Pan American IST Congress, Charlottesville, VI, USA, March 2001.
Conference abstract
Graudins, A., Padula, M.P., 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)', XX International Congress of the European Association of Poisons Centres and Clinical Toxicologists, May 2000 in Journal of Toxicology-Clinical Toxicology, ed Nicholas Bateman, Taylor & Francis, London, pp. 205-206.

Journal Articles

Touchard, A., Dauvois, M., Arguel, M., Petitclerc, P.F., Leblanc, M., Dejean, A., Orivel, J., Nicholson, G.M. & Escoubas, P. 2014, 'Elucidation of the unexplored biodiversity of ant venom peptidomes by MALDI-TOF mass spectrometry and its application for chemotaxonomy', Journal of Proteomics, vol. 80, pp. 292-310.
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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.
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 a-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.
Touchard, A., Labrire, N., Roux, O., Petitclerc, P.F., Orivel, J., Escoubas, P., Koh, J., 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.
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, Pseudomyrmex penetrator and Pseudomyrmex gracilis. 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 homodi- meric 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.
Bende, N.S., Dziemborowicz, S.A., Mobli, M., Herzig, V., Gilchrist, J., Wagner, J., Nicholson, G.M., King, G.K. & Bosmans, F. 2014, 'A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a', Nature Communications, vol. 5, no. 11 July, pp. 4350-4359.
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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 Nav channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identified two residues within the BgNav1 domain II S1-S2 loop that when mutated to their PaNav1 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.
Palagi, A., Koh, J., 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.
Bende, N.S., Kang, E.J., 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 b 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.
Marcon, F., Purtell, L.A., 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 a-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 a-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 a-subunit of P-EPTX-As1a shows high homology to taipoxin a-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.
de Araujo, A.D., Herzig, V., Windley, M.J., Dziemborowicz, S.A., 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.
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 alpha-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 alpha-latrotoxin (alpha-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 alpha-LTX, alpha-LIT and delta-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-alpha-LTX monoclonal antibody 4C4.1, raised against alpha-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 alpha-LTXs in theridiid spider venoms. We therefore cloned and sequenced the alpha-LTX from the Australian red-back spider L hasselti (alpha-LTX-Lh1a). The deduced amino acid sequence of the mature alpha-LTX-Lh1a comprises 1180 residues (similar to 132 kDa) with similar to 93% sequence identity with alpha-LTX-Lt1a. alpha-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 alpha-LTX-Lt1a, indicates that alpha-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that alpha-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.
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.
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. SI4, pp. 478-491.
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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 pA2 value of 7.902 (KB = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal +7 nAChRs, with an IC50 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 Ala28 and Arg36 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
Marcon, F., Leblanc, M., Vetter, I., Lewis, R.J., Escoubas, P. & Nicholson, G.M. 2012, 'Pharmacological characterization of a-elapitoxin-Al2a from the venom of the Australian pygmy copperhead (Austrelaps labialis): An atypical long-chain a-neurotoxin with only weak affinity for a7 nicotinic receptors', Biochemical Pharmacology, vol. 84, pp. 851-863.
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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, a-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 pA2 value of 7.902 (KB = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal +7 nAChRs, with an IC50 of 1.2 mM, 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 Ala28 and Arg36 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
Blacklow, B.J., Kornhauser, R., Hains, P.G., Loiacono, R., Escoubas, P., Graudins, A. & Nicholson, G.M. 2011, 'Alpha-Elapitoxin-Aa2a, a long-chain snake alpha-neurotoxin with potent actions on muscle (alpha1)2betagammadelta nicotinic receptors, lacks the classical high affinity for neuronal alpha7 nicotinic receptors', Biochemical Pharmacology, vol. 81, no. 2, pp. 314-325.
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In contrast to all classical long-chain alpha-neurotoxins possessing the critical fifth disulfide bond, alpha-elapitoxin-Aa2a (alpha-EPTX-Aa2a), a novel long-chain alpha-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal alpha7-type nicotinic acetylcholine receptors (nAChRs). alpha-EPTX-Aa2a (8850 Da; 0.1-1 microM) 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. alpha-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 alpha-neurotoxin, alpha-bungarotoxin. In contrast, alpha-EPTX-Aa2a produced complete, but weak, inhibition of 125I-alpha-bungarotoxin binding to rat hippocampal a7 nAChRs (pKI = 3.670), despite high sequence homology and similar mass to a wide range of long-chain alpha-neurotoxins. The mostly likely cause for the loss of alpha7 binding affinity is a leucine substitution, in loop II of alpha-EPTX-Aa2a, for the highly conserved Arg33 in long-chain alpha-neurotoxins. Arg33 has been shown to be critical for both neuronal and muscle activity. Despite this substitution, alpha-EPTX-Aa2a retains high affinity for muscle (alpha1)2betagammadelta nAChRs. This is probably as a result of an Arg29 residue, previously shown to be critical for muscle (alpha1)2betagammadelta nAChR affinity, and highly conserved across all short-chain, but not long-chain, alpha-neurotoxins. alpha-EPTX-Aa2a therefore represents a novel atypical long-chain alpha-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.
Herzig, V., Wood, D.L., Newell, F., Chaumeil, P., 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, pp. D653-D657.
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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.
Smith, J.J., Hill, J.M., Little, M.J., Nicholson, G.M., Alewood, P.F. & King, G.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 beta-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.
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 signifi- cant 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 A2 (PLA2) 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 PLA2 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.
Windley, M.J., Escoubas, P., Valenzuela, S. & 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 (kappa-TRTX-Ec2a, kappa-TRTX-Ec2b, and kappa-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 kappa-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K+ (BKCa) channel currents with IC50 values of 3 to 25 nM. In addition, kappa-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K+ currents, but only at significantly higher concentrations. kappa-TRTX-Ec2a and kappa-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous kappa-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, kappa-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, kappa-TRTX-Ec2a and kappa-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 BKCa channels. This study therefore establishes these toxins as tools for studying the role of BKCa channels in insects and lead compounds for the development of novel insecticides.
Blacklow, B.J., 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 (44,698 Da) 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.
Blacklow, B.J., 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 some- times 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 post-synaptic alpha-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 ant- arcticus. Venoms were separated by size-exclusion liquid chromatography under non- denaturing conditions and fractions corresponding to proteins in the range of 22 to >60 kDa 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.
Mobli, M., de Araujo, A.D., Lambert, L.K., Pierens, G.K., Windley, M.J., Nicholson, G.M., Alewood, P.F. & King, G.F. 2009, 'Direct Visualisation of Disulfide Bonds through Diselenide Proxies Using 77Se NMR Spectroscopy', Angewandte Chemie-international Edition, vol. 48, no. 49, pp. 9312-9314.
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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.
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.
Escoubas, P., Quinton, L. & Nicholson, G.M. 2008, 'Venomics: unraveling 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 aswell as pharmacological diversity is immense, andmass 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.
Nicholson, G.M. 2008, 'Member of Toxicon Editorial Board', Toxicon.
Nicholson, G.M. 2008, 'Member of Frontiers in Cellular Neuroscience Editorial Board', Frontiers in Cellular Neuroscience.
Nicholson, G.M. 2008, 'Member of the Open Toxicology Journal Editorial Board', The Open Toxicology Journal.
http://www.bentham.org/open/totoxij/EBM.htm
Gunning, S.J., Maggio, F.J., Windley, M.J., Valenzuela, S., 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 calcium activated K+ (KCa) channels. 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 potassium (KV), calcium (CaV), or sodium (NaV) channel subtypes. J ACTX-Hv1c blocked heterologously expressed cockroach BKCa (pSlo) channels without a significant shift in the voltage-dependence of activation. However, the block was voltage-dependent, indicating that the toxin likely 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 IK(Ca). This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate KCa channels and suggests that KCa channels might be a potential insecticide target.
King, G.K., Escoubas, P. & Nicholson, G.M. 2008, 'Peptide toxins that selectively target insect NaV and CaV 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 certainvertebrate 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.
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.
Herzig, V., Khalife, 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, 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 signi+cant 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 d-missulenatoxin-Mb1a, the toxin responsible for the effects of male M. bradleyi venom in vertebrates. These +ndings 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.
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 iof effective control by conventional agrochemical insecticides, a number of factors aere 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 recobitant baculoviruses as delivery systems for a variety of insect-selective toxins. Additional approaches for the development of foliar sprays include the rational dsign of peptidomimetics based on the key residues of thes toxins that inetract 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 weakenesses of biopepticides int he global control of arthropod pests.
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 (Na(v)) 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 Na(v) 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 Na(v) channel conductance. Magi 2 and Tx4(6-1) slow Na(v) channel inactivation via an interaction with site-3. The delta-palutoxins, and most likely mu-agatoxins and curtatoxins, target site-4. However, their action is complex with the mu-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion beta-toxins, but with both delta-palutoxins and mu-agatoxins slowing Na(v) channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as delta-atracotoxins, are known to target both insect and vertebrate Na(v) 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.
Nicholson, G.M. 2007, 'Member of Toxicon Editorial Board', Toxicon, vol. 49-50.
Chong, Y., Hayes, J.L., Sollod, B., Wen, S., Wilson, D., Hains, P.G., Hodgson, W.C., Broady, K.W., King, G.K. & Nicholson, G.M. 2007, 'The omega-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 w-atracotoxins (w-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, w-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the w-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 mM. To further characterize the target of the w-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 w-ACTX-Ar1a, and its homolog w-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 w-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 mM w-ACTX-Ar1a failed to significantly affect global Kv channel currents. However, 1 mM w-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 w-agatoxin IVA. These findings validate both M-LVA and HVA Cav channels as potential targets for insecticides
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. & Lewis, R.J. 2006, 'Ciguatoxins: Cyclic polyether modulators of voltage-gated Iion 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
Nicholson, G.M., Graudins, A., Wilson, H.I., Little, M.J. & Broady, K.W. 2006, 'Arachnid toxinology in Australia: clinical toxicology to potential applications', Toxicon, vol. 48, no. 7, pp. 872-898.
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The unique geographical isolation of Australia has resulted i the evolution of a distinctive range of Australian arachnid fauna. Through the pioneering work of a number of Asurtalian arachnologists 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, paraysis ticks and buthid scorpions that contain nerotoxins capable of causing deht 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 efficious 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 fromt he venom of Asutralian arachnids will make them invaluable molecular toold for studies of neurotransmitter release and vesicle exocytosis as well a sion 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 biopeptides and the characterisation of previously unvalidated insecticide targets
Nicholson, G.M. 2006, 'Member of Toxicon Editorial Board', Toxicon, vol. 47-48.
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 applic
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
Nicholson, G.M. & Little, M.J. 2005, 'Spider neurotoxins targeting voltage-gated sodium channels', Toxin Reviews, vol. 24, no. 38810, 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 nu
Wen, S., Wilson, D., Kuruppu, S., Korsinczky, M.L., 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 sequences homology but not pharmacological properties with AVIT 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 fro the venom of two Australian funnerl-web spiders, Hadronyche sp. 20 and H. infesa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral prtein 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).
Nicholson, G.M. 2005, 'Member of Toxicon Editorial Board', Toxicon, vol. 45-46.
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 delta-atracotoxins: lethal neurotoxins targeting the voltage-gates sodium channel', Toxicon, vol. 43, no. 5, pp. 587-599.
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funnel-web spider; &atracotoxin; voltage-gated sodium channel; inhibitor cystine-knot; scorpion X-toxin; sea anemone toxin
Nicholson, G.M. 2004, 'Member of Toxicon Editorial Board', Toxicon, vol. 43-44.
Gunning, S.J., Chong, Y., Khalife, A., Hains, P.G., Broady, K.W. & Nicholson, G.M. 2003, 'Isolation of delta-missulenatoxin-Mb1a, the major vertebrate-active spider delta-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.
Alewood, D., Birinyi-Strachan, L.C., Pallaghy, P.K., Norton, R.S., Nicholson, G.M. & Alewood, P.F. 2003, 'Synthesis and characterization of delta-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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Nicholson, G.M. & Graudins, A. 2003, 'Antivenoms for treatment of spider envenomation', Journal of Toxicology, vol. 22, no. 1, pp. 35-59.
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Nicholson, G.M. 2003, 'Member of Toxicon Editorial Board', Toxicon, vol. 41-42.
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.
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Graudins, A., Wilson, D., Alewood, P.F., Broady, K.W. & Nicholson, G.M. 2002, '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.
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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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.
Gilles, N., Harrison, G., Karbat, I., Gurevitz, M., Nicholson, G.M. & Gordon, D. 2002, 'Variations in receptor site-3 on rat brain and insect sodium channels highlighted by binding of a funnel-web spider delta-atracotoxin', European Journal of Biochemistry, vol. 269, no. 5, pp. 1500-1510.
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Delta-atracotoxins (delta-ACTXs) from Australian funnel-web spiders differ structurally from scorpion alpha-toxins (Sc(alpha)Tx) but similarly slow sodium current inactivation and compete for their binding to sodium channels at receptor site-3. Characterization of the binding of 125I-labelled delta-ACTX-Hv1a to various sodium channels reveals a decrease in affinity for depolarized (0 mV; Kd=6.5 +/- 1.4 nm) vs.polarized (-55 mV; Kd=0.6 +/- 0.2 nm) rat brain synaptosomes. The increased Kd under depolarized conditions correlates with a 4.3-fold reduction in the association rate and a 1.8-increase in the dissociation rate. In comparison, Sc(alpha)Tx binding affinity decreased 33-fold under depolarized conditions due to a 48-fold reduction in the association rate.
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.
Nicholson, G.M. 2002, 'Member of Toxicon Editorial Board', Toxicon, vol. 40.
Graudins, A., Padula, M.P., Broady, K.W. & Nicholson, G.M. 2001, 'Red Back Spider (Latrodectus haselti) Antivenom Prevents the Toxicity of Widow Spider Venoms', Annals of Emergency Medicine, vol. 37, no. 2, pp. 154-160.
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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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Grolleau, F., Birinyi-Strachan, L.C., Stankiewicz, M., Wang, X., Nicholson, G.M., Pelhate, M. & Lapied, B. 2001, 'Electrophysiological Analysis of the Neurotoxic Action of a Funnel-Web Spider Toxin, d-atracotoxin-Hv1a on Insect Voltage-Gated Sodium Channels', Journal of Experimental Biology, vol. 204, pp. 711-721.
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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.
Rash, L.D., Birinyi-Strachan, L.C., Nicholson, G.M. & Hodgson, W.C. 2000, 'Neurotoxic activity of venom from the Australian Eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating', British Journal of Pharmacology, vol. 130, no. 8, pp. 1817-1824.
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Szeto, T., Wang, X., Smith, R., Connor, M., Christie, M., Nicholson, G.M. & King, G.F. 2000, 'Isolation of a Funnel-Web Spider Polypeptide with Homology to Mamba Intestinal Toxin 1 and the Embryonic Head Inducer Dickkopf-1', Toxicon, vol. 38, no. 3, pp. 429-442.
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Torres, A.M., De Plater, G.M., Doverskog, M., Birinyi-Strachan, L.C., Nicholson, G.M., Gallagher, C.H. & Kuchel, P.W. 2000, 'Defensin-like Peptide-1 from Platypus Venom: Member of a Class of Peptides with a Distinct Structural Fold', Biochemical Journal, vol. 348, no. 3, pp. 649-656.
The venom of the male Australian duck-billed platypus contains a family of four polypeptides of appox. 5 kDa, which are referred to as defensin-like peptides (DLPs). They are unique in that their amino acid sequences have no significant similarities to those of any known peptides; however, the tertiary structure of one of them, DLP-1, has recently been shown to be similar to b-defensin-12 and to the sodium neurotoxin peptide ShI (Stichodactyla helianthus neurotoxin I). Although DLPs are the major peptides in the platypus venom, little is known about their biological roles. In this study, we determined the three-dimensional structure of DLP-2 by NMR spectroscopy, with the aim of gaining insights into the natural function of the DLPs in platypus venom. The DLP-2 structure was found to incorporate a short helix that spans residues 9 12, and an antiparallel b-sheet defined by residues 15 18 and 37 40. The overall fold and cysteine-pairing pattern of DLP-2 were found to be similar to those of DLP-1, and hence b-defensin-12; however, the sequence similarities between the three molecules are relatively small. The distinct structural fold of the DLP-1, DLP-2, and b-defensin-12 is based upon several key residues that include six cysteines. DLP-3 and DLP-4 are also likely to be folded similarly since they have high sequence similarity with DLP-2. The DLPs, and b-defensin-12 may thus be grouped together into a class of polypeptide molecules which have a common or very similar global fold. The fact that the DLPs did not display antimicrobial, myotoxic, or cell-growth-promoting activities implies that the nature of the side chains in this group of peptides is likely to play an important role in defining the biological function(s).
Wang, X., Connor, M., Smith, R., Maciejewski, M.W., Howden, M.E., Nicholson, G.M., Christie, M. & King, G.F. 2000, 'Discovery and Characterisation of a Family of Insecticidal Neurotoxins with a Rare Vicinal Disulfide Bridge', Nature Structural Biology, vol. 7, no. 6, pp. 505-513.
Szeto, T., Birinyi-Strachan, L.C., Smith, R., Connor, M., Christie, M., King, G.F. & Nicholson, G.M. 2000, 'Isolation and pharmacological characterisation of delta-atracotoxin-Hv1b, a vertebrate selective sodium channel toxin', FEBS Letters, vol. 470, no. 3, pp. 293-299.
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delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.

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)