What's New in 2006

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April 2006 "Pain Relief Drugs from the Sea" [Format: streaming audio MP3].
Desley Blanch interviews Dr. Livett about his research and progress with the development of the cone shell analgesic, ACV1, from Conus victoriae. This interview was broadcast on Radio Australia's Innovations program, April 10, 2006. (Be patient as it takes a while to download).
Alternatively, for a printable transcript click here

For further information see Bruce Livett's Research on cone shell venom peptides for treatment of chronic pain conditions at the Department of Biochemistry and Molecular Biology and the Bio21 Institute for Innovation and Entrepreureship at the University of Melbourne.

  • For a free article on theories about pain, click here: Encyclopedia Britannica.

    Analgesic Component of Venom (ACV1) from Cone Snails :
    see Nature Science Update "Snail toxin could ease chronic pain" by Ingrid Holmes


    An "Internet Interview" with Bruce Livett: conducted in February 2001 about his scientific work with cone shells and conotoxins (and his interaction with other malacologists and shell collectors), is now available as a downloadable Adobe pdf file. This extensive Intervista web "interview" conducted by Eduardo Moreira for Callostoma was subsequently published (in condensed form) in American Conchologist Volume 30, Number 1, 2002, pp. 5 & 14.

    For a one-page description of Cone Shells and their Conotoxins click here

    Bruce Livett's more recent publications (1998-2006)

    Site Map of Cone Shells and Conotoxins HomePage

       


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    HAPPY NEW YEAR - and good shelling !!


    Conus victoriae, Broome, Western Australia. Collected by Sally Johnsen and Bruce Livett, 7 October 2006. Photographed by David Paul, in marine aquarium, Dept. Zoology, University of Melbourne

    31 December, 2006

      Some Newly Described Species of Conus

      Source: VISAYA

        See also:
      • Links to Images of Recent Conidae maintained by Ross Mayhew at Schooner Specimen Shells
      • "List of Species Described after 1999" compiled by Paul Kersten, in 'The Cone Collector #0', October 2006 (Editor, Antonio Monteiro), pp. 15-16.
      • "List of Recently Described Species (after 1999)" in 'The Cone Collector #1', January 2007 (Editor, Antonio Monteiro). The corrected list on p.19 contains in addition 3 fossil species from 2003 and an additional 4 species not listed in 'The Cone Collector #0'. On page 6-8, John Tucker from Great Rivers Field Station, Illinois Natural History Survey, has provided "A bibliography of cone shells described after 1999".
      • The above lists have been expanded in 'The Cone Collector #1', January 2007, to include alphabetic listings of "Species described from 1995 to 2000" pp. 20-23 and "Species described from 2000 to 20006" pp. 23-28. These listings are illustrated with photos of selected cones.

      VISAYA I
      Conus frausseni n. sp. Tenorio, M.J. & G.T. Poppe, 2004, Philippines
      Conus grohi n. sp. Tenorio, M.J. & G.T. Poppe, 2004, Philippines
      Conus terryni n. sp. Tenorio, M.J. & G.T. Poppe, 2004, Philippines

      VISAYA II
      Conus medoci sp. nov. Lorenz, F. Jr., 2004, Madagascar
      Conus chiapponorum sp. nov. Lorenz, F. Jr., 2004, Madagascar
      Conus vulcanus n. sp. Tenorio, M.J. & C.M.L. Afonso, 2004, Cape Verdes
      Conus claudiae n. sp. Tenorio, M.J. & C.M.L. Afonso, 2004, Cape Verdes
      Conus isabelarum n. sp. Tenorio, M.J. & C.M.L. Afonso, 2004, Cape Verdes
      Conus crioulus n. sp. Tenorio, M.J. & C.M.L. Afonso, 2004, Cape Verdes
      Conus suduirauti n. sp. Raybaudi Massilia, G., 2004, Philippines

      VISAYA IV
      Conus betulinus rufoluteus n. ssp. Bozzetti, L. & M. Ferrario, 2005, Madagascar

      VISAYA V
      Conus guidopoppei sp. nov. Raybaudi Massilia, L., 2005. Philippines

      Pain, Calcium channels, Prialt - and more

      Cao YQ. (2006) Voltage-gated calcium channels and pain. Pain. 126: 5-9. Epub 2006 Nov 7. Review.
      Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine, MO 63110, United States. caoy@morpheus.wustl.edu

      Introduction: Virtually all excitable cells express plasma membrane voltage-gated Ca2+ channels (VGCCs) that transduce electrical activity into intracellular biochemical signals. Membrane depolarization triggers the opening of VGCCs to allow rapid influx of extracellular Ca2+, which, in turn, regulates numerous physiological processes: the release of neurotransmitters and neuropeptides, neuronal excitability and plasticity, gene expression, development as well as cell survival and death (Tsien and Wheeler, 1999). Studies of Ca2+ currents in dorsal root ganglion (DRG) neurons date back to the early days of VGCC research and in 1985 led to the functional discovery of N-type Ca2+ channel (Nowycky et al., 1985). Remarkably, only twenty years later, the N-type Ca2+ channel blocker Prialt, also known as SNX111 or ziconotide, was approved in both United States and Europe to treat intractable pain. It is anticipated that additional treatment modalities will derive from further understanding of the contribution of multiple VGCCs to nociceptive processing, in both the normal and the disease setting.

      Conotoxin ImI used to characterize acetylcholine binding protein from Aplasia californica

      Hansen SB, Sulzenbacher G, Huxford T, Marchot P, Bourne Y, Taylor P. (2006) Structural Characterization of Agonist and Antagonist-Bound Acetylcholine-Binding Protein From Aplysia californica. J Mol Neurosci. 30: 101-102.
      Departments of Pharmacology; Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093-0636; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093-0636.

      Abstract: Nicotinic acetylcholine receptors (nAChRs) are well-characterized allosteric transmembrane proteins involved in the rapid gating of ions elicited by ACh. These receptors belong to the Cys-loop superfamily of ligand-gated ion channels, which also includes GABAA and GABAC, 5-HT3, and glycine receptors. The nAChRs are homo- or heteromeric pentamers of structurally related subunits that encompass an extracellular N-terminal ligand-binding domain, four transmembrane-spanning regions that form the ion channel, and an extended intracellular region between spans 3 and 4. Ligand binding triggers conformational changes that are transmitted to the transmembrane-spanning region, leading to gating and changes in membrane potential. The four transmembrane spans on each of the five subunits create a substantial region of hydrophobicity that precludes facile crystallization of this protein. However the freshwater snail, Lymnaea stagnalis, produces a soluble homopentameric protein, termed the ACh-binding protein (AChBP), which binds ACh (Smit et al., 2001). Its structure was determined recently (Brejc et al., 2001) at high resolution, revealing the structural scaffold for nAChR, and has become a functional and structural surrogate of the nAChR ligand-binding domain. We have characterized an AChBP from Aplysia californica and determined distinct ligand-binding properties when compared to those of L. stagnalis, including ligand specificity for the nAChR alpha7 subtype-specific alpha-conotoxin ImI (Hansen et al., 2004).

      alpha-Conotoxin analogues with high affinity

      Tsetlin VI, Kasheverov IE, Zhmak MN, Utkin YN, Vulfius CA, Smit AB, Bertrand D. (2006) alpha-Conotoxin Analogs With Enhanced Affinity for Nicotinic Receptors and Acetylcholine-Binding Proteins. J Mol Neurosci. 30: 77-78.
      Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia.

      Abstract: alpha-Conotoxins, neurotoxic peptides from poisonous Conus marine snails, can be subdivided into several groups targeting distinct subtypes of nicotinic acetylcholine receptors (nAChRs). Such alpha-conotoxins as, for example, GI, MI, or SIA potently block muscle-type nAChRs from muscles and from the electric organ of Torpedo ray, whereas others target distinct neuronal nAChRs: alpha-conotoxins ImI and PnIB block pentaoligomeric alpha7 nAChRs, and alpha-conotoxins MII or PnIA inhibit heteromeric nAChRs made of combinations of alpha3 or alpha6 subunits with beta2 subunit. alpha-Conotoxins interact with N-terminal extracellular ligand-binding domains of nAChRs and are indispensable tools for distinguishing various subtypes of AChRs at normal and pathological states. Although many alpha-conotoxins have been isolated from Conus venoms, there is still a great need in more potent and selective tools, which in principle can be obtained by design and synthesis of novel alpha-conotoxin analogs.

      Short alpha-neurotoxins bind to Torpedo receptor

      Mordvintsev DY, Polyak YL, Kuzmine DA, Levtsova OV, Tourleigh YV, Kasheverov IE. (2006) A Model for Short alpha-Neurotoxin Bound to Nicotinic Acetylcholine Receptor From Torpedo californica. J Mol Neurosci. 30: 71-72.
      Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, GSP-7, Moscow, Russia.

      Abstract: Short- and long-chain alpha-neurotoxins from snake venoms are potent blockers of nicotinic acetylcholine receptors (nAChRs). Short alpha-neurotoxins consist of 60-62 amino acid residues and include 4 disulfide bridges, whereas long alpha-neurotoxins have 66-75 residues and 5 disulfides. The spatial structure of these toxins is built by three loops, I-III "fingers," confined by four disulfide bridges; the fifth disulfide of long-chain alpha-neurotoxins is situated close to the tip of central loop II. An accurate knowledge of the mode of alpha-neurotoxin-nAChR interaction is important for rational design of new nAChR agonists and antagonists for medical purposes. Ideas on the topography of toxin-nAChR complexes were based until recently on nAChR interactions with selectively labeled alpha-neurotoxins, mutations in toxins, nAChR, or both. Recently, crystal structures have been solved for the Torpedo marmorata nAChR (4A[Unwin, 2005]) and for the acetylcholine-binding protein (AChBP) complexed with mollusk alpha-conotoxin (2.4 A[Celie et al., 2005]) or alpha-cobratoxin, long-chain alpha-neurotoxin (4 A [Bourne et al., 2005]). However, there were no angstrom-resolution models for complexes of shortchain alpha-neurotoxins. Here, we report the model of the Torpedo californica nAChR extracellular domain complexed to a short-chain alpha-neurotoxin II (NTII) from Naja oxiana cobra venom.

      delta-Conotoxin Am2766 from Conus amadis inhibits sodium channel inactivation

      Sarma SP, Kumar GS, Sudarslal S, Iengar P, Ramasamy P, Sikdar SK, Krishnan KS, Balaram P. (2005) Solution structure of delta-Am2766: a highly hydrophobic delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels. Chem Biodivers. 2: 535-556.
      Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka-560012, India. sidd@mbu.iisc.ernet.in

      Abstract: The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.

      Ziconotide - a new medication: Review

      Cohen AF, van Bronswijk H. (2006) [New medications; ziconotide] Ned Tijdschr Geneeskd. 150: 2427-2428. Dutch.
      Centre for Human Drug Research, Zernikedreef 10, 2333 CL Leiden. ac@chdr.nl

      Abstract: Ziconotide is a synthetic analogue of a peptide found in the poison of the marine snail Conus magus. Ziconotide blocks N-type calcium channels, which play an important role in the transmission of pain signals in the dorsal ganglia of the spinal cord. The drug is indicated for 'severe chronic pain' and is administered intrathecally.

    22 December, 2006

      Conus egg sacks on display

      Katie Grove-Velasquez from Maui Ocean Center, Hawaii and I have been exchanging information about our respective Conus species (Conus textile and Conus victoriae) over the last three months as we have each had the opportunity to observe them depositing egg sacks in an aquarium setting and both species have now hatched veligers. It has not been possible to photograph the veligers (now hidden in the sand at the bottom of the tank we surmise) but we were able to capture some nice photos of the cone shells depositing their egg sacks, thanks to our professional photographers, Tim Filler of F Stop Productions, Hawaii, and David Paul of University of Melbourne, Australia. So here they are - our cones, strutting their stuff for you to view. Enjoy !

      Conus textile
      (Katie Grove-Velasquez, Maui Ocean Center, Hawaii)
      Photography by Tim Filler of F Stop Productions, Hawaii, USA


      Conus victoriae
      (Bruce Livett, Melbourne, Australia)
      Photography by David Paul, Dept. Zoology, University of Melbourne, Australia


    21 December, 2006

      O-superfamily Conotoxins from C. betulinus, C. lividus and C. caracteristicus

      Zhangsun D, Luo S, Wu Y, Zhu X, Hu Y, Xie L. (2006) Novel O-superfamily Conotoxins Identified by cDNA Cloning From Three Vermivorous Conus Species. Chem Biol Drug Des. 68:256-265.
      Key Laboratory for Tropical Biology Resources (MOE), Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou Hainan 570228, China.

      Abstract: The O-superfamily of conotoxins includes several subfamilies with different pharmacological targets, all of which are voltage-gated ion channels and distributed widely in varied Conus species. The venom components from any Conus species are quite distinct from those of other species. Seven novel O-superfamily peptides were identified by cDNA cloning from the three vermivorous Conus species of C. betulinus, C. lividus and C. caracteristicus native to Hainan. They share three common signal sequences, and a conserved arrangement of cysteine residues (C-C-CC-C-C). Phylogenetic analysis of newly found conotoxins in this study and known homologue O-superfamily sequences from the other Conus species was performed systematically. Divergence and percentage identity of the amino acid sequences of the signal regions suggest that the novel conotoxins described in this investigation belong to the three broad clades: MSGL, ME-QK and MKLT, each of which has its own characteristic signature signal sequence and cysteine codon conservation. Relative to this work, it is noted that O-superfamily conotoxins are not well represented from vermivorous species. The elucidated cDNAs of these newly found vermivorous toxins would facilitate a better understanding for basic research and drug discovery.

    18 December, 2006

    16 December, 2006

      Conotoxins from Conus spurius

      Aguilar MB, Lopez-Vera E, de la Cotera EP, Falcon A, Olivera BM, Maillo M. (2006) I-conotoxins in vermivorous species of the West Atlantic: Peptide sr11a from Conus spurius. Peptides. 2006 Dec 11; [Epub ahead of print]
      Laboratorio de Neurofarmacologia Marina, Departamento de Neurobiologia Celular y Molecular, Instituto de Neurobiologia, Universidad Nacional Autonoma de Mexico, Campus Juriquilla, Queretaro 76230, Mexico.

      Abstract: Peptide sr11a was purified from the venom of Conus spurius, a vermivorous cone snail collected in the Yucatan Channel, in the Western Atlantic. Its primary structure was determined by automatic Edman degradation after reduction and alkylation. Its molecular mass, as determined by MALDI-TOF mass spectrometry (average mass 3650.77Da), confirmed the chemical data (calculated average mass, 3651.13Da). The sequence of peptide sr11a (CRTEGMSCgammagammaNQQCCWRSCCRGECEAPCRFGP&; gamma, gamma-carboxy-Glu; &, amidated C-terminus) shows eight Cys residues arranged in the pattern that defines the I-superfamily of conotoxins. Peptide sr11a contains two gamma-carboxy-Glu residues, a post-translational modification that has been found in other I-conotoxins from species that live in the West Pacific: r11e from the piscivorous Conus radiatus, and kappa-BtX from the vermivorous Conus betulinus. Peptide sr11a is the eighth I-conotoxin isolated from a Conus venom and the first I-conotoxin from a species from the Western Atlantic. Peptide sr11a produced stiffening of body, limbs and tail when injected intracranially into mice.

      Luna-Ramirez KS, Aguilar MB, Falcon A, Heimer de la Cotera EP, Olivera BM, Maillo M (2006). An O-conotoxin from the vermivorous Conus spurius active on mice and mollusks. Peptides. 2006 Dec 11; [Epub ahead of print]
      Laboratorio de Neurofarmacologia Marina, Departamento de Neurobiologia Celular y Molecular, Instituto de Neurobiologia, Universidad Nacional Autonoma de Mexico, Campus Juriquilla, Queretaro 76230, Mexico.

      Abstract: Here, we report the purification, amino acid sequence and a preliminary biological characterization of a peptide, sr7a, from the venom of Conus spurius, a vermivorous species collected in the Yucatan Channel, Mexico. The peptide consists of 32 amino acid residues (CLQFGSTCFLGDDDICCSGECFYSGGTFGICS&; &, amidated C-terminus) and contains six cysteines arranged in the pattern (C-C-CC-C-C) that characterizes the O-superfamily of conotoxins. This superfamily includes several pharmacological families (omega-, kappa-, muO-, delta- and gamma-conotoxins) that target Ca(2+), K(+), Na(+) and pacemaker voltage-gated ion channels. Compared with other O-conotoxins that were purified from venoms, this peptide displays sequence similarity with omega-SVIA (from Conus striatus), delta-TxVIA/B (from Conus textile), omega-CVID (from Conus catus) and kappa-PVIIA (from Conus purpurascens). At a dose of 250pmol, peptide sr7a elicited hyperactivity when injected intracranially into mice and produced paralysis when injected into the pedal muscle of freshwater snails, Pomacea paludosa, but it had no apparent effect after intramuscular injection into the limpet Patella opea or the freshwater fish Lebistes reticulatus.

      Conotoxin kappaA-SIVA from Conus striatus

      Kelley WP, Schulz JR, Jakubowski JA, Gilly WF, Sweedler JV. (2006) Two toxins from Conus striatus that individually induce tetanic paralysis. Biochemistry 45: 14212-14222.
      Department of Chemistry and The Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and Hopkins Marine Station of Stanford University, Department of Biological Sciences, Pacific Grove, California 93950

      Abstract: We describe structural properties and biological activities of two related O-glycosylated peptide toxins isolated from injected (milked) venom of Conus striatus, a piscivorous snail that captures prey by injecting a venom that induces a violent, spastic paralysis. One 30 amino acid toxin is identified as kappaA-SIVA (termed s4a here), and another 37 amino acid toxin, s4b, corresponds to a putative peptide encoded by a previously reported cDNA. We confirm the amino acid sequences and carry out structural analyses of both mature toxins using multiple mass spectrometric techniques. These include electrospray ionization ion-trap mass spectrometry and nanoelectrospray techniques for small volume samples, as well as matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis as a complementary method to assist in the determination of posttranslational modifications, including O-linked glycosylation. Physiological experiments indicate that both s4a and s4b induce intense repetitive firing of the frog neuromuscular junction, leading to a tetanic contracture in muscle fiber. These effects apparently involve modification of voltage-gated sodium channels in motor axons. Notably, application of either s4a or s4b alone mimics the biological effects of the whole injected venom on fish prey.

    15 December, 2006

      Peptide synthesis of conotoxins

      Brust A, Tickle AE. (2006) High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format. J Pept Sci. 2006 Dec 7; [Epub ahead of print]
      Xenome Ltd, 120 Meiers Road, Indooroopilly 4068, Australia.

      Abstract: Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides.Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO.This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of >95% purity.

      alpha-conotoxins, mini-M conotoxins, conophans, and gamma-hydroxyconophans.

      Franco A, Pisarewicz K, Moller C, Mora D, Fields GB, Mari F. (2006)Hyperhydroxylation: a new strategy for neuronal targeting by venomous marine molluscs. Prog Mol Subcell Biol. 43: 83-103.
      Department of Chemistry & Biochemistry, Centre of Excellence in Biomedical and Marine Biotechnology, Florida Atlantic University, 777 Glades Rd., Boca Raton, 33431 Florida, USA.

      Venomous marine molluscs belonging to the genus Conus (cone snails) utilize a unique neurochemical strategy to capture their prey. Their venom is composed of a complex mixture of highly modified peptides (conopeptides) that interact with a wide range of neuronal targets. In this chapter, we describe a set of modifications based upon the hydroxylation of polypeptidic chains that are defining within the neurochemical strategy used by cone snails to capture their prey. In particular, we present a differential hydroxylation strategy that affects the neuronal targeting of a new set of alpha-conotoxins, mini-M conotoxins, conophans, and gamma-hydroxyconophans. Differential hydroxylation, preferential hydroxylation and hyperhydroxylation have been observed in these conopeptide families as a means of augmenting the venom arsenal used by cone snails for neuronal targeting and prey capture.

    14 December, 2006

      Ziconotide for Severe Complex Regional Pain Syndrome Type 1

      Michael Stanton-Hicks, Leonardo Kapural (2006) An Effective Treatment of Severe Complex Regional Pain Syndrome Type 1 in a Child Using High Doses of Intrathecal Ziconotide. Journal of Pain and Symptom Management, 32:6:509-511 (December 2006)

      Ziegler D. (2006) Treatment of diabetic polyneuropathy: update 2006. Ann N Y Acad Sci. 1084:250-266.
      FRCPE, Deutsche Diabetes-Klinik, Deutsches Diabetes-Zentrum, Leibniz-Zentrum an der Heinrich-Heine-Universitat Dusseldorf, Auf'm Hennekamp 65, 40225 Dusseldorf, Germany. dan.ziegler@ddz.uni-duesseldorf.de.

      Abstract: At least one of four diabetic patients is affected by distal symmetric polyneuropathy (DSP), which represents a major health problem, as it may present with partly excruciating neuropathic pain and is responsible for substantial morbidity, increased mortality, and impaired quality of life. Treatment is based on four cornerstones: (a) causal treatment aimed at (near)-normoglycemia, (b) treatment based on pathogenetic mechanisms, (c) symptomatic treatment, and (d) avoidance of risk factors and complications. Recent experimental studies suggest a multifactorial pathogenesis of diabetic neuropathy. From the clinical point of view it is important to note that, on the basis of these pathogenetic mechanisms, therapeutic approaches could be derived, some of which are currently being evaluated in clinical trials. Among these agents only alpha-lipoic acid is available for treatment in several countries and epalrestat in Japan. Although several novel analgesic drugs, such as duloxetine and pregabalin, have recently been introduced into clinical practice, the pharmacological treatment of chronic painful diabetic neuropathy remains a challenge for the physician. Individual tolerability remains a major aspect in any treatment decision. Epidemiological data indicate that not only increased alcohol consumption but also the traditional cardiovascular risk factors, such as hypertension, smoking, and visceral obesity, play a role in development and progression of diabetic neuropathy and, hence, need to be prevented or treated.

    13 December, 2006

      Sea snail key to future of pain relief

      Dr Jenny Ekberg and colleagues from the University of Queensland (see entry below for 29 October) announced in PNAS that they had identified a toxin (uO-Conotoxin MrVIB) in the venom of Conus marmoreus that targets Nav1.8 sensory neuron specific sodium channels and which in animal tests is effective in providing pain relief. Intrathecal administration of MrVIB into rats selectively blocked sodium channels in sensory neurons and prevented chronic pain behaviour without motor deficits.

      Ekberg, J., Jayamanne, A., Vaughan, C.W., Aslan, S., Thomas, L.,, Mould, J., Drinkwater, R., Baker, M.D., Abrahamsen, B., Wood, J.N., Adams, D.J., Christie, M.J., Lewis RJ.(2006) {micro}O-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits. Proc Natl Acad Sci U S A. 103: 17030-17035

    12 December, 2006

      Pausing Pain with Poison

      Venom-based pain medication gives woman a new lease on life
      http://www.clevelandclinic.org/painmanagement/newsletter/newsletter_sum05.pdf

      Transcript: Cheryl McDowell had just remarried after an unhappy first marriage and was looking forward to a new life with her “prince charming,” Johnnie. Then, two weeks after their wedding, she thought her life was over.

      She was suddenly immobilized with deep pain In her legs. “It was unbearable,” McDowell recalls. “I couldn’t get out of bed.”McDowell was diagnosed with Dercum’s Disease, a rare disease that causes small, fatty tumors to grow in the trunk, arms and legs. The tumors press on nerves, causing chronic – and often excruciating – pain.

      In the three years since then, McDowell had six surgeries to remove the tumors, but new ones kept growing back.

      “We haven’t had a marriage; my husband has been a full-time caregiver,” McDowell says. “Sometimes I get really depressed and ask him why he stays with me. He says ‘Honey, I’m not going anywhere.’ He’s just wonderful.” The couple slept on the sofa together for nearly a year, with her head in his lap. The pain in her legs was so intense that she couldn’t stand to have sheets or blankets touching them.

      McDowell’s doctors at Wright-Patterson Air Force Base Medical Center in Dayton, Ohio, tried a host of different pain medications such as Vicodin and Percocet, but McDowell found no relief. She then sought help from a local pain management clinic, where she was prescribed methadone and morphine.

      The medications did not relieve her pain, and it was suspected that they caused McDowell to suffer grave health consequences on two occasions. Both times, she nearly died.

      Miracle in Cleveland

      In December 2004, McDowell asked her doctors to refer her to The Cleveland Clinic, where she met Emad Mikhail, M.D., Medical Director of the Pain Management Center at Euclid Hospital, a Cleveland Clinic Health System hospital.

      Dr. Mikhail suggested a brand-new drug just approved by the FDA that month. McDowell was the first patient in Ohio to try Ziconotide – more commonly known as Prialt – made from a component of Conus magus

      Marine snail venom. The venom paralyses the snail’s prey by blocking nerve signals that normally transmit pain signals to the brain.

      Prialt works in much the same way. Delivered to the spinal cord via a pump that is surgically implanted under the skin in the abdomen, the drug blocks the brain’s reception of pain signals. “Unlike narcotics and opioids, Prialt is nonaddictive, and patients don’t develop a tolerance for it over time,” explains Dr. Mikhail, “So we don’t have to keep increasing the dosage for it to remain effective.”

      Dr. Mikhail believes that Prialt could make a big difference for people with cancer pain and other chronic pain, such as back pain caused by bulging or herniated disks.

      McDowell was the first patient in Ohio to try Ziconotide – more commonly known as Prialt

      Ziconotide / Prialt

      Made from a component of Conus magus marine snail venom. The venom paralyzes the snail’s prey by blocking nerve signals that normally transmit pain signals to the brain.

      McDowell is thrilled with the results.Within a few days of the trial, the Prialt reduced her pain from eight to zero on the pain scale (10 being the worst), and with no side effects. Dr. Mikhail implanted a permanent pump in her abdomen in March, and he refills it for her about every two or three months. She’s eliminated all her other pain medications.

      “It’s a miracle,” she says. “I’m finally able to get out in the world and do things. We’re planning a vacation for the first time since we’ve been married. I’ve planted a garden for the first time, too. And my daughter is happy because we can spend more time together.”

    Follow these links to view pain management videos:

    10 December, 2006

      'Conus furiosus' makes its mark

      Sandra Rodriguez Barron has included a quest for 'Conus furiosus' in the plot of her recent novel "The Heiress of Water" HarperCollins Publishers, New York. 2006. ISBN13: 9780061142819; ISBN: 0061142816; Imprint: Rayo ; On Sale: 9/5/2006; Format: Trade PB; Trimsize: 5 5/16 x 8; Pages: 320; $13.95; Ages: 12 and Up

      Synopsis: When young Monica Winters Borrero loses her luminous mother in an accident at sea, she is exiled from the tropical paradise that was her home. Grieving and cut off from a life among El Salvador's elite, Monica and her American father move to Connecticut, vowing never to look back.

      Years later, an intriguing stranger, who has endured a terrible loss of his own, enters Monica's life, bearing an unusual request. Monica is propelled back to her lost world, retracing the shadowy last days of her mother, a marine scientist who had been on the brink of understanding the therapeutic applications of a rare, venomous sea creature. Now, her research is being corrupted by a secret clinic that claims the power to restore consciousness to the comatose.

      What Monica discovers will shatter the family's delicate truce with the past, and compel everyone involved to challenge their deepest notions of what it means to be alive. Atmospheric, thought-provoking, and timely. The Heiress of Water is a stunning parable of paradise lost and found.

      Text Excerpt (edited): Chapter One. Bivalves El Salvador, 1981.
      Monica concluded, in the manner of a miniature research assistant, by asserting that this particular tide pool didn't contain anything out of the ordinary, a circumstance that she was expected to report to her mother. "Nothing unusual," she said. "But one day, we'll find the Conus furiosus, even if it's the last one in the world. We'll find it, Mami, you'll see."

      Click here and scroll down to read the full Text Excerpt from Chapter One, plus Critical Praise, and Reader Reviews for this book. For further information about the book, including where to buy the book online click here. For an interesting interview with the author and insight into the influence of CONIDAE on the plot click here.

    9 December, 2006

      mu-Conotoxin TIIIA from Conus tulipa

      Lewis RJ, Schroeder CI, Ekberg J, Nielsen KJ, Loughnan M, Thomas L, Adams DA, Drinkwater R, Adams DJ, Alewood PF (2007) Isolation and structure-activity of {micro}-conotoxin TIIIA, a potent inhibitor of TTX-sensitive voltage-gated sodium channels. Mol Pharmacol. 71:(3) 676-
      University of Queensland.

      Abstract: micro-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels (VGSCs) during prey capture. Using PCR techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new micro-conotoxin TIIIA. A (125)I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of C. striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and < 35% homology to other micro-conotoxins. TIIIA potently displaced (3)H-STX and (125)I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, which were critical for high affinity binding to TTX-sensitive Na(+) channels. Surprisingly, [Glu15Ala]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50 15 pM vs 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and 1.4 over Nav1.3, 1.5, 1.7 and 1.8 expressed in Xenopus oocytes and was without effect on rat dorsal root ganglion neuron Na(+) current. (1)H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for micro-conotoxin PIIIA. TIIIA and analogues provide new biochemical probes as well insights into the structure-activity of micro-conotoxins.

      Bromotryptophan-containing Conotoxin

      Nair SS, Nilsson CL, Emmett MR, Schaub TM, Gowd KH, Thakur SS, Krishnan KS, Balaram P, Marshall AG (2006). De Novo Sequencing and Disulfide Mapping of a Bromotryptophan-Containing Conotoxin by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal Chem. 78: 8082-8088.
      National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India, and Tata Institute of Fundamental Research, Mumbai- 400005, India.

      Abstract: T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.

      O-superfamily conotoxins from Conus miles

      Luo S, Zhangsun D, Feng J, Wu Y, Zhu X, Hu Y.(2007) Diversity of the O-superfamily conotoxins from Conus miles. J Pept Sci. 13: 44-53.
      Key Laboratory for Tropical Biological Resources, (MOE); Ocean College, Center for Experimental Biotechnology, Hainan University; Haikou Hainan, 570228 China.

      Abstract: Conopeptides display prominent features of hypervariability and high selectivity of large gene families that mediate interactions between organisms. Remarkable sequence diversity of O-superfamily conotoxins was found in a worm-hunting cone snail Conus miles. Five novel cDNA sequences encoding O-superfamily precursor peptides were identified in C. miles native to Hainan by RT-PCR and 3'-RACE. They share the common cysteine pattern of the O-superfamily conotoxin (C--C--CC--C--C, with three disulfide bridges). The predicted peptides consist of 27-33 amino acids. We then performed a phylogenetic analysis of the new and published homologue sequences from C. miles and the other Conus species. Sequence divergence (%) and residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were analyzed. Percentage divergence of the amino acid sequences of the prepro region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical with to highly divergent from inter- and intra-species. Despite the O-superfamily being a large and diverse group of peptides, widely distributed in the venom ducts of all major feeding types of Conus and discovered in several Conus species, it was for the first time that the newly found five O-superfamily peptides in this research came from the vermivorous C. miles. So far, conotoxins of the O-superfamily whose properties have been characterized are from piscivorous and molluscivorous Conus species, and their amino acid sequences and mode of action have been discussed in detail. The elucidated cDNAs of the five toxins are new and of importance and should attract the interest of researchers in the field, which would pave the way for a better understanding of the relationship of their structure and function. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

      Conotoxin MII from Conus magus: Flourescent probe

      Vishwanath VA, McIntosh JM (2006) Synthesis of Fluorescent Analogs of alpha-Conotoxin MII. Bioconjug Chem. 17: 1612-1617.
      Interdepartmental Program in Neuroscience and Departments of Psychiatry and Biology, University of Utah, Salt Lake City, Utah 84112.

      Abstract: alpha-Conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3alpha2 and rat chimeric alpha6/alpha3alpha2alpha3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.

    7 December, 2006

      Acceleration of development of the oral version of ACV1 for neuropathic pain

      Dr. Roland Scollay, CEO of Metabolic Pharmaceuticals, said "proceeds from the (A$10.5 million) share placement will be used to accelerate the development of the recently announced oral version of the Company's neuropathic pain drug, ACV1 (synthetic conotoxin Vc1.1), and to actively progress its Oral Peptide Delivery Platform - the technology platform used to convert ACV1 from an injectable to an oral drug.The proceeds will be used, in part, to determine to what extent this technology can be applied to other currently injected peptide drugs on the market or in develoment."
      For further details view or download the ASX announcement of 7 December.

    30 November, 2006

      Clinical trials with ACV1 to treat sciatica

      Sciatic pain trial: The Great Barrier Reef could hold the key to curing sciatic pain. Australian scientists have developed a drug, called ACV1, using an extract from a venomous sea snail (Conus victoriae). Researchers are looking for participants who have suffered sciatic pain for more than three months. To take part in this Phase II Clinical Trial call Adelaide University: +61-8-8222 5422.

      Clinical trials with ACV1 are being conducted at the Royal Adelaide Hospital, South Australia, by CMAX - Clinical Studies Unit (A Division of IDT Australia Ltd), under the direction of Prof. Guy Ludbrook, Professor and Head of Anaesthesia and Intensive Care, University of Adelaide.

      Clicking on the following link activates a video segment from the Channel 7 News in which Prof. Ludbrook describes the Clinical Trials to treat sciatica with ACV1 at Royal Adelaide Hospital.

      If you experience trouble with this link, paste the following URL directly into your browser.
      http://cosmosweb02.bcst.scd.yahoo.com/scp_v3/viewer/index.php?pid=16471&rn=248153&cl=1318724&ch=248154

      (Wait for any advertisements that may precede the ACV1 news item)


      • In addition, this report from Roger Highfield from the Daily Telegraph, London. You may recall that Roger wrote about the cone shell venom studies in his book the "Science of Harry Potter". Now he has featured the recent preclinical work on ACV1 in this report "Snails venom signals a pain free future". A powerful toxin gives new hope to the millions who suffer from nerve pain, reports Roger Highfield in his regular science column in the Daily Telegraph, London.

      • The following excerpt is from Medical News Today. "A New Target For Painkillers: Snail Toxins Show The Way" : A brand new approach to treating severe nerve pain - by aiming drugs at a previously unrecognized molecular target - has been discovered by University of Utah scientists who study the venoms of deadly, sea-dwelling cone snails...

    29 November, 2006

      ACV1 Clinical Trials Update

      Metabolic's neuropathic pain drug, ACV1 - additional clinical studies reveal:

      • First patients have been treated with ACV1 in the Phase 2A sciatica trial
      • A separate trial to test safety of a higher dose level in healthy volunteers has commenced – important information for regulatory authorities and potential licensing partners
      • The three clinical trials for ACV1 in progress or final planning are progressing on time

      24 November, 2006

        Excitatory short kappaA-conotoxins from C. purpurascens

        Teichert RW, Jacobsen R, Terlau H, Yoshikami D, Olivera BM.(2006) Discovery and characterization of the short kappaA-conotoxins: A novel subfamily of excitatory conotoxins. Toxicon. 2006 Oct 14; [Epub ahead of print]
        Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

        Abstract: We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.

      23 November, 2006

        ACV1 PreClinical Update

        Metabolic's neuropathic pain drug ACV1 - additional preclinical studies reveal greater potential This ASX Announcement by Metabolic Pharmaceuticals reports that the:

        • Latest oral version of ACV1 works as well as the injected version in new animal studies
        • Provides proof-of-concept for Metabolic's Oral Peptide Delivery Platform
        • An independent study provides new knowledge about how ACV1 works and reveal the likely biochemical target for ACV1

        I-conotoxins revisisted

        Mondal S, Bhavna R, Mohan Babu R, Ramakumar S. (2006) Pseudo amino acid composition and multi-class support vector machines approach for conotoxin superfamily classification. J Theor Biol. 243:252-260.
        Department of Physics, Indian Institute of Science, Bangalore 560 012, India.

        Abstract: Conotoxins are disulfide rich small peptides that target a broad spectrum of ion-channels and neuronal receptors. They offer promising avenues in the treatment of chronic pain, epilepsy and cardiovascular diseases. Assignment of newly sequenced mature conotoxins into appropriate superfamilies using a computational approach could provide valuable preliminary information on the biological and pharmacological functions of the toxins. However, creation of protein sequence patterns for the reliable identification and classification of new conotoxin sequences may not be effective due to the hypervariability of mature toxins. With the aim of formulating an in silico approach for the classification of conotoxins into superfamilies, we have incorporated the concept of pseudo-amino acid composition to represent a peptide in a mathematical framework that includes the sequence-order effect along with conventional amino acid composition. The polarity index attribute, which encodes information such as residue surface buriability, polarity, and hydropathy, was used to store the sequence-order effect. Several methods like BLAST, ISort (Intimate Sorting) predictor, least Hamming distance algorithm, least Euclidean distance algorithm and multi-class support vector machines (SVMs), were explored for superfamily identification. The SVMs outperform other methods providing an overall accuracy of 88.1% for all correct predictions with generalized squared correlation of 0.75 using jackknife cross-validation test for A, M, O and T superfamilies and a negative set consisting of short cysteine rich sequences from different eukaryotes having diverse functions. The computed sensitivity and specificity for the superfamilies were found to be in the range of 84.0-94.1% and 80.0-95.5%, respectively, attesting to the efficacy of multi-class SVMs for the successful in silico classification of the conotoxins into their superfamilies.

        Mondal S, Babu RM, Bhavna R, Ramakumar S. (2006) I-conotoxin superfamily revisited. J Pept Sci. 12:679-685.
        Department of Physics, Indian Institute of Science, Bangalore 560012, India.

        Abstract: The I-conotoxin superfamily (I-Ctx) is known to have four disulfide bonds with the cysteine arrangement C-C-CC-CC-C-C, and the members inhibit or modify ion channels of nerve cells. Recently, Olivera and co-workers (FEBS J. 2005; 272: 4178-4188) have suggested that the previously described I-Ctx should now be divided into two different gene superfamilies, namely, I(1) and I(2), in view of their having two different types of signal peptides and exhibiting distinct functions. We have revisited the 28 entries presently grouped as I-Ctx in UniProt Swiss-Prot knowledgebase, and on the basis of in silico analysis have divided them into I(1) and I(2) superfamilies. The sequence analysis has provided a framework for in silico annotation enabling us to carry out computer-based functional characterization of the UniProtKB/TrEMBL entry Q59AA4 from Conus miles and to predict it as a member of the I(2) superfamily. Furthermore, we have predicted the mature toxin of this entry and have proposed that it may be an inhibitor of voltage-gated potassium channels. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

        Mondal S, Vijayan R, Shichina K, Babu RM, Ramakumar S. (2005) I-superfamily conotoxins: sequence and structure analysis. In Silico Biol. 5:557-571.
        Department of Physics, Indian Institute of Science, Bangalore 560 012, India.

        Abstract: I-superfamily conotoxins have four-disulfide bonds with cysteine arrangement C-C-CC-CC-C-C, and they inhibit or modify ion channels of nerve cells. They have been characterized only recently and are relatively less well studied compared to other superfamily conotoxins. We have detected selective and sensitive sequence pattern for I-superfamily conotoxins. The availability of sequence pattern should be useful in protein family classification and functional annotation. We have built by homology modeling, a theoretical structural 3D model of ViTx from Conus virgo, a typical member of I-superfamily conotoxins. The modeling was based on the available 3D structure of Janus-atracotoxin-Hv1c of Janus-atracotoxin family whose members have been suggested as possible biopesticides. A study comparing the theoretically modeled structure of ViTx, with experimentally determined structures of other toxins, which share functional similarity with ViTx, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels.

      15 November, 2006

        alpha-conotoxin RgIA from Conus regius interacts with alpha9/alpha10 nAChRs and suppresses pain

        Snail toxin may help nerve pain relief: Associated Press, November 14 - Toxins from cone snail venom may help point the way to better relief of severe nerve pain in people, researchers report. Results were promising in tests on rats with a type of nerve pain similar to sciatica, according to researchers.

        Michelle Vincler, Shannon Wittenauer, Renee Parker, Michael Ellison, Baldomero M. Olivera, and J. Michael McIntosh (2006) Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. PNAS 103: 17880-17884
        *Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157; and Departments of Psychiatry, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112

        Abstract: alpha10 nicotinic acetylcholine receptors (nAChRs) have been identified in a variety of tissues including lymphocytes and dorsal root ganglia; except in the case of the auditory system, the function of alpha10 nAChRs is not known. Here we show that selective block (rather than stimulation) of alpha10 nAChRs is analgesic in an animal model of nerve injury pain. In addition, blockade of this nAChR subtype reduces the number of choline acetyltransferase-positive cells, macrophages, and lymphocytes at the site of injury. Chronic neuropathic pain is estimated to affect up to 8% of the world's population; the numerous analgesic compounds currently available are largely ineffective and act through a small number of pharmacological mechanisms. Our findings not only suggest a molecular mechanism for the treatment of neuropathic pain but also demonstrate the involvement of alpha10 nAChRs in the pathophysiology of peripheral nerve injury.

        Opoiorphin, a natural painkiller in saliva

        Natural chemical, "beats morphine": BBC (UK), November 14 - The human body produces a natural painkiller several times more potent than morphine, research suggests. When given to rats, the chemical, called opiorphin, was able to curb pain at much lower concentration than the powerful painkiller morphine.

        Anne Wisner, Evelyne Dufour, Michaël Messaoudi, Amine Nejdi, Audrey Marcel, Marie-Noelle Ungeheuer, and Catherine Rougeot (2006) Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. PNAS 103: 17979-17984

        Abstract: Mammalian zinc ectopeptidases play important roles in turning off neural and hormonal peptide signals at the cell surface, notably those processing sensory information. We report here the discovery of a previously uncharacterized physiological inhibitor of enkephalin-inactivating zinc ectopeptidases in humans, which we have named Opiorphin. It is a QRFSR peptide that inhibits two enkephalin-catabolizing ectoenzymes, human neutral ecto-endopeptidase, hNEP (EC 3.4.24.11), and human ecto-aminopeptidase, hAP-N (EC 3.4.11.2). Opiorphin displays potent analgesic activity in chemical and mechanical pain models by activating endogenous opioid-dependent transmission. Its function is closely related to the rat sialorphin peptide, which is an inhibitor of pain perception and acts by potentiating endogenous µ- and delta-opioid receptor-dependent enkephalinergic pathways. Here we demonstrate the functional specificity in vivo of human Opiorphin. The pain-suppressive potency of Opiorphin is as effective as morphine in the behavioral rat model of acute mechanical pain, the pin-pain test. Thus, our discovery of Opiorphin is extremely exciting from a physiological point of view in the context of endogenous opioidergic pathways, notably in modulating mood-related states and pain sensation. Furthermore, because of its in vivo properties, Opiorphin may have therapeutic implications.

      6 November, 2006

        alpha3/alpha6beta2* nAChRs identified using (125)I-alpha-conotoxinMII

        Bordia, T., Parameswaran, N., Fan, H., Langston, J.W., McIntosh, J.M. and Quik, M. (2006) Partial recovery of striatal nicotinic receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys with chronic oral nicotine. J Pharmacol Exp Ther. 2006 Oct;319(1):285-92. Epub 2006 Jul 12.
        The Parkinson's Institute, 1170 Morse Ave, Sunnyvale, CA 94089-1605, USA.

        Abstract: Recent studies in nonhuman primates show that chronic nicotine treatment protects against nigrostriatal degeneration, with a partial restoration of neurochemical and functional measures in the striatum. The present studies were done to determine whether long-term nicotine treatment also protected against striatal nicotinic receptor (nAChR) losses after nigrostriatal damage. Monkeys were administered nicotine in the drinking water for 6 months and subsequently lesioned with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over several months while nicotine was continued. (125)I-Epibatidine, [(125)I]5-[(125)I]iodo-3(2(S)-azetidinylmethoxy)-pyridine (A85380), and (125)I-alpha-conotoxinMII autoradiography was performed to evaluate changes in alpha4beta2* and alpha3/alpha6beta2* nAChRs, the major striatal subtypes. Nicotine treatment increased alpha4beta2* nAChRs by > or =50% in striatum of both unlesioned and lesioned animals. This increase in alpha4beta2* nAChRs was significantly greater in lesioned compared with unlesioned monkey striatum. Chronic nicotine treatment led to a small decrease in alpha3/alpha6beta2* nAChR subtypes. The decline in alpha3/alpha6beta2* subtypes, defined using alpha-conotoxinMII-sensitive (125)I-epibatidine or [(125)I]A85380 binding, was significantly smaller in striatum of nicotine-treated lesioned monkeys compared with unlesioned monkeys. This difference was not observed for alpha3/alpha6beta2* nAChRs identified using (125)I-alpha-conotoxinMII. These data suggest that there are at least two striatal alpha3/alpha6beta2* subtypes that are differentially affected by chronic nicotine treatment in lesioned animals. In addition, the results showing an improvement in striatal alpha4beta2* and select alpha3/alpha6beta2* nAChR subtypes, combined with previous work, demonstrate that chronic nicotine treatment restores and/or protects against the loss of multiple molecular markers after nigrostriatal damage. Such findings suggest that nicotine or nicotinic agonists may be of therapeutic value in Parkinson's disease.

      29 October, 2006

        muConotoxin MrVIB from Conus marmoreus selectively blocks sodium channels on sensory neurons and chronic pain behavior

        Ekberg, J., Jayamanne, A., Vaughan, C.W., Aslan, S., Thomas, L.,, Mould, J., Drinkwater, R., Baker, M.D., Abrahamsen, B., Wood, J.N., Adams, D.J., Christie, M.J., Lewis RJ.(2006) {micro}O-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits. Proc Natl Acad Sci U S A. 103: 17030-17035
        Institute for Molecular Bioscience and School of Biomedical Sciences, University of Queensland, QLD 4072, Australia; Pain Management Research Institute and Kolling Institute, Northern Clinical School, University of Sydney at Royal North Shore Hospital, St Leonards, NSW 2065, Australia.

        Abstract: The tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Nav1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that microO-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Nav1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetrodotoxin-resistant current characteristic of Nav1.8 but not Nav1.9 or tetrodotoxin-sensitive VGSC currents. MrVIB blocked human Nav1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03-3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Nav1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.

        Novel muO-conotoxin lt7a, from Conus litteratus

        Pi, C., Liu, J., Wang, L., Jiang, X., Liu, Y., Peng, C., Chen, S. and Xu, A. (2006) Soluble expression, purification and functional identification of a disulfide-rich conotoxin derived from Conus litteratus. J Biotechnol. 2006 Sep 27; [Epub ahead of print]
        State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Therapeutic Functional Genes, The Open Laboratory for Marine Functional Genomics, of State High-Tech Development Program, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingangxi Road, Guangzhou 510275, People's Republic of China.

        Abstract: Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Usually, the analogue of natural conotoxins is produced by means of chemical synthesis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. By fused with thioredoxin and His tag, a novel O-superfamily conotoxin lt7a was successfully expressed in Escherichia coli and purified, resulting in a high yield of recombinant lt7a about 6mg/l. The purity of target protein is up to 95% as identified by HPLC results. Whole cell patch-clamp recording revealed that the new conotoxin blocked voltage-sensitive sodium channels in rat dorsal root ganglion neurons, indicating it might be a novel muO-conotoxin.

        Disulfide bond rearrangement during synthesis of alpha-Conotoxin GI

        Szabo, I., Schlosser, G., Hudecz, F., Mezo, G. (2006). Disulfide bond rearrangement during regioselective oxidation in PhS(O)Ph/CH(3)SiCl(3) mixture for the synthesis of alpha-conotoxin GI. Biopolymers. 2006 Oct 24.; [Epub ahead of print]
        Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos L. University, Budapest, Hungary.

        Abstract: Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of Acm and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We had to conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds lead up to the appropriate compound in case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the non-native disulfide isomer. (c) 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2006.

        Omega conotoxins for pain treatment: Review

        Schroeder CI, Doering CJ, Zamponi GW, Lewis RJ. N-type calcium channel blockers: novel therapeutics for the treatment of pain. Med Chem. 2006 Sep;2(5):535-43.
        Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia.

        Abstract: Highly selective Ca(v)2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (omega-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Ca(v)2.2 calcium channel have met with varied success. This review focuses on the properties of the Ca(v)2.2 calcium channel in different pain states, the action of omega-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Ca(v)2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.

      28 October, 2006

        Bromotryptophan, a post-translational modification of conotoxins, is a feature of many marine species

        Bittner, S., Scherzer, R. and Harlev, E. (2006) The five bromotryptophans. [Review] Amino Acids. 2006 Oct 12; [Epub ahead of print]
        Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

        Abstract: The five regioisomeric bromotryptophans (BrTrps) play an important role in the life of sponges and lower marine invertebrates. These bromo-amino acids, which are formed by post-translational modifications, are not found in nature in their free state, but rather are involved in more complex structures. Any of the BrTrps can be part of a peptide, a cyclic peptide, an indole alkaloid, an ergot alkaloid, a macrocycle and others. The present review covers the synthesis, physical and spectroscopic properties of the five BrTrps. It also describes the many exiting pharmacological and biological activities played by the BrTrps and by various secondary metabolites containing brominated tryptophan moieties. Of special interest are cyclic peptides containing the 2-BrTrp unit, which were isolated from marine sponges e.g. konbamide, orbiculamide A, the various keramamides, jaspamide eusynstyelamide and more. Important families of non-cyclic peptides containing the 6-BrTrp, include the styelins, the conotoxins, the cathelicidins and several constrained macrocyclic peptides. Many marine secondary BrTrp-containing, non-peptidic metabolites also display a remarkable spectrum of bioactivities, which can be harnessed for therapeutic and other purposes. Examples are: barettin, bromotryptanthrin, tetraacetyl clionamide, cyclocinamide A, clavicipitic acid, various brominated beta-carbolines. In this review we have presented the various synthetic routes leading to the preparation of the five BrTrps and many of its derivatives. Also, we have introduced the reader to many synthetic routes leading to BrTrp-containing non-peptidic natural products. Although the functional role of the various compounds in the human body is only poorly understood, its effects were extensively studied. Almost all of these compounds exhibit important therapeutic properties e.g. antifungal, antimicrobial, antihelmintic, insecticidal ichthyotoxic and anticancer activity. In the present review attempts have been made to provide synopsis, synthesis and symbiosis of chemical and biological actions, which may provide future guidance and facilitate further research in this area.

        Development of omega-conotoxins GVIA, MVIIA, CVID for the treatment of chronic pain

        Schroeder, C.I., Doering, C.J., Zamponi, G.W. and Lewis, R.J. (2006) N-type calcium channel blockers: novel therapeutics for the treatment of pain. Med Chem. 2: 535-543.
        Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia.

        Abstract: Highly selective Ca(v)2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (omega-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Ca(v)2.2 calcium channel have met with varied success. This review focuses on the properties of the Ca(v)2.2 calcium channel in different pain states, the action of omega-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Ca(v)2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.

      28 October, 2006

        Redox-active agents influence oxidative folding of conotoxins GI, PVIIA and r11a

        Buczek, O., Green, B.R. and Bulaj, G. (2006) Albumin is a redox-active crowding agent that promotes oxidative folding of cysteine-rich peptides. Biopolymers. 2006 Oct 23; [Epub ahead of print]
        Department of Biology, University of Utah, Salt Lake City, Utah 84112.

        Abstract: Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin and polysaccharides influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13-residues (1.4 kDa) to 58-residues (6.5 kDa): conotoxins: GI, PVIIA, r11a, and bovine pancreatic trypsin inhibitor. Our results indicate that the excluded volume effect does not significantly alter the folding rates nor equilibria for these peptides. In stark contrast, folding reactions were dramatically accelerated, when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone was as effective as glutathione in promoting the oxidative folding of GI conotoxin at concentrations typically found in the ER. To best of our knowledge, this is the first report and quantitative characterization of oxidative folding of peptides mediated by other than thioredoxinbased protein disulfide bonds. Our work raises a possibility that concurrent secretory and ER resident proteins may influence the oxidative folding of small, cysteine-rich peptides not as crowding agents, but as redox-active factors. (c) 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2006.

        Ziconotide a treatment option for neuraxial anaesthesia.

        Schug, S.A., Saunders, D., Kurowski, I. and Paech, M.J. (2006) Neuraxial drug administration : a review of treatment options for anaesthesia and analgesia. CNS Drugs. 20: 917-933.
        Pharmacology Unit, School of Medicine and Pharmacology, UWA Anaesthesia, University of Western Australia, Perth, Western Australia, AustraliaDepartment of Anaesthesia and Pain Medicine, Royal Perth Hospital, Perth, Western Australia, Australia.

        Abstract: Neuraxial drug administration describes techniques that deliver drugs in close proximity to the spinal cord, i.e. intrathecally into the CSF or epidurally into the fatty tissues surrounding the dura, by injection or infusion. This approach was initially developed in the form of spinal anaesthesia over 100 years ago. Since then, neuraxial drug administration has evolved and now includes a wide range of techniques to administer a large number of different drugs to provide anaesthesia, but also analgesia and treatment of spasticity in a variety of acute and chronic settings.This review concentrates on the pharmacological agents used and the clinical basis behind currently utilised approaches to neuraxial drug administration. With regard to local anaesthetics, the main focus is on the development of the enantiomer-specific compounds ropivacaine and levobupivacaine, which provide similar efficacy to bupivacaine with a reduced risk of severe cardiotoxicity. Opioids are the other group of drugs widely used neuraxially, in particular to provide analgesia alone or more commonly in combination with other agents. The physicochemical properties of the various opioids explain the main differences in efficacy and safety between these drugs when used intrathecally, of which morphine, fentanyl and sufentanil are most commonly used. Another group of drugs including clonidine, dexmedetomidine and epinephrine (adrenaline) provide neuraxial analgesia via alpha-adrenergic receptors and are used mainly as adjuvants to local anaesthetics and opioids. Furthermore, intrathecal baclofen is in routine clinical use to treat spasticity in a number of neurological conditions.Beside these established approaches, a wide range of other drugs have been assessed for neuraxial administration to provide analgesia; however, most are in various early stages of investigation and are not used routinely. These drugs include neostigmine, ketamine, midazolam and adenosine, and the conotoxin ziconotide. The latter is possibly the most unusual compound here; it has recently gained registration for intrathecal use in specific chronic pain conditions.

      27 October, 2006

        A synthetic cyclic version of Conotoxin MrIA, from Conus marmoreus, retains biological activity

        Lovelace, E.S., Armishaw, C.J., Colgrave, M.L., Wahlstrom, M.E., Alewood, P.F., Daly, N.L. and Craik, D.J. (2006) Cyclic MrIA: A Stable and Potent Cyclic Conotoxin with a Novel Topological Fold that Targets the Norepinephrine Transporter. J Med Chem. 2006 Nov 2;49(22):6561-8.
        Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.

        Abstract: Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally hampered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.

        Craik, D. (2006) Animal, plant and microbial toxins - 15th world congress. (Meeting Report). IDrugs. 2006 Oct;9(10):679-81.
        Institute for Molecular Bioscience, Australian Research Council Special Research Center for Functional and Applied Genomics, University of Queensland, Brisbane, QLD 4072, Australia. d.craik@imb.uq.edu.au.

        No abstract available; an edited extract follows.

        Extract: Update on the development of the analgesic conotoxin ACV1.
        Several other presentations at the meeting described preclinical and clinical studies on conopeptides, revealing the apparent importance of these promising disufide-rich molecules as drug leads. Bruce Livett [Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Australia] presented two posters that updated progress on the analgesic peptide ACV1 (Metabolic Pharmaceuticals Ltd) and related conotoxins derived from the venom of Conus victoriae. ACV1 is a synthetic peptide corresponding to the non-post-translationally modified venom peptide alpha-conotoxin Vc1.1. The conotoxin compound is currently undergoing Phase II clinical trials for neuropathic pain. In the first poster, Dr. Livett and colleagues described a chemical analysis of the C. victoriae venom in combination with the specific chemical synthesis of some post-translationally modified analogs of ACV1. In several animal models of human neuropathic pain, previous research had established that the 16-amino acid peptide Vc1.1 is active, but that the major post-translational form of Vc1.1 present in the venom, the peptide vc1a, which contains a hydroxyproline at position 6 and a gamma-carboxyglutamic acid at position 14, is inactive. The new research detected the presence of a previously unreported dimeric version of the post-translationally modified peptide, 6-Hyp Vc1.1. This dimeric form of the 6-Hyp peptide exhibited 36% of the activity of the monomeric alpha-conotoxin Vc1.1 (ACV1) in a neuronal nicotinic receptor assay. The second poster provided the first report that ACV1 is effective at alleviating neuropathic pain in an animal model of diabetic neuropathy. In the streptozotocin-induced diabetic rat model of peripheral neuropathy, an antiallodynic effect was observed at doses of 30 and 300 ug/kg within 1 h of dosing. ACV1 attenuated allodynia for up to 1 week following cessation of treatment, and also reduced oxidative stress markers such as lipid hydroperoxidases and nitrotyrosine. These findings broaden the indications for the development of ACV1 to treat neuropathic pain and diabetic neuropathy.

      26 October, 2006

        Rearrangement of disulfide bonds during synthesis of alpha-conotoxin GI from Conus geographus

        Szabo, I., Schlosser, G., Hudecz, F. and Mezo, G.(2006). Disulfide bond rearrangement during regioselective oxidation in PhS(O)Ph/CH(3)SiCl(3) mixture for the synthesis of alpha-conotoxin GI. Biopolymers. 2006 Oct 24; [Epub ahead of print]
        Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos L. University, Budapest, Hungary.

        Abstract: Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of Acm and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We had to conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds lead up to the appropriate compound in case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the non-native disulfide isomer. (c) 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2006.

      24 October, 2006

        O-conotoxin from Conus marmoreus from Hainan

        Luo, S., Zhangsun, D., Lin, Q., Xie, L., Wu, Y. and Zhu, X. (2006) Sequence diversity of O-superfamily conopetides from Conus marmoreus native to Hainan. Peptides. 2006 Sep 29; [Epub ahead of print]
        Key Laboratory for Tropical Biology Resources, Ministry of Education, Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou, Hainan 570228, China.

        Abstract: The full-length cDNAs of six new O-superfamily conotoxins (CTX) were cloned and sequenced from Conus marmoreus native to Hainan in China South Sea using RT-PCR and 3'-RACE. Six novel conotoxin precursors encoded by these cDNAs consist of three typical regions of signal, pro-peptide and mature peptide. All the six toxin regions share a common O-superfamily cysteine pattern (C-C-CC-C-C, with three disulfide bridges). The predicted precursors are composed of 73-88 amino acids, and the predicted mature peptides consist of 26-34 amino acids. Phylogenetic analysis of new conotoxins from C. marmoreus from the present study and published homologue T-superfamily sequences from other Conus species was performed systematically. Patterns of sequence divergence for three regions of signal, pro-region and mature peptides, as well as Cys codon usage define the major O-superfamily branches and suggest how these separate branches arose. Percent identities of the amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the pro-region was also high with intermediate divergence between that observed in signal and toxin regions. Amino acid sequences and their mode of action (target) of previously identified conotoxins from molluscivorous C. marmoreus for the known conotoxins classes are discussed in detail. The data presented are new and should pave the way for chemical synthesis of these unique conotoxins for to allow determination of the molecular targets of these peptides, and also to provide clues for a better understanding of the phylogeny of these peptides.

        T-conotoxins from Conus lividus and Conus litteratus

        Luo, S., Zhangsun, D., Wu, Y., Zhu, X., Xie, L., Hu, Y., Zhang, J. and Zhao, X. (2006) Identification and Molecular Diversity of T-superfamily Conotoxins from Conus lividus and Conus litteratus.Chem Biol Drug Des. 2006 Aug;68(2):97-106.
        Key Laboratory for Tropical Biological Resources (MOE), Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou, Hainan 570228 China.

        Abstract: The T-superfamily conotoxins comprise a large and diverse group of biologically active peptides and are widely distributed in venom ducts of all major feeding types of Conus. Six novel T-superfamily peptides from the two worm-hunting cone snail species of Conus lividus and Conus litteratus native to Hainan were identified and determined to share a common signal sequence as well as a conserved arrangement of cysteine residues (CC-CC). The predicted mature peptides consist of 11-15 amino acids only. Phylogenetic analyses of new conotoxins from C. lividus and C. litteratus in present study and published homologue T-superfamily sequences from the other Conus species was systematically performed. Phylogenetic trees, residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were explored, as well as residue frequency component and cystine codon usage. Percent divergence of the amino acid sequences of the signal-region exhibited high conservation, whereas the sequences of the mature peptides ranged from high similarity to high divergence between inter- and intro-species. Notably, diversity of pro-peptide region was also high with intermediate percent divergence between that observed in signal and toxin-regions. Consensus hydrophobic residues Leu, Val, Ala, Ile and Pro of signal regions were abundant, whereas among propeptides, basic residues Arg and Lys and acidic residue Asp, addition of hydrophilic residues Thr and Ser were abundant. Residue frequency components were hypervariable in mature toxin region except for highly conservative cystine frame residues. The T-superfamily conotoxins have been previously found mainly in piscivorous and molluscivorous cone snails. The newly identified six T-superfamily peptides described in this investigation exemplify the first to be found from vermivorous C. lividus and C. litteratus. The elucidated cDNAs of the six toxins will facilitate a better understanding of the relationship between structure and function as well as provide a framework for their further research and development.

        alpha-conotoxins from Cone shells native to Hainan

        Luo, S., Zhangsun, D., Zhang, B., Quan, Y. and Wu, Y. (2006) Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006 Nov;12(11):693-704.
        Key Laboratory for Tropical Aquatic and Terrestrial Biological Resources (MOE), Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou Hainan, 570228 China.

        Abstract: Conotoxins (CTX) from the venom of marine cone snails (genus Conus) represent large families of proteins, which show a similar precursor organization with surprisingly conserved signal sequence of the precursor peptides, but highly diverse pharmacological activities. By using the conserved sequences found within the genes that encode the alpha-conotoxin precursors, a technique based on RT-PCR was used to identify, respectively, two novel peptides (LiC22, LeD2) from the two worm-hunting Conus species Conus lividus, and Conus litteratus, and one novel peptide (TeA21) from the snail-hunting Conus species Conus textile, all native to Hainan in China. The three peptides share an alpha4/7 subfamily alpha-conotoxins common cysteine pattern (CCX(4)CX(7)C, two disulfide bonds), which are competitive antagonists of nicotinic acetylcholine receptor (nAChRs). The cDNA of LiC22N encodes a precursor of 40 residues, including a propeptide of 19 residues and a mature peptide of 21 residues. The cDNA of LeD2N encodes a precursor of 41 residues, including a propeptide of 21 residues and a mature peptide of 16 residues with three additional Gly residues. The cDNA of TeA21N encodes a precursor of 38 residues, including a propeptide of 20 residues and a mature peptide of 17 residues with an additional residue Gly. The additional residue Gly of LeD2N and TeA21N is a prerequisite for the amidation of the preceding C-terminal Cys. All three sequences are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The properties of the alpha4/7 conotoxins known so far were discussed in detail. Phylogenetic analysis of the new conotoxins in the present study and the published homologue of alpha4/7 conotoxins from the other Conus species were performed systematically. Patterns of sequence divergence for the three regions of signal, proregion, and mature peptides, both nucleotide acids and residue substitutions in DNA and peptide levels, as well as Cys codon usage were analyzed, which suggest how these separate branches originated. Percent identities of the DNA and amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the proregion was also high, with an intermediate percentage of divergence between that observed in the signal and in the toxin regions. The data presented are new and are of importance, and should attract the interest of researchers in this field. The elucidated cDNAs of these toxins will facilitate a better understanding of the relationship of their structure and function, as well as the process of their evolutionary relationships. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

        Discovery and development of alpha-conotoxins as novel analgesics

        Livett, B.G., Sandall, D.W., Keays, D., Down, J., Gayler, K.R., Satkunanathan, N. and Khalil Z. (2006) Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor. Toxicon. 2006 Jul 15; [Epub ahead of print]
        Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.

        Abstract: Pain therapeutics discovered by molecular mining of the expressed genome of Australian predatory cone snails are providing lead compounds for the treatment of neurological diseases such as multiple sclerosis, shingles, diabetic neuropathy and other painful neurological conditions. The high specificity exhibited by these novel compounds for neuronal receptors and ion channels in the brain and nervous system indicates the high degree of selectivity that this class of neuropeptides can be expected to show when used therapeutically in humans. A lead compound, ACV1 (conotoxin Vc1.1 from Conus victoriae), has entered Phase II clinical trials and is being developed for the treatment for neuropathic pain. ACV1 will be targeted initially for the treatment of sciatica, shingles and diabetic neuropathy. The compound is a 16 amino acid peptide [Sandall et al., 2003. A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo. Biochemistry 42, 6904-6911], an antagonist of neuronal nicotinic acetylcholine receptors. It has potent analgesic activity following subcutaneous or intramuscular administration in several preclinical animal models of human neuropathic pain [Satkunanathan et al., 2005. Alpha conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurons. Brain. Res. 1059, 149-158]. ACV1 may act as an analgesic by decreasing ectopic excitation in sensory nerves. In addition ACV1 appears to accelerate the recovery of injured nerves and tissues.

        Unusual structure of alpha-conotoxin BuIA from Conus bullatus

        Chi SW, Kim DH, Olivera BM, McIntosh JM, Han KH. (2006) NMR structure determination of alpha-conotoxin BuIA, a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold. Biochem Biophys Res Commun. 349:1228-1234.
        Molecular Cancer Research Center, Division of Molecular Therapeutics, KRIBB, Daejeon 305-806, Republic of Korea.

        Abstract: We have determined a high-resolution three-dimensional structure of alpha-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout alpha-conotoxin BuIA exhibits strong antagonistic activity at alpha6/alpha3beta2beta3, alpha3beta2, and alpha3beta4 nAChR subtypes like some alpha4/7 conotoxins. alpha-Conotoxin BuIA lacks the C-terminal beta-turn present within the second disulfide loop of alpha4/7 conotoxins, having only a "pseudo omega-shaped" molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in alpha4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of alpha-conotoxin BuIA to alpha4/7 conotoxins. Structural comparison of alpha-conotoxin BuIA with alpha4/7 conotoxins and alpha4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in alpha4/7 and alpha4/4 conotoxins may be important for binding to the alpha3 and/or alpha6 subunit of nAChR.

      23 October, 2006

        Novel M-superfamily conotoxins from Conus marmoreus, C.textile, C. leopardus and C. quercinus

        Han YH, Wang Q, Jiang H, Liu L, Xiao C, Yuan DD, Shao XX, Dai QY, Cheng JS, Chi CW. (2006) Characterization of novel M-superfamily conotoxins with new disulfide linkage. FEBS J. 273: 4972-4982
        Key laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China.

        Abstract: The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

      22 October, 2006

        3-D Model of alpha-Conotoxin Vc1.1


        The PDB coordinates of alpha-conotoxin Vc1.1 have been released and can be found under ID code # 2H8S following the link:

        http://www.rcsb.org/pdb/explore.do?structureId=2H8S

        Clark, R.J., Fischer, H., Nevin, S.T., Adams, D.J. and Craik, D.J. (2006) The synthesis, structural chracterisation and receptor specificity of the alpha-conotoxin Vc1.1. J Biol Chem. 281: 23254-23263.


      16 October, 2006

        Roman Coligula spawns modern counterpart

        'An image and a thousand words' is a regular column at the Toronto Star written by Peter Calamai, National Science Editor, Canada. This article about Bruce Livett's cone shell research appeared the Toronto Star on October 16 (Bruce Livett's 30th wedding anniversary as it happens !).

        To read the article in the Toronto Star Click here

      16 October, 2006

        Recombinant conotoxin MVIIA expressed in E. coli is 800 times stronger than morphine

        Xia, Z., Chen, Y., Zhu, Y., Wang, F., Xu, X. and Zhan, J. (2006) Recombinant omega-Conotoxin MVIIA possesses strong analgesic activity.BioDrugs 20:275-281.
        Department of Biochemistry and Molecular Biology, Zhejiang University Medical School, Hangzhou, People's Republic of China.

        Abstract: omega-Conotoxin (CTX) MVIIA is a specific antagonist of N-type voltage-sensitive calcium channels. A synthetic peptide version of CTX MVIIA (ziconotide) has been approved by the US FDA for severe and chronic pain. Given the high cost and complexity of the synthetic process of the disulfide-rich peptide, the genetic recombinant approach may simplify the development of this potent therapeutic agent. AIM: In this study, we report a new method for production of the recombinant CTX MVIIA. METHOD: A novel DNA fragment encoding CTX MVIIA was designed using Escherichia coli-preferred codons, and the fragment was cloned into the expression vector pGEX(2T). The fusion protein, CTX MVIIA and glutathione-S-transferase (GST) [GST-CTX MVIIA], was expressed in E. coli and purified by affinity chromatography on a glutathione-agarose column. After digestion with thrombin, the CTX MVIIA fragment was purified on a Sephacryltrade mark S-100 HR column and identified by mass spectrometry. The bioactivity of the peptide was evaluated by the hot tail-flick assay, in which the CTX MVIIA was intracerebroventricularly administered into Sprague-Dawley rats and its antinociceptive effect measured. RESULTS: The analgesic activity of the conotoxin was about 800 times stronger than that of morphine. CONCLUSION: The recombinant CTX MVIIA expressed in E. coli has shown marked analgesic activity, which may have potential in clinical application.


      15 October, 2006

        Conotoxins provide protection to the rapidly diversifying species of Conus. REVIEW

        Olivera BM.(2006) Conus peptides, biodiversity-based discovery and exogenomics..J Biol Chem. 2006 Aug 11; [Epub ahead of print] .
        Dept. of Biology, University of Utah, Salt Lake City, UT 84112-0840

        Abstract: The 700 different predatory cone snails (genus Conus) have evolved complex venoms, each species with it's own distinct set of 100 to 200 biologically active venom peptides. These are specified by a small number of gene superfamilies that rapidly diversify as speciation occurs. Most conopeptides are multiply disulfide cross-linked, with peptides in a superfamily having a characteristic disulfide framework, resulting in generally similar structures, but diverging function. Conopeptides have direct therapeutic applications - at least five are in clinical trials, and one (Prialt) has been approved as a drug for severe pain. Many conopeptides have exquisite targeting selectivity, and have the potential to be diagnostic ligands for a particular molecular isoform of an ion channel or receptor (the majority of conopeptide targets fall into these categories). The rapid diversification observed in conopeptide genes may be a general feature of all genes used by organisms to interact with other organisms in their environment. Across megadiverse taxa, each individual species has it's own complement of predators, prey and competitors; those genes whose products are used to mediate the biotic interactions of that particular species should thus be rapidly diversifying, if compared to homologous genes of other species. For conopeptides, an integrated approach to discovery that incorporates phylogenetics has been highly successful for identifying peptides with differing subtype-specificity.

        3-D Structure of alpha-conotoxin BuIA from Conus bullatus

        Chi, S.W., Kim, D.H., Olivera, B.M., McIntosh, J.M. and Han, K.H.(2006). NMR structure determination of alpha-conotoxin BuIA, a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold. Biochem Biophys Res Commun. 349:1228-1234.
        Molecular Cancer Research Center, Division of Molecular Therapeutics, KRIBB, Daejeon 305-806, Republic of Korea.

        Abstract: We have determined a high-resolution three-dimensional structure of alpha-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout alpha-conotoxin BuIA exhibits strong antagonistic activity at alpha6/alpha3beta2beta3, alpha3beta2, and alpha3beta4 nAChR subtypes like some alpha4/7 conotoxins. alpha-Conotoxin BuIA lacks the C-terminal beta-turn present within the second disulfide loop of alpha4/7 conotoxins, having only a "pseudo omega-shaped" molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in alpha4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of alpha-conotoxin BuIA to alpha4/7 conotoxins. Structural comparison of alpha-conotoxin BuIA with alpha4/7 conotoxins and alpha4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in alpha4/7 and alpha4/4 conotoxins may be important for binding to the alpha3 and/or alpha6 subunit of nAChR.

      6 September, 2006

        Selectivity of alpha-conotoxin BuIA from Conus bullatus

        Shiembob, D.L., Roberts, R.L., Luetje, C.W., McIntosh, J.M. (2006) Determinants of alpha-conotoxin BuIA selectivity on the nicotinic acetylcholine receptor beta subunit. Biochemistry. 45:11200-11207.

        Department of Biology, University of Utah, Salt Lake City, Utah, Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, Florida, and Department of Psychiatry, University of Utah, Salt Lake City, Utah.

        Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers composed of alpha and beta subunits. Different molecular compositions of these subunits constitute various receptor subtypes that are implicated in the pathophysiology and/or treatment of several disease states but are difficult to distinguish pharmacologically. alpha-Conotoxins are a group of small, structurally defined peptides that may be used to molecularly dissect the nAChR-binding site. Heteromeric nAChRs generally contain either a beta2 or beta4 subunit in addition to an alpha subunit at the ligand-binding interface. alpha-Conotoxin BuIA kinetically distinguishes between beta2- and beta4-containing nAChRs, with long off times for the latter. Mutational studies were used to assess the influence of residues that line the putative acetylcholine-binding pocket but differ between beta2 and beta4 subunits. Residues Thr/Lys59, Val/Ile111, and Phe/Gln119 of the respective beta2 and beta4 subunits are critical to off-rate differences. Among these residues, Thr59 of nAChR beta2 may interfere with effective access to the binding site, whereas Lys59 may facilitate this binding.

        Kasheverov, I.E., Zhmak, M.N., Vulfius, C.A., Gorbacheva, E.V., Mordvintsev, D.Y., Utkin, Y.N., van Elk, R., Smit, A.B., Tsetlin, V.I. (2006) alpha-Conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors.
        Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

        Abstract: alpha-Conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.

        Norton, R.S. and Olivera, B.M.(2006) Conotoxins down under. Toxicon 2006 Jul 15; [Epub ahead of print]
        Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Victoria, Australia. Department of Biology, University of Utah, Salt Lake City, Utah, Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, Florida, and Department of Psychiatry, University of Utah, Salt Lake City, Utah.

        Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers composed of alpha and beta subunits. Different molecular compositions of these subunits constitute various receptor subtypes that are implicated in the pathophysiology and/or treatment of several disease states but are difficult to distinguish pharmacologically. alpha-Conotoxins are a group of small, structurally defined peptides that may be used to molecularly dissect the nAChR-binding site. Heteromeric nAChRs generally contain either a beta2 or beta4 subunit in addition to an alpha subunit at the ligand-binding interface. alpha-Conotoxin BuIA kinetically distinguishes between beta2- and beta4-containing nAChRs, with long off times for the latter. Mutational studies were used to assess the influence of residues that line the putative acetylcholine-binding pocket but differ between beta2 and beta4 subunits. Residues Thr/Lys59, Val/Ile111, and Phe/Gln119 of the respective beta2 and beta4 subunits are critical to off-rate differences. Among these residues, Thr59 of nAChR beta2 may interfere with effective access to the binding site, whereas Lys59 may facilitate this binding.

        Hawgood, B. (2006) The marine biologist-Bob Endean. Toxicon. 2006 Jul 14; [Epub ahead of print]
        26 Cloister Road, Acton, London W3 0DE, UK.

        Abstract: Bob Endean was a dedicated marine biologist with an extensive knowledge of coral reef communities in the Great Barrier Reef and fauna in subtropical Queensland waters. He commenced a study of venomous and poisonous marine animals dangerous to man at a time when the field was new, employing a variety of techniques to investigate the venom apparatus, mode of delivery of venom or toxin, mode of toxic action on excitable tissues, and biochemistry of venom or toxin. Determination of the pharmacological properties of crude venom from Conus marine snails advanced characterization of conotoxins by later workers. A study of four types of nematocysts from the box-jellyfish Chironex fleckeri provided information as to their structure, function, and mechanism of discharge; myotoxins T1 and T2 were isolated from microbasic mastigophores. Endean studied poisonous stonefish (Synanceia trachynis) and, with Ann Cameron, scorpionfish (Notesthes robusta); investigations of ciguatera and of paralytic shellfish poisoning were initiated. He organized the collection of Australian frogs which led to the isolation of caerulein by Erspamer in Italy. Endean highlighted the ecological danger of the population explosion of the crown-of-thorns starfish (Acanthaster planci) and provided the impetus for the creation of the Great Barrier Reef Marine Park.

        In the four decades since toxinologists in Australia and elsewhere started to investigate the active constituents of venomous cone snails, a wealth of information has emerged on the various classes of peptides and proteins that make their venoms such potent bioactive cocktails. This article provides an overview of the current state of knowledge of these venom constituents, several of which are of interest as potential human therapeutics as a consequence of their high potency and exquisite target specificity. With the promise of as many as 50,000 venom components across the entire Conus genus, many more interesting peptides can be anticipated.

        Dewan, K.K. (2006) Secondary structure formations of conotoxin genes: A possible role in mediating variability. Biochem Biophys Res Commun. 2006 Aug 23; [Epub ahead of print]
        Unichem Biosciences R and D Centre, Society for Innovation and Development, Indian Institute of Science, Bangalore 560 012, Karnataka, India.

        Abstract: Small venomous peptides called conotoxins produced by the predatory marine snail (genus Conus) present an interesting case for mutational studies. They have a high degree of amino acid variability among them yet they possess highly conserved structural elements that are defined by cysteine residues forming disulfide bridges along the length of the mature peptide. It has been observed that codons specifying these cysteines are also highly conserved. It is unknown how such codon conservation is maintained within the mature conotoxin gene since this entire region undergoes an accelerated rate of mutation. There is evidence suggesting that nucleic acids wield some influence in mechanisms that dictate the region and frequency where mutations occur in DNA. Nucleic acids exert this effect primarily through secondary structures that bring about local peaks and troughs in the energy relief of these transient formations. Secondary structure predictions of several conotoxin genes were analyzed to see if there was any correspondence between the highly variable regions of the conotoxin. Regions of the DNA encompassing the conserved Cys codons (and several other conserved amino acid codons) have been found to correspond to predicted secondary structures of higher stabilities. In stark contrast the regions of the conotoxin that have a higher degree of variation correlate to regions of lower stability. This striking co-relation allows for a simple model of inaccessibility of a mutator to these highly conserved regions of the conotoxin gene allowing them a relative degree of resistance towards change.

        Wang, X., Engisch, K.L., Teichert, R.W., Olivera, B.M., Pinter, M.J., Rich, M.M.(2006) Prolongation of evoked and spontaneous synaptic currents at the neuromuscular junction after activity blockade is caused by the upregulation of fetal acetylcholine receptors. J. Neurosci. 2006 Aug 30;26(35):8983-7.
        Department of Neuroscience, Cell Biology, and Physiology, Wright State University, Dayton, Ohio 45435, USA.

        Abstract: It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (alphaA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.

      6 September, 2006

        Conkunizin-S1, a peptide neurotoxin from Conus striatus

        Dy, C.Y., Buczek, P., Imperial, J.S., Bulaj, G., Horvath, M.P. (2006) Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail. Acta Crystallogr D Biol Crystallogr.62 (Pt 9):980-990.
        Biology, University of Utah, 257 S 1400 E, Salt Lake City, Utah 84112-0840, USA.

        Abstract: Cone snails (Conus) are predatory marine mollusks that immobilize prey with venom containing 50-200 neurotoxic polypeptides. Most of these polypeptides are small disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine-cysteine disulfides. Conkunitzin-S1, a potassium-channel pore-blocking toxin isolated from C. striatus venom, is a member of a newly defined conotoxin family with sequence homology to Kunitz-fold proteins such as alpha-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and alpha-dendrotoxin are 42% identical in amino-acid sequence, conkunitzin-S1 has only four of the six cysteines normally found in Kunitz proteins. Here, the crystal structure of conkunitzin-S1 is reported. Conkunitzin-S1 adopts the canonical 3(10)-beta-beta-alpha Kunitz fold complete with additional distinguishing structural features including two completely buried water molecules. The crystal structure, although completely consistent with previously reported NMR distance restraints, provides a greater degree of precision for atomic coordinates, especially for S atoms and buried solvent molecules. The region normally cross-linked by cysteines II and IV in other Kunitz proteins retains a network of hydrogen bonds and van der Waals interactions comparable to those found in alpha-dendrotoxin and BPTI. In conkunitzin-S1, glycine occupies the sequence position normally reserved for cysteine II and the special steric properties of glycine allow additional van der Waals contacts with the glutamine residue substituting for cysteine IV. Evolution has thus defrayed the cost of losing a disulfide bond by augmenting and optimizing weaker yet nonetheless effective non-covalent interactions.

        Conopeptides Lo959 from Conus loroisii and Am975 from Conus amadis, two new contryphans

        Sabareesh, V., Gowd, K.H., Ramasamy, P., Sudarslal, S., Krishnan, K.S., Sikdar, S.K., Balaram, P. (2006) Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels. Peptides. 2006 Aug 29; [Epub ahead of print]
        Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.

        Abstract: Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.

        Case studies with Prialt (Ziconotide)

        Wermeling, D.P. and Berger, J.R. (2006) Ziconotide infusion for severe chronic pain: case series of patients with neuropathic pain. Pharmacotherapy. 2006 Mar;26(3):395-402.
        Department of Pharmacy Practice and Science, College of Pharmacy, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA. dwermel@uky.edu

        Abstract: Ziconotide intrathecal infusion was recently approved by the United States Food and Drug Administration for the treatment of intractable severe chronic pain. Patients with neuropathic pain make up a significant population among those who experience chronic pain for which there are less than optimal pharmacotherapeutic options. Published clinical trials provide a global view of ziconotide efficacy and safety. A subset of patients in clinical trials obtained complete pain relief, a remarkable finding given the history of drug treatment for neuropathic pain. To provide more information regarding those who respond to ziconotide therapy, we discuss three patients with neuropathic pain who received ziconotide infusion. Two patients with longstanding neuropathic pain, one with complex regional pain syndrome (formerly known as reflex sympathetic dystrophy) of the leg and one with lumbar radiculitis, achieved temporary but complete pain relief from single 5- and 10-microg epidural test doses. In the third case, a patient with longstanding bilateral leg and foot neuropathic pain from acquired immunodeficiency syndrome and antiretroviral drug therapy achieved considerable pain relief from a long-term continuous intrathecal infusion. The patients who received a single dose had mild central nervous system adverse effects such as sedation, somnolence, nausea, headache, and lightheadedness. The patient who received the intrathecal infusion experienced mild-to-severe adverse effects depending on the rate of infusion; these effects included sedation, confusion, memory impairment, slurred speech, and double vision. This patient could sense impending adverse effects and made rate adjustments or suspended infusion to avert untoward symptoms. In all three cases, patients achieved considerable pain relief that was long-lasting and persisted well after dose administration or suspension of infusion.

      5 September, 2006

        Conotoxin pl14a from Conus planorbis defines the J-conotoxin superfamily

        Imperial, J.S., Bansal, P.S., Alewood, P.F., Daly, N.L., Craik, D.J., Sporning, A., Terlau, H., Lopez-Vera, E., Bandyopadhyay, P.K. and Olivera, B.M. (2006) A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins. Biochemistry. 45: 8331-8340. PMID: 16819832 [PubMed - indexed for MEDLINE]
        Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA. imperial@biology.utah.edu

        Abstract: Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels. PMID: 16819832 [PubMed - indexed for MEDLINE]

        Complete mitochondrial genome of a venomous mollusc

        Bandyopadhyay, P.K., Stevenson, B.J., Cady, M.T., Olivera, B.M., Wolstenholme, D.R. (2006) Complete mitochondrial DNA sequence of a Conoidean gastropod, Lophiotoma (Xenuroturris) cerithiformis: gene order and gastropod phylogeny. Toxicon. 48: 29-43.
        Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

        Abstract: We have determined the first complete nucleotide sequence of the mitochondrial genome of a venomous mollusc, the Conoidean gastropod, Lophiotoma (Xenuroturris) cerithiformis. It is 15,380 nucleotide pairs (ntp) and encodes 13 proteins, two ribosomal RNAs and 22 tRNAs of the mitochondrion's own protein synthesizing system. The protein mRNAs, ribosomal RNAs and 13 of the tRNAs are transcribed from the same strand, the remaining tRNAs from the other strand. The longest segment of unassigned sequence is 139 ntp and includes a 82 ntp segment that is a perfect inverted repeat sequence of 37 ntp separated by 8 nt. The gene arrangement of L. cerithiformis mtDNA shows remarkable similarity to the gene arrangements of mtDNAs of the vetigastropod Haliotis rubra, the polyplacophoran Katharina tunicata and the cephalopod Octopus vulgaris, but differs dramatically from the gene arrangements found in the mtDNAs of pulmonate and opisthobranch gastropods, as well as mtDNAs of bivalves and scaphopods. A single sixteen gene inversion that distinguishes L. cerithiformis mtDNA from mtDNAs of H. rubra, K. tunicata and O. vulgaris is shared by mtDNA of a littorinomorph gastropod Littorina saxitalis, suggesting a close relationship of conoidean and littorinomorph gastropods.

      4 September, 2006

        A New Tool Against Brain Disease.

        Conus omaria.

        Snail toxin may spur new medications for Alzheimer's, Parkinson's and Depression.
        Lee Siegel, Science News Specialist with the University of Utah Public Relations writes to tell of recent research by Dr. J. Michael McIntosh, Research Professor of Biology and Professor of Psychiatry at the University of Utah who, together with his collaborators, discovered a new toxin from Conus omaria. This venomous cone snail, lives in the Pacific and Indian oceans and eats other snails. It ranges from 1ľ to 3˝ inches long. The novel toxin, alpha-conotoxin OmIA "may enable scientists to more effectively develop medications for a wide range of nervous system disorders including Parkinson's disease, Alzheimer's disease, depression, nicotine addiction and perhaps even schizophrenia", said McIntosh. The new toxin – named alpha conotoxin OmIA. McIntosh discovered and analyzed the new toxin with help from University of Utah cone snail research pioneer Baldomero "Toto" Olivera, who is a distinguished professor of biology, and lab technicians Sean B. Christensen and Cheryl Dowell. Other coauthors of the study are Palmer Taylor, professor and dean of pharmacology at the University of California, San Diego, and his associates – Todd Talley, Igor Tsigelny and Kwok-Yiu Ho – as well as Kyou-Hoon Han at the Korea Research Institute of Bioscience and Biotechnology. Discovery of the new cone snail toxin was published on Friday, Aug. 25 in The Journal of Biological Chemistry.
        Talley, T.T., Olivera, B.M., Han, K.H., Christensen, S.B., Dowell, C., Tsigelny, I., Ho, K.Y, Taylor, P. and McIntosh, J.M.(2006) alpha-conotoxin OMIA is a potent ligand for the acetylcholine binding protein as well as alpha 3 beta 2 and alpha 7 nicotinic acetylcholine receptors. J Biol Chem. 281: 24678-24686 (See entry for 30 June below) PubMed entry

      1 September, 2006

        alpha-A conotoxin OIVA[K15N] from Conus obscurus selectively targets fetal-type nicotinic receptors.

        Wang, X., Engisch, K.L., Teichert, R.W., Olivera, B.M., Pinter, M.J. and Rich, M.M. (2006) Prolongation of evoked and spontaneous synaptic currents at the neuromuscular junction after activity blockade is caused by the upregulation of fetal acetylcholine receptors.
        (1)Department of Neuroscience, Cell Biology, and Physiology, Wright State University, Dayton, Ohio 45435, (2)Department of Biology, University of Utah, Salt Lake City, Utah 84112, and (3)Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
        Correspondence should be addressed to Dr. Mark M. Rich, Room 014, M & M Building, Wright State University, 3640 Colonel Glenn, Dayton, OH 45435. Email: mark.rich@wright.edu

        Abstract: It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (alphaA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.


      31 August, 2006

        w-conotoxins CVID ad CVIB from Conus catus modulate nociception

        Motin L., Lewis R.J. and Adams D.J.(2006) Modulation of synaptic trnasmission by omega-conotoxins CVID and CVIB in spinal superficial dorsal horn.Proc. ComBio2006, 24-28 September, Brisbane, Australia
        School of Biomedical Sciences, University of Queensland.

        Abstract: The selective blocker of N-type voltage-gated Ca2+ channels (VGCCs) CVID (Lewis et al., 2000) and non-selective N- and P/Q- types antagonist, CVIB, are likely to be of interest as both research tools and potential anti-nociceptive agents. In the present study, the effects of these omega-conotoxins on synaptic transmission between primary A? and C primary afferents and dorsal horn superficial laminae neurons of rat spinal cord were tested by comparison of evoked excitatory postsynaptic currents (EPSCs) amplitude distributions in the absence and the presence of toxin. CVID (200 nM) completely inhibited both poly- and mono-synaptic EPSCs. CVIB (235 nM) reduced the mean of monosynaptic EPSCs amplitude distribution on average to 48 ± 8% (n=5, p<0.02) of control, without affecting the area of the distributions. In contrast, for polysynaptic transmission the area in the presence of CVIB was on average 55 ± 15% (n=7, p<0.05) of control. Given that N-type VGCCs are localised both pre- and post-synaptically and P/Q- VGCCs presynaptically (Murakami et al. (2004) Brain Res. 1024, 122-129.), it can be hypothesised that (i) CVID has higher affinity to postsynaptic N-type VGCCs in interneurons receiving nociceptive information than CVIB, (ii) CVIB predominantly blocks presynaptic P/Q-type VGCCs reducing the probability of transmitter release from interneurons, and (iii) synaptic transmission between primary afferents and superficial laminae neurons is mainly mediated by N-type VGCCs. Further investigation on the modulation of synaptic transmission in interneurons by omega-conotoxins is likely to reveal new information on mechanisms of propagation of nociception in superficial dorsal horn. Lewis et al. (2000) J. Biol. Chem. 275, 35335-35344. Murakami et al. (2004) Brain Res. 1024, 122-129.

        Cyclization of conotoxin GS from Conus geographus

        Yulyaningsih E., Cemazar M. and Craik D.J. (2006) Structural stabilization of GS conotoxin by means of cyclization. Proc. ComBio2006, 24-28 September, Brisbane, Australia
        Institute for Molecular Bioscience, University of Queensland.

        Abstract: Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus. This molecule interacts with the extra cellular interface of muscle sodium channels to inhibit the transport of sodium ion. Its structural framework contains four loops that arise from the three disulfide bonds formed between cysteine residues 1-4, 2-5 and 3-6. GS, along with other conotoxins, has an exceptional affinity and selectivity, making it an invaluable neurophysiological probe and drug lead, as well as a useful tool to study the mechanism and function of ion transport in membranes. However, functional exploration of GS conotoxin is limited by its susceptibility to in vivo proteolytic degradation. This limitation, as observed with a number of linear peptides, could be substantially overcome by cyclization of the peptide backbone as exemplified by cyclic proteins. We have synthesized cyclic GS conotoxin and its variant, which contained two additional glycine residues, using Boc solid phase peptide synthesis (Boc-SPPS) with in situ neutralization and a thioester linker between the resin and peptide chain. The peptides were purified by reversed phase high performance liquid chromatography (RP-HPLC) in conjunction with electro spray ionization mass spectroscopy (ESI MS) to verify the purity and molecular weight of the peptides. Oxidations of the molecules were determined through a set of small scale folding trials and confirmation of the folded peptide conformation is obtained through 1 dimensional NMR analysis. This study will hopefully provide valuable insight towards the development of GS conotoxin for pharmaceutical applications.


      18 August, 2006

        Globular and Ribbon Structures of the chi/lambda conotoxin, CmrVIA from Conus marmoreus

        Kang, T.S., Jois, S.D. and Kini, R.M.(2006) Solution Structures of Two Structural Isoforms of CMrVIA chi/lambda-Conotoxin. Biomacromolecules. 7(8):2337-2346.

        Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Block S3 #03-17 Singapore 117543, and Department of Pharmacy, National University of Singapore, Singapore 117543.

        Abstract: alpha-Conotoxins possess a conserved four-cysteine framework and disulfide linkages (C(1)(-)(3), C(2)(-)(4)) that fold toward the globular conformation with absolute fidelity. Despite the presence of a similar conserved set of cysteine framework, chi/lambda-conotoxins adopt an alternate disulfide-pairing (C(1)(-)(4), C(2)(-)(3)) and its consequent ribbon conformation, exhibiting distinct biological activities from alpha-conotoxins. chi/lambda-Conotoxin CMrVIA (VCCGYKLCHOC-COOH) isolated from the venom of Conus marmoreus natively exists in the ribbon conformation and induces seizures in mice at a potency that is of three orders higher than the non-native globular form. We have chemically synthesized two isoforms of CMrVIA conotoxin in the ribbon and globular conformation and determined their structures by (1)H NMR spectroscopy. The ribbon (PDB ID 2B5P) and globular conformations (PBD ID 2B5Q) were calculated to have paired-wise backbone RMSDs of 0.48 +/- 0.1 and 0.58 +/- 0.1 A respectively. Unlike the native globular alpha-conotoxins, the globular canonical form of CMrVIA chi/lambda-conotoxin exhibited heterogeneity in its solution structure as noted by the presence of minor conformers and poorer RMSD of structure calculation. Paired-wise backbone comparison between the native ribbon and the non-native globular form of CMrVIA conotoxin revealed an RMSD of 4.73 A, emphasizing their distinct conformational differences. These structural data are essential for the understanding of the structure-function activity of chi/lambda-conotoxins, as well as unraveling the folding propensities of these short peptide toxins.

        Pi C, Liu Y, Peng C, Jiang X, Liu J, Xu B, Yu X, Yu Y, Jiang X, Wang L, Dong M, Chen S, Xu AL. (2006) Analysis of expressed sequence tags from the venom ducts of Conus striatus: focusing on the expression profile of conotoxins. Biochimie 88:131-140.
        State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Therapeutic Functional Genes, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingangxi Road, 510275 Guangzhou, China.

        Abstract: Cone snails (genus Conus) are predatory marine gastropods that use venom peptides for interacting with prey, predators and competitors. A majority of these peptides, generally known as conotoxins demonstrate striking selectivity in targeting specific subtypes of ion channels and neurotransmitter receptors. So they are not only useful tools in neuroscience to characterize receptors and receptor subtypes, but offer great potential in new drug research and development as well. Here, a cDNA library from the venom ducts of a fish-hunting cone snail species, Conus striatus is described for the generation of expressed sequence tags (ESTs). A total of 429 ESTs were grouped into 137 clusters or singletons. Among these sequences, 221 were toxin sequences, accounting for 52.1% (corresponding to 19 clusters) of all transcripts. A-superfamily (132 ESTs) and O-superfamily conotoxins (80 ESTs) constitute the predominant toxin components. Some non-disulfide-rich Conus peptides were also found. The expression profile of conotoxins also explained to some extent the pharmacological and physiological reactions elicited by this typical piscivorous species. For the first time, a nonstop transcript of conotoxin was identified, which is suggestive that alternative polyadenylation may be a means of post-transcriptional regulation of conotoxin production. A comparison analysis of these conotoxins reveals the different variation and divergence patterns in these two superfamilies. Our investigations indicate that focal hyper-mutation, block substitution and exon shuffling are three main mechanisms leading to the conotoxin diversity in a species. The comprehensive set of Conus gene sequences allowed the identification of the representative classes of conotoxins and related components, which may lay the foundation for further research and development of conotoxins.

        Pi, C., Liu, J., Peng, C., Liu, Y., Jiang, X., Zhao, Y., Tang, S., Wang, L., Dong, M., Chen, S., Xu, A.(2006) Diversity and evolution of conotoxins based on gene expression profiling of Conus litteratus. Genomics. 2006 Aug 11; [Epub ahead of print]
        State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Therapeutic Functional Genes, The Open Laboratory for Marine Functional Genomics of the State High-Tech Development Program, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingangxi Road, Guangzhou 510275, People's Republic of China.

        Abstract: Cone snails are attracting increasing scientific attention due to their unprecedented diversity of invaluable channel-targeted peptides. As arguably the largest and most successful evolutionary genus of invertebrates, Conus also may become the model system to study the evolution of multigene families and biodiversity. Here, a set of 897 expressed sequence tags (ESTs) derived from a C. litteratusvenom duct was analyzed to illuminate the diversity and evolution mechanism of conotoxins. Nearly half of these ESTs represent the coding sequences of conotoxins, which were grouped into 42 novel conotoxin cDNA sequences (seven superfamilies), with T-superfamily conotoxins being the dominant component. The gene expression profile of conotoxin revealed that transcripts are expressed with order-of-magnitude differences, sequence divergence within a superfamily increases from the N to the C terminus of the open reading frame, and even multiple scaffold-different mature peptides exist in a conotoxin gene superfamily. Most excitingly, we identified a novel conotoxin superfamily and three novel cysteine scaffolds. These results give an initial insight into the C. litteratus transcriptome that will contribute to a better understanding of conotoxin evolution and the study of the cone snail genome in the near future. PMID: 16908117 [PubMed - as supplied by publisher]

      12 August, 2006

        REVIEW: Cone shell peptides - Discovery and Exogenomics

        Olivera, B.M. (2006) Conus peptides, biodiversity-based discovery and exogenomics J. Biol. Chem, 10.1074/jbc.R600020200. Papers In Press, published online ahead of print August 11, 2006
        Dept. of Biology, University of Utah, Salt Lake City, UT 84112-0840

        Abstract: The 700 different predatory cone snails (genus Conus) have evolved complex venoms, each species with it’s own distinct set of 100 to 200 biologically active venom peptides. These are specified by a small number of gene superfamilies that rapidly diversify as speciation occurs. Most conopeptides are multiply disulfide cross-linked, with peptides in a superfamily having a characteristic disulfide framework, resulting in generally similar structures, but diverging function. Conopeptides have direct therapeutic applications – at least five are in clinical trials, and one (Prialt) has been approved as a drug for severe pain. Many conopeptides have exquisite targeting selectivity, and have the potential to be diagnostic ligands for a particular molecular isoform of an ion channel or receptor (the majority of conopeptide targets fall into these categories). The rapid diversification observed in conopeptide genes may be a general feature of all genes used by organisms to interact with other organisms in their environment. Across megadiverse taxa, each individual species has it’s own complement of predators, prey and competitors; those genes whose products are used to mediate the biotic interactions of that particular species should thus be rapidly diversifying, if compared to homologous genes of other species. For conopeptides, an integrated approach to discovery that incorporates phylogenetics has been highly successful for identifying peptides with differing subtype-specificity.

      10 August, 2006

        Venom Apparatus of the Superfamily: Conacea

        For a very clear depiction of the venom appratus used by molluscs in the Conacea superfamily, consisting of the Cone, Turrid and Auger families, click on this link Venom Apparatus of the Superfamily: Conacea

        Conotoxin-like sequence in a baculovirus

        Ikeda, M., Shikata, M., Shirata, N., Chaeychomsri, S., Kobayashi, M. (2006). Gene organization and complete sequence of the Hyphantria cunea nucleopolyhedrovirus genome. J Gen Virol. 87(Pt 9):2549-2562.
        Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

        Abstract: The whole-genome sequence of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) was analysed. The entire nucleotide sequence of the HycuNPV genome was 132 959 bp long, with a G+C content of 45.1 mol%. A total of 148 open reading frames (ORFs) consisting of more than 50 aa were encoded by the genome. HycuNPV shares more than 122 ORFs with other lepidopteran group I NPVs, including Autographa californica MNPV, Bombyx mori NPV, Choristoneura fumiferana MNPV (CfMNPV), Choristoneura fumiferana defective NPV, Epiphyas postvittana MNPV and Orgyia pseudotsugata MNPV (OpMNPV). Six ORFs are identified as being unique to HycuNPV. Most of the HycuNPV ORFs showed higher similarity to CfMNPV and OpMNPV ORFs than to those of the other group I NPVs. HycuNPV encodes two conotoxin-like homologues (ctls), which are observed only in OpMNPV in group I NPVs. HycuNPV encodes three inhibitors of apoptosis (iaps), hycu-iap-1, hycu-iap-2 and hycu-iap-3, a feature that it shares only with CfMNPV. In addition, six homologous regions (hrs) are identified in the HycuNPV genome. These hrs are located in regions similar to those of the OpMNPV hrs, but different from most of the CfMNPV hrs. Based on the close phylogenetic relationship and conservation of group I NPV-specific genes, such as gp64, ie-2 and ptp-1, it is concluded that HycuNPV belongs to the group I NPVs and is most similar to CfMNPV or OpMNPV.

        Action of conantokin G from Conus geographus on NMDA receptor responses in cultured neurons

        Alex, A.B., Baucum, A.J., Wilcox, K.S.(2006). Effect of Conantokin G on NMDA Receptor-Mediated Spontaneous EPSCs in Cultured Cortical Neurons. J Neurophysiol. 96: 1084-1092. Epub 2006 Jun 7.
        Anticonvulsant Drug Development Program, Dept. of Pharmacology and Toxicology, 417 Wakara Way, Suite 3211, Univ. of Utah, Salt Lake City, UT 84108. .

        Abstract: Conantokin G (Con G), derived from the venom of Conus geographus, is the most characterized natural peptide antagonist targeted to N-methyl-d-aspartate (NMDA) receptors. Although Con G is known to bind to the glutamate binding site on the NR2 subunit of the receptor, it is unclear whether it can allosterically modulate the function of the receptor through the glycine binding site on the NR1 subunit. This study was designed to evaluate the action of Con G on NMDA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) and its modulation by glycine in cultured cortical neurons (13-19 days in vitro) using the whole cell patch-clamp technique. Con G inhibited NMDA receptor-mediated sEPSCs in a concentration-dependent manner. Also, the potency of Con G decreased as a function of time in culture. The inhibition of EPSCs observed after application of Con G in the presence of high (10 muM) and nominal (no added) concentrations of glycine was not different at 13 days in vitro (DIV). Furthermore, similar results were obtained with experiments on Con G-induced inhibition of NMDA-evoked whole cell currents. These results indicate that glycine concentrations do not have a direct effect on Con G-induced inhibition of NMDA currents. In addition, age dependency in the action of Con G on cortical neurons in vitro suggests that this model system would be useful in examining the effects of different agonists/antagonists on native synaptic NMDA receptors.

      2 August, 2006

        Ziconotide: new option for refratory pain

        Prommer, E. (2006) Ziconotide: a new option for refractory pain. Drugs Today (Barc). 42: 369-378.
        Division of Hematology/Oncology, Veterans Integrated Palliative Care Program, UCLA School of Medicine, Los Angeles, California, USA. Email: Eric Prommer.

        Abstract: Ziconotide has been introduced as a new nonopioid treatment for chronic pain. Structurally, it is a peptide, the synthetic analog of the omega-conotoxin, derived from the marine snail, Conus magus. N-type voltage-sensitive calcium channels play a role in the transmission of nociceptive stimuli and also are involved in the release of neurotransmitters important in pain transmission. Ziconotide's therapeutic benefit derives from its potent and selective blockade of neuronal-type voltage-sensitive calcium channels. Blockade of the channels results in suppression of abnormal ectopic discharges from the injury site or the dorsal root ganglia, possibly resulting in decreased neuroplasticity, and decreased synaptic transmission that leads to the generation of chronic pain syndromes. The advantage of ziconotide is that tolerance does not occur, while disadvantages associated with ziconotide are the need for intrathecal administration and significant neurotoxicites associated with its use. When tested in clinical trials, ziconotide has been shown to have synergistic or additive value to the effect of morphine. Ziconotide, formerly known also as SNX- 111, represents a new class of agents, the N-type calcium channel blockers. These may represent another option for patients with refractory pain and refractory pain syndromes. (c) 2006 Prous Science. All rights reserved.

      1 August, 2006

        Simulation of conantokin-T folding

        Wei, C.C., Ho, M.H., Wang, W.H. and Sun, Y.C. (2006) Molecular dynamics simulation of folding of a short helical peptide with many charged residues.J Phys Chem B Condens Matter Mater Surf Interfaces Biophys 2005; 109:19980-6.
        Department of Chemistry, National Taiwan Normal University, 88, TingChow Road Section 4, Taipei 116, Taiwan.

        Abstract: A molecular dynamics simulation of the folding of conantokin-T (con-T), a short helical peptide with 5 helical turns of 21 amino acids with10 charged residues, was carried out to examine folding pathways for this peptide and to predict the folding rate. In the 18 trajectories run at 300 K, 16 trajectories folded, with an averaged folding time of approximately 50 ns. Two trajectories did not fold in up to 200 ns simulation. The folded structure in folded trajectories is in good agreement with experimental structure (Skjaerbaek; et al. J. Biol. Chem. 1997, 272, 2291. Lin; et al. FEBS Lett. 1997, 407, 243). An analysis of the trajectories showed that, at the beginning of a few nanoseconds, helix formation started from residues 5-9 with assistance of a hydrophobic clustering involving Tyr5, Met8, and Leu9. The peptide formed a U-shape mainly due to charge-charge interactions between charged residues at the N- and C-terminus segments. In the next approximately 10 ns, several nonnative charge-charge interactions were broken and nonnative Gla10-Lys18 (this denotes a salt bridge between Gal10 and Lys18) and/or Gla10-Lys19 interactions appeared more frequently in this folding step and the peptide became a fishhook J-shape. From this structure, the peptide folded to the folded state in 7 of all 16 folded trajectories in approximately 15 ns. Alternatively, in approximately 30 ns, the con-T went to a conformation in an L-shape with 4 helical turns and a kink at the Arg13 and Gla14 segment in the other 9 trajectories. Con-T in the L-shape then required another approximately 15 ns to fold into the folded state. In addition, in overall folding times, the former 7 trajectories folded faster with the total folding times all shorter than 45 ns, while the latter 9 trajectories folded at a time longer than 45 ns, resulting in an average folding time of approximately 50 ns. Two major folding intermediates found in 2 nonfolded trajectories are stabilized by charge clusters of 5 and 6 charged residues, respectively. With inclusion of friction and solvent-solvent interactions, which were ignored in the present GB/SA solvation model, the folding time obtained above should be multiplied by a factor of 1.25-1.7 according to a previous, similar simulation study. This results in a folding time of 65-105 ns, slightly shorter than the folding time of 127 ns for an alanine-based peptide of the same length. This suggests that the energy barrier of folding for this type of peptides with many charged residues is slightly lower than alanine-based helical peptides by less than 1 kcal/mol.

      31 July, 2006

        Conopeptides at the I.S.T. 15th International Toxinology Congress, Glasgow (23-28 July 2006)

        Visit the Congress Web Site.
        Some abstract titles of interest were:
        Monday 23 July
        Toxins and Drug Discovery Symposium

      • Miljanich, G. PRIALT: Ziconotide intrathecal infusion: a conotoxin for treating severe chronic pain.
        Elsevier-Toxicon Plenary Lecture
      • Stemmer, Willem Pim (CEO and Founder of Amunix, Inc. California). The advantages of disulfide-rich microproteins as scaffolds for the construction of antibody-mimetic pharmaceuticals.

        Free Oral Communications

      • Tsetlin, V.I. alpha-conotoxins and their targets: From photolabeling to the three-dimensional structures.
      • Adams, D.J. : The structural characterisation and receptor specificity of alpha-conotoxin Vc1.1

        Tuesday
        Free Oral Communications

      • Mari, F. Novel Conotoxin Frameworks. [various Atlantic cones]
      • Kauferstein, S. A novel family of conotoxins target to nACh-receptors. [Conus capitaneous]

        Posters:

          #
        • 2. Aguilar, MB et al. O-conotoxin-like Cyst framework and hydroxyproline in peptides of the venom of the turrid Polystira albida from the Bay of Campeche, Mexico.
        • 26. Norton, RS. et al. Post-translational amino acid epimerization in an excitatory conotoxin. Structures of the L- and D-forms.
        • 41. Saminathan, R. et al Discovery of a novel conopeptide - AmIXA: Prediction of functional pharmacophres and mechanism of action.
        • 46. Croker, D. et al. Discovery and pharmacology of Conopressin-T, a novel Vasopressin-like peptide from Conus tulipa.
        • 47. Dutertre, S. et al Isolation and characterisation of Conomap-Vt, a D-aminoacid-containing excitatory peptide from the venom of a vermivorous cone snail.
        • 50. Townsend, A. et al. Post-translational modifications of venom components in Conus victoriae.

        Thursday 27 July
        Posters #

        • 13. Crocker, D.E. et al. Xen2174: A novel NET inhibitor that enhances alpha2-adrenoceptor inhibition of spinal pain pathways
        • 45. Satkunanathan, N. et al. : An alpha-conotoxin from Conus victoriae is effective at alleviating neuropathic pain in an animal model of diabetic neuropathy.

        Friday 28 July

      11 July, 2006

        Structural features of conotoxins

        Marx, U.C., Daly, N.L., Craik, D.J.(2006) NMR of conotoxins: structural features and an analysis of chemical shifts of post-translationally modified amino acids. Magn Reson Chem. 2006 Jul 6;44(S1):41-50 [Epub ahead of print]
        Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane QLD 4072, Australia.

        Abstract:Conotoxins are small conformationally constrained peptides found in the venom of marine snails of the genus Conus. They are usually cysteine rich and frequently contain a high degree of post-translational modifications such as C-terminal amidation, hydroxylation, carboxylation, bromination, epimerisation and glycosylation. Here we review the role of NMR in determining the three-dimensional structures of conotoxins and also provide a compilation and analysis of (1)H and (13)C chemical shifts of post-translationally modified amino acids and compare them with data from common amino acids. This analysis provides a reference source for chemical shifts of post-translationally modified amino acids. Copyright (c) 2006 John Wiley & Sons, Ltd.

        Snail venom painkiller launched

        The cone shell analgesic Ziconotide (Prialt) was launched in the UK today (click here) by Eisai Pharmaceuticals for treatment of painful neuropathies.

      9 July, 2006

        Clinical Toxinology Resources Website

        The Clinical Toxinology Resources Website is a premier site for information on venomous animals and poisonous animals, plants and mushrooms. It covers the whole World, with both general information and information about particular organisms, located through a searchable database, that allows users to look for an animal, plant or mushroom, based on a common name, a scientific name or family, a country or region. Most of the world's cone shells are listed though specific information is still to be included about many. A "Subscribers" section with a subscription of approx. $180 per year entitles the viewer to more complete information about treatment of envenomation and other materials not covered in the free web version. The site is maintained by the Women's & Children's Hospital, The University of Adelaide, Adelaide, South Australia, Australia.

      30 June, 2006

        alpha-conotoxin GI from Conus geographus used to profile receptor function in living cells.

        Schreiter C, Gjoni M, Hovius R, Martinez KL, Segura JM, Vogel H.(2005). Reversible sequential-binding probe receptor-ligand interactions in single cells. Chembiochem. 6 : 2187-2194.
        Ecole Polytechnique Federale de Lausanne (EPFL), Laboratoire de Chimie Physique des Polymeres et Membranes, 1015 Lausanne, Switzerland.

        Abstract:With the reversible sequential (ReSeq) binding assay,we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast association-dissociation kinetics. We chose the nicotinic-acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equilibrium, kinetic, and competition-binding assays using fluorescent derivatives of the antagonist alpha-conotoxin GI (alpha-CnTx). Thereby, we determined the binding constants of unlabeled alpha-CnTx and d-tubocurarine. The high selectivity of alpha-CnTx for muscle-type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand-binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq-binding assays even at low receptor-expression levels. We expect our approach to be of generic importance for functional screening of compounds or membrane receptors, and for the detailed characterization of rare primary cells. PMID: 16270372 [PubMed - in process]
        [Note: The use of CnTx as an abbreviation for 'conotoxin' is likely to lead to confusion. This paper is about the muscle-type alpha-conotoxin GI from Conus geographus, and NOT about alpha-CnIA an alpha conotoxin from Conus caonsors. BGL]

      30 June, 2006

        Novel alpha-conotoxin from Conus omaria.

        Talley, T.T., Olivera, B.M., Han, K.H., Christensen, S.B., Dowell, C., Tsigelny, I., Ho, K.Y, Taylor, P. and McIntosh, J.M.(2006) alpha-conotoxin OMIA is a potent ligand for the acetylcholine binding protein as well as alpha 3 beta 2 and alpha 7 nicotinic acetylcholine receptors. J Biol Chem. 281: 24678-24686.
        Department of Biology, University of Utah, Salt Lake City, UT 84112-0840. Abstract:The molluskan acetylcholine binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the alpha subunit of nicotinic acetylcholine receptors and in particular the homomeric alpha7 nicotinic receptor. We report the isolation and characterization of an alpha-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the alpha7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the alpha3beta2 nAChR indicating that alpha-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of alpha3beta2 nAChRs. alpha-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17 amino acid, two-disulfide bridge peptide. The ligand is the first alpha-conotoxin with higher affinity for the closely related receptor subtypes, alpha3beta2 vs. alpha6beta2 and selectively blocks these two subtypes when compared with alpha2beta2, alpha4beta2 and alpha1betadeltaepsilon nAChRs.

        Conotoxin-like peptide in a marine sponge.

        Takada, K., Hamada, T., Hirota, H., Nakao, Y., Matsunaga, S., van Soest, R.W. and Fusetani, N. (2006) Asteropine A, a Sialidase-Inhibiting Conotoxin-like Peptide from the Marine Sponge Asteropus simplex. Chem Biol.13: 569-574.
        Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.

        Abstract:Marine sponges contain structurally intriguing and biologically active peptides of nonribosomal peptide synthase origin, often containing amino acids with novel structures. Here we report the discovery of asteropine A (APA), a cystine knot to be isolated from marine sponges. The solution structure of APA as determined by NMR belongs to the four-loop class of cystine knots similar to those of some conotoxins and spider toxins. However, the highly negatively charged surface of APA is uncommon among other cystine knots. APA competitively inhibits bacterial sialidases, but not a viral sialidase. APA was inactive against all other enzymes tested and did not have any apparent antitumor activity. Our data suggest that APA and other knotting peptides may be important leads for antibacterial and even antiviral drug development.

      29 June, 2006

        Ziconotide(Prialt) - results of a study with 220 patients.

        NEW YORK (Reuters Health) Jun 21 - Dr. Richard L. Rauck and colleagues reported that intrathecal Ziconotide appears to be a safe and effective therapy for severe chronic pain refractory to other agents. To minimize adverse effects, slow titration of the drug to a low maximum dose is required. See J Pain Symptom Manage 31: 393-406

        Summary:Intrathecal opioid therapy often fails to provide relief for patients with severe chronic pain, lead author Dr. Richard L. Rauck, from Wake Forest University School of Medicine in Winston-Salem, North Carolina, and colleagues note. Ziconotide was specifically designed to address this problem. It is a nonopioid analgesic that is the synthetic equivalent of a 25-amino acid peptide found in the venom of Conus magus, a marine snail. Two previous studies showed that ziconotide was significantly better than placebo in providing relief to patients with severe chronic pain. However, in both studies, high rates of serious adverse events and drug discontinuation were noted.The present study aimed to improve the safety profile and tolerability of intrathecal ziconotide by using a slower titration schedule than that used in the previous trials and by trying to achieve a lower maximum dose. A total of 220 patients were randomized to receive placebo or ziconotide, starting at a rate of 0.1 micrograms/hour and increasing gradually over 3 weeks by 0.05-0.1 microgram/hour increments. The final mean dose was 0.29 micrograms/hour, or 6.96 micrograms/day. The researchers' findings appear in the Journal of Pain and Symptom Management for May.
        Based on visual analogue scale results, ziconotide was significantly better than placebo at providing pain relief, the report indicates.Dizziness, confusion, ataxia, abnormal gait, and memory impairment were side effects seen with ziconotide. However, the drug was no more likely than placebo to cause serious adverse events or discontinuation. The degree of pain relief achieved with the slower titration schedule in the current study "was less than that noted in the two previous controlled trials of ziconotide, but better patient retention and in improved safety profile were observed," the authors note. "As the most comprehensively studied intrathecal analgesic in controlled trials, Ziconotide appears to have a place in the management of severe chronic pain."
        The study was funded by Elan Pharmaceuticals, which markets intrathecal ziconotide as Prialt.

      24 June, 2006

        Dimeric alpha-conotoxins VxXIIA, VxXIIB and VxXIIC from Conus vexillum

        Loughnan, M., Nicke, A., Jones, A., Schroeder, C.I., Nevin, S.T., Adam,s D.J., Alewood, P.F., Lewis, R.J. (2006) Identification of a novel class of nicotinic receptor antagonists: dimeric conotoxins VxXIIA, VxXIIB and VxXIIC from Conus vexillum. J Biol Chem. 281: 24745-24755.
        Institute for Molecular Bioscience, Brisbane, Queensland 4072.

        Abstract: The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, alphaD-VxXIIA, alphaD-VxXIIB and alphaD-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an ~11 kDa protein by LC/MS, size exclusion chromatography and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homo-dimers of paired 47-50 residue peptides. The toxin subunits exhibited a novel arrangement of ten conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two electrode voltage clamp analyses showed that alphaD-VxXIIA, alphaD-VxXIIB and alphaD-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for alpha7 and beta2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.

      16 June, 2006

        Conantokin G (from Conus geographus) actions on NMDA glutamate receptor

        Alex, A.B., Baucum, A.J. and Wilcox, K.S. (2006) The Effect of Conantokin G on NMDA Receptor-Mediated Spontaneous EPSCs in Cultured Cortical Neurons. J Neurophysiol. 2006 Jun 7; [Epub ahead of print]
        Anticonvulsant Drug Development Program, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, United States.

        Abstract: Conantokin G (Con G), derived from the venom of Conus geographus, is the most characterized natural peptide antagonist targeted to NMDA receptors. Although Con G is known to bind to the glutamate binding site on the NR2 subunit of the receptor, it is unclear whether it can allosterically modulate the function of the receptor through the glycine binding site on the NR1 subunit. The present study was designed to evaluate the action of Con G on NMDA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) and its modulation by glycine in cultured cortical neurons (13-19 days in vitro) using the whole-cell patch clamp technique. Con G inhibited NMDA receptor-mediated spontaneous EPSCs in a concentration-dependent manner. Also, the potency of Con G decreased as a function of time in culture. The inhibition of EPSCs observed following application of Con G in the presence of high (10 microM) and nominal (no added) concentrations of glycine, were not different at 13 days in vitro (DIV). Further, similar results were obtained with experiments on Con G-induced inhibition of NMDA-evoked whole cell currents. The present results indicate that glycine concentrations do not have a direct effect on Con G-induced inhibition of NMDA currents. In addition, age-dependency in the action of Con G on cortical neurons in vitro suggests that this model system would be useful in examining the effects of different agonists/antagonists on native synaptic NMDA receptors.

        omega-conotoxins GVIA and MVIIC and the spider toxin PhTx3 provide a level of neuroprotection against brain ischemia

        Pinheiro, A.C., Gomez, R.S., Massensini, A.R., Cordeiro, M.N., Richardson, M., Romano-Silva, M.A., Prado, M.A., Marco, L.D. and Gomez, M.V. (2006) Neuroprotective effect on brain injury by neurotoxins from the spider Phoneutria nigriventer. Neurochem Int. 2006 Jun 4; [Epub ahead of print]
        Department of Pharmacology, ICB-UFMG, Av. Antonio Carlos 6627, Belo Horizonte 21270-901, Minas Gerais, Brazil.

        Abstract: The role of calcium channels blockers in ischemic condition has been well documented. The PhTx3 neurotoxic fraction of the spider Phoneutria nigriventer venom is a broad-spectrum calcium channel blocker that inhibits glutamate release, calcium uptake and also glutamate uptake in synaptosomes. In the present study we describe the effect of PhTx3 (1.0mug/mL), omega-conotoxin GVIA (1.0mumol/L) and omega-conotoxin MVIIC (100nmol/L) on neuroprotection of hippocampal slices and SN56 cells subjected to ischemia by oxygen deprivation and low glucose insult (ODLG). After the insult, cell viability in the slices and SN56 cells was assessed by confocal microscopy and epifluorescence, using live/dead kit containing calcein-AM and ethidium homodimer. Confocal images of CA1 region of the rat hippocampal slices subjected to ischemia insult and treated with omega-conotoxin GVIA, omega-conotoxin MVIIC and PhTx3 showed a percentage of dead cells of 68%, 54% and 18%, respectively. The SN56 cells subjected to ischemia were almost completely protected from damage by PhTx3 while with omega-conotoxin GVIA or omega-conotoxin MVIIC the cell protection was only partial. Thus, PhTx3 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of brain ischemia.

      9 June, 2006

        Novel conotoxins from Conus leopardus

        Chen, W.H.(1,2), Han, Y.H.(2,3), Wang, Q.(2,3), Miao, X.W.(2), Ou, L.(1) and Shao X.X.(2) (2006) cDNA cloning of two novel T-superfamily conotoxins from Conus leopardus. Acta Biochim Biophys Sin (Shanghai). 38: 287-291.
        1 Bioengineering Institute, East China University of Science and Technology, Shanghai 200237, China
        2. Institute of Protein Research, Tongji University, Shanghai 200092, China.
        3 Institute of Biochemistry and Cell Biology, Shanghai Institute of Biochemical Sciences, Chinese Academy of Sciences, Shanghai 200031, China
        4 Department of Biological Engineering, School of Life Science, Shanghai University, Shanghai 200436, China

        Abstract: The full-length cDNAs of two novel T-superfamily conotoxins, Lp5.1 and Lp5.2, were cloned from a vermivorous cone snai l Conus leopardus using 3'/5'-rapid amplification of cDNA ends. The cDNA of Lp5.1 encodes a precursor of 65 residues, including a 22-residue signal peptide, a 28-residue propeptide and a 15-residue mature peptide. Lp5.1 is processed at the common signal site-X-Arg- immediately before the mature peptide sequences. In the case of Lp5.2, the precursor includes a 25-residue signal peptide and a 43-residue sequence comprising the propeptide and mature peptide, which is probably cleaved to yield a 29-residue propeptide and a 14-residue mature toxin. Although these two conotoxins share a similar signal sequence and a conserved disulfide pattern with the known T-superfamily, the pro-region and mature peptides are of low identity, especially Lp5.2 with an identity as low as 10.7% compared with the reference Mr5.1a. The elucidated cDNAs of these two toxins will facilitate a better understanding of the species distribution, the sequence diversity of T-superfamily conotoxins, the special gene structure and the evolution of these peptides.

        Characterization of alpha-conotoxin Vc1.1 from Conus victoriae

        Clark, R.J., Fischer, H., Nevin, S.T., Adams, D.J. and Craik, D.J. (2006) The synthesis, structural chracterisation and receptor specificity of the alpha -conotoxin Vc1.1. J Biol Chem. 2006 281(32): p. 23254-23263.
        Chemistry and Structural Biology, Institute for Molecular Bioscience, Brisbane , QLD 4072.

        Abstract: The alpha-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. This study describes the synthesis, determination of the disulfide connectivity and the determination of the three dimensional structure of Vc1.1 using NMR spectroscopy. Vc1.1 was shown to inhibit nicotine-evoked membrane currents in isolated bovine chromaffin cells in a concentration dependant manner and preferentially targets peripheral nAChR subtypes over central subtypes. Specifically, Vc1.1 is selective for a3-containing nAChR subtypes. The three dimensional structure of Vc1.1 comprises a small a-helix spanning residues P6 to D11 and is braced by the I-II, III-IV disulfide connectivity seen in other a-conotoxins. A comparison of the structure of Vc1.1 with other a-conotoxins, taken together with nAChR selectivity data, suggests that the conserved proline at position 6 is important for binding while a number of residues in the C-terminal portion of the peptide contribute towards the selectivity. The structure reported here should open new opportunities for further development of Vc1.1 or analogues as analgesic agents.

        3-D Model of alpha-Conotoxin Vc1.1

        The PDB coordinates of Vc1.1 have been released and can be found under ID code # 2H8S following the link:

        http://www.rcsb.org/pdb/explore.do?structureId=2H8S

      7 June, 2006

        Hyperhydroxylation in cone shells: Conotoxins, mini-M-conotoxins, conophans and g-hydroxyconophans

        Aldo Franco, Carolina Moller, Katarzyna Pisarewicz, David Mora, Gregg B. Fields, and Frank Mari* (2006), “Hyperhydroxylation: A New Strategy for Neuronal Targeting by Venomous Marine Molluscs” in Progress in Molecular and Subcellular Biology, Subseries Marine Molecular Biotechnology: Molluscs” Chapter 4, pp: 83-103, G. Cimino and M. Gavagnin (Eds.), Springer-Verlag, Berlin Heidelbeerg, Germany.
        Department of Chemistry & Biochemistry and Center of Excellence in Biomedical & Marine Biotechnology, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL, 33431.U.S.A.
        *Corresponding author, email: mari@fau.edu, Fax 001-561-2972759, Tel. 001-561-2973316

        Abstract: Venomous marine molluscs belonging to the genus Conus (cones snails) utilize a unique neurochemical strategy to capture their prey. Their venom is composed of a complex mixture of highly modified peptides (conopeptides) that interact with a wide range of neuronal targets. Here we describe an unprecedented set of modifications based upon hydroxylation of polypeptidic chains that are defining within the neurochemical strategy used by cone snails to capture their prey. We present a differential hydroxylation strategy that affects the neuronal targeting of a new set of a-conotoxins, mini-M-conotoxins, conophans and g-hydroxyconophans. We have observed differential hydroxylation, preferential hydroxylation and hyperhydroxylation in these conopeptide families as means of augmenting the venom arsenal used by cone snails for neuronal targeting and prey capturing.

      1 June, 2006

        alpha-conotoxin BuIA used to reveal novel alpha3beta4 nAChRs in hippocampus

        Layla Azam 1 J. Michael McIntosh 2* (2006) Characterization of nicotinic acetylcholine receptors that modulate nicotine-evoked [3H]norepinephrine release from mouse hippocampal synaptosomes.
        1 University of Utah, Dept. of Biology 2 University of Utah, Departments of Biology and Psychiatry
        * Address correspondence to: E-mail: mcintosh.mike@gmail.com

        Abstract: Nicotine's modulation of hippocampal noradrenergic neurotransmission may contribute to its mnemonic properties, but the nAChR subtypes that modulate terminal release of norepinephrine are unknown. In the present study, we have used a number of subtype selective alpha-conotoxins in combination with nicotinic receptor subunit deficient mice to characterize nAChRs that modulate [3H]norepinephrine release from synaptosomes.The results indicate that at least 2 populations of nAChRs contribute to this release. These are a novel novel alpha6(alpha4)beta2,3,4 subtype and an alpha6alpha4beta2beta3 subtype. These are distinct from subtypes that modulate synaptosomal norepinephrine release in the rat hippocampus where a beta2 and/or beta4 ligand-binding interface is not present. Whereas alpha-conotoxin BuIA, a toxin that kinetically distinguishes between beta4-containing nAChRs, was partially reversible in mouse but irreversible in rat. This indicates that in contrast to rat, mouse nAChRs are made of both beta4-containing populations. Results from beta4 null mutant mice confirmed this conclusion, indicating the presence of the beta4 subunit in a subpopulation of nAChRs. Additionally, both beta3 subunits are essential for formation of functional nAChRs on mouse noradrenergic terminals. Cytisine, a ligand formerly believed to be alpha6beta2-containing nAChRs. The sum of these results suggests a possible novel nAChR subtype that modulates noradrenergic neurotransmission within the mouse hippocampus.

      31 May, 2006

        Analgesia produced by a conotoxin that blocks sodium channels

        Grzegorz Bulaj, Min-Min Zhang, Brad R. Green, Brian Fiedler, Richard T. Layer, Sue Wei, Jacob S. Nielsen, Scott J. Low, Brian D. Klein, John D. Wagstaff, Linda Chicoine, T. Patrick Harty, Heinrich Terlau, Doju Yoshikami and Baldomero M. Olivera (2006) Synthetic muO-conotoxin MrVIB blocks TTX-resistant sodium channel NaV1.8 and has a long-lasting analgesic activity. Biochemistry. 45 :7404-7414.
        Department of Biology, The UniVersity of Utah, Salt Lake City, Utah 84112, Cognetix, Inc., 421 Wakara Way, Salt Lake City, Utah 84108, and Molecular and Cellular Neuropharmacology Group, Max Plank Institute for Experimental Medicine, Goettingen, Germany

        Abstract: muO-Conotoxin MrVIB is a blocker of voltage-gated sodium channels, including TTX-sensitive and -resistant subtypes. A comprehensive characterization of this peptide has been hampered by the lack of sufficient synthetic material. Here, we describe the successful chemical synthesis and oxidative folding of MrVIB that has made an investigation of the pharmacological properties and therapeutic potential of the peptide feasible. We show for the first time that synthetic MrVIB blocks rat NaV1.8 sodium channels and has potent and long-lasting local anesthetic effects when tested in two pain assays in rats. Furthermore, MrVIB can block propagation of action potentials in A- and C-fibers in sciatic nerve as well as skeletal muscle in isolated preparations from rat. Our work provides the first example of analgesia produced by a conotoxin that blocks sodium channels. The emerging diversity of antinociceptive mechanisms targeted by different classes of conotoxins is discussed.

        Oncomodulin - a new central and peripheral nerve growth factor

        Yin Y, Henzl MT, Lorber B, Nakazawa T, Thomas TT, Jiang F, Langer R, Benowitz LI. (2006) Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells. Nat Neurosci. 9: 843-852.
        Department of Neurosurgery and Neurobiology Program, Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA.

        Abstract: The optic nerve, like most mature CNS pathways, does not regenerate after injury. Through unknown mechanisms, however, macrophage activation in the eye stimulates retinal ganglion cells (RGCs) to regenerate long axons beyond the site of optic nerve injury. Here we identify the calcium (Ca(2+))-binding protein oncomodulin as a potent macrophage-derived growth factor for RGCs and other neurons. Oncomodulin binds to rat RGCs with high affinity in a cyclic AMP (cAMP)-dependent manner and stimulates more extensive outgrowth than other known trophic agents. Depletion of oncomodulin from macrophage-conditioned media (MCM) eliminates the axon-promoting activity of MCM. The effects of oncomodulin involve downstream signaling via Ca(2+)/calmodulin kinase and gene transcription. In vivo, oncomodulin released from microspheres promotes regeneration in the mature rat optic nerve. Oncomodulin also stimulates outgrowth from peripheral sensory neurons. Thus, oncomodulin is a new growth factor for neurons of the mature central and peripheral nervous systems.

        See also Nature Neuroscience 9, 715-717 (01 Jun 2006) News and Views "How inflammation promotes regeneration" by Marie T Filbin. SUMMARY: Macrophages near neuronal cell bodies can promote regeneration in an otherwise inhibitory environment. Now Yin et al. Nat Neurosci. 9: 843-852 identify oncomodulin as a factor secreted from macrophages. (Click for Figure 1, Table 1).

      30 May, 2006

        Intrathecal Ziconotide (Prialt) Trial

        Rauck, R.L., Wallace, M.S., Leong, M.S., Minehart, M., Webster, L.R., Charapata, S.G., Abraham, J.E., Buffington, D.E., Ellis, D., Kartzinel, R., The Ziconotide 301 Study Group. (2006) A Randomized, Double-Blind, Placebo-Controlled Study of Intrathecal Ziconotide in Adults with Severe Chronic Pain. J Pain Symptom Manage. 31: 393-406.
        Wake Forest University School of Medicine (R.L.R.), Winston-Salem, North Carolina; Center for Pain and Palliative Medicine (M.S.W.), University of California, La Jolla, California; Bay Area Pain Center (M.S.L.), Los Gatos, California; Advanced Pain Institute (M.M.), Duarte, California; Lifetree Clinical Research and Pain Clinic (L.R.W.), Salt Lake City, Utah; Pain Management Associates (S.G.C.), Research Medical Center, Pain Institute, Kansas City, Missouri; Hot Springs Pain Clinic (J.E.A.), Hot Springs, Arkansas; University of South Florida College of Medicine (D.E.B.), Tampa, Florida; and Elan Pharmaceuticals Inc. (D.E., R.K.), San Diego, California.

        Abstract: Safety and efficacy data from a study of slow intrathecal (IT) ziconotide titration for the management of severe chronic pain are presented. Patients randomized to ziconotide (n=112) or placebo (n=108) started IT infusion at 0.1mug/hour (2.4mug/day), increasing gradually (0.05-0.1mug/hour increments) over 3 weeks. The ziconotide mean dose at termination was 0.29mug/hour (6.96mug/day). Patients' baseline Visual Analogue Scale of Pain Intensity (VASPI) score was 80.7 (SD 15). Statistical significance was noted for VASPI mean percentage improvement, baseline to Week 3 (ziconotide [14.7%] vs. placebo [7.2%; P=0.036]) and many of the secondary efficacy outcomes measures. Significant adverse events (AEs) reported in the ziconotide group were dizziness, confusion, ataxia, abnormal gait, and memory impairment. Discontinuation rates for AEs and serious AEs were comparable for both groups. Slow titration of ziconotide, a nonopioid analgesic, to a low maximum dose resulted in significant improvement in pain and was better tolerated than in two previous controlled trials that used a faster titration to a higher mean dose.

        Ziconotide (Prialt): A REVIEW

        Lyseng-Williamson, K.A. and Perry, C. (2006) Ziconotide. CNS Drugs. 20:331-338; discussion 339-41.
        Adis International Limited, Auckland, New Zealand. demail@adis.co.nz

        Abstract: Ziconotide, an intrathecal analgesic for the management of chronic intractable pain, binds with high affinity to N-type calcium channels in neuronal tissue and obstructs neurotransmission. In three pivotal, well designed trials of 5-6 or 21 ays' duration, titrated ziconotide was significantly more effective than placebo in treating chronic malignant or nonmalignant pain as assessed by mean percentage improvements from baseline in Visual Analogue Scale Pain Intensity scores. Improvements in secondary endpoints (e.g. proportion of patients who responded or achieved pain relief and the change in opioid use) generally support the efficacy of ziconotide over placebo. Ziconotide maintains its analgesic efficacy in preliminary results from long-term, open-label trials (data available for up to 12 months). Most ziconotide-related adverse events are neurological, mild to moderate in severity, resolve over time and reverse without sequelae on drug discontinuation. Low initial doses of ziconotide and gradual titration to onset of analgesia reduces the incidence and severity of dverse events. No evidence of respiratory depression has been reported with intrathecal ziconotide.

        alpha-conotoxin AuIB used to identify alpha3beta4 subunit combination as major functional nicotinic receptor in pelvic ganglion

        Park, K.S., Cha, S.K., Kim, M.J., Kim, D.R., Jeong, S.W., Lee, J.W., Kong, I.D. (2006) An alpha3beta4 subunit combination acts as a major functional nicotinic acetylcholine receptor in male rat pelvic ganglion neurons. Pflugers Arch. 2006 May 20; [Epub ahead of print]
        Department of Physiology and Institute of Basic Medical Science, Yonsei University Wonju College of Medicine, Wonju, South Korea, kong@yonsei.ac.kr.

        Abstract: We identified major subunits of the nicotinic acetylcholine receptor (nAChR) involved in excitatory postsynaptic potential and intracellular Ca(2+) ([Ca(2+)]( i )) increase in the major pelvic ganglion (MPG) neurons of the male rat. ACh elicited fast inward currents in both sympathetic and parasympathetic MPG neurons. Mecamylamine, a selective antagonist for alpha3beta4 nAChR, potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons (IC(50); 0.53 and 0.22 muM, respectively). Furthermore, alpha-conotoxin AuIB (10 muM), a new selective antagonist for alpha3beta4 nAChR, blocked more than 80% of the ACh-induced currents in MPG neurons. Conversely, alpha-bungarotoxin, alpha-methyllycaconitine, and dihydro-beta-erythroidine, known as blockers of the alpha7 or alpha4beta2, did not show selective blocking effects on MPG neurons. ACh transiently increased [Ca(2+)]( i ) which was subsequently abolished in the extracellular Ca(2+)-free environment. Simultaneous recording of [Ca(2+)]( i ) and ionic currents revealed that ACh increased [Ca(2+)]( i ) under the conditions of the voltage-clamped (at -80 mV) state, and this resulted from the influx through nAChR itself. ACh-induced [Ca(2+)]( i ) increase was blocked by mecamylamine (10 muM), but was not affected by atropine (1 muM). RT-PCR analysis showed that, among subunits of nAChR, alpha3 and beta4 were predominantly expressed in MPG. We suggest that activation of alpha3 and beta4 nAChR subunits in MPG neurons induce fast inward currents and [Ca(2+)]( i ) increase, possibly mediating a major role in pelvic autonomic synaptic transmission.

        alpha-conotoxin MII and alpha-conotoxin AuIB used to identify functional nicotinic receptors involved in excitation of cardiac vagal neurons

        Kamendi, H., Stephens, C., Dergacheva, O., Wang, X., Huang, Z.G., Bouairi, E., Gorini, C., McIntosh, J.M., Mendelowitz, D.(2006) Prenatal nicotine exposure alters the nicotinic receptor subtypes that modulate excitation of parasympathetic cardiac neurons in the nucleus ambiguus from primarily alpha3beta2 and/or alpha6betaX to alpha3beta4. Neuropharmacology. 2006 May 8; [Epub ahead of print]
        Department of Pharmacology and Physiology, George Washington University, 2300 Eye Street, N.W. Washington, DC 20037, USA.

        Abstract: Nicotinic receptors play an essential role in central cardiorespiratory function, however, the types of nicotinic receptors responsible for activating cardiac vagal neurons in the nucleus ambiguus that control heart rate are unknown. This study tests whether alpha-conotoxin MII and alpha-conotoxin AuIB sensitive nicotinic receptors are involved in augmentation of glutamatergic neurotransmission and changes in holding current in cardiac vagal neurons, and whether exposure to nicotine in the prenatal period alters these responses. The nicotinic agonist cytisine significantly increased the holding current and amplitude of glutamatergic mEPSCs. In unexposed animals alpha-conotoxin MII (100nM) significantly reduced the increase in mEPSC amplitude and change in holding current evoked by cytisine. However, in animals prenatally exposed to nicotine, alpha-conotoxin MII blunted but did not block the increase in mEPSC amplitude but blocked the increase in holding current evoked by cytisine. In unexposed animals, alpha-conotoxin AuIB (10muM) blocked the cytisine evoked increase in mEPSC amplitude and inhibited but did not abolish the increase in holding current. In contrast, in animals exposed to nicotine, alpha-conotoxin AuIB blunted the increase in mEPSC amplitude, and completely abolished the cytisine evoked increase in holding current. These data demonstrate that the prenatal nicotine exposure alters the nicotinic receptors involved in excitation of cardiac vagal neurons.

        NMR structures of the interaction of conotoxin GI with the muscle-type nicotinic acetylcholine receptor

        Kasheverov, I.E., Chiara, D.C., Zhmak, M.N., Maslennikov, I.V., Pashkov, V.S., Arseniev, A.S., Utkin, Y.N., Cohen, J.B., Tsetlin, V.I. (2006) alpha-Conotoxin GI benzoylphenylalanine derivatives. (1)H-NMR structures and photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor. FEBS J. 273:1373-1388.
        Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

        Abstract: alpha-Conotoxins are small peptides from cone snail venoms that function as nicotinic acetylcholine receptor (nAChR)-competitive antagonists differentiating between nAChR subtypes. Current understanding about the mechanism of these selective interactions is based largely on mutational analyses, which identify amino acids in the toxin and nAChR that determine the energetics of ligand binding. To identify regions of the nAChR involved in alpha-conotoxin binding by use of photoactivated cross-linking, two benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI, GI(Bpa12) and GI(Bpa4), were synthesized by replacing the respective residues with Bpa, and their (1)H-NMR structures were determined. Both analogs preserved the GI conformation, but only GI(Bpa12) displaced (125)I-labeled GI from the Torpedo californica nAChR. (125)I-labeled GI(Bpa12) bound to two sites on the receptor (K(d) 13 and 1800 nM), and on UV irradiation specifically photolabeled the alpha, gamma and delta subunits. Photolabeling sites were mapped by selective proteolysis and enzymatic deglycosylation, combined with SDS/PAGE, HPLC and Edman degradation. In the alpha subunit, cobratoxin-inhibited incorporation was limited to the 22-kDa fragment beginning at alphaSer173 and containing the agonist-binding site segment C. In the gamma subunit, radioactivity was localized to two distinct peptides containing agonist-binding site segments F and D: nonglycosylated 24-kDa and glycosylated 13-kDa fragments starting at gammaAla167 and gammaAla49, respectively. The labeling of these fragments is discussed in terms of a model of GI(Bpa12) bound to the extracellular domain of the Torpedo nAChR homology model derived from the cryo-electron microscopy structure of Torpedo marmorata nAChR and X-ray crystal structures of snail acetylcholine-binding protein complexes with agonists and antagonists.

        alpha-conotoxins - a REVIEW

        [No authors listed] [Natural alpha-conotoxins and their synthetic analogues in study of nicotinic acetylcholine receptors] [REVIEW Article in Russian] Bioorg Khim. 2006 Mar-Apr;32(2):115-29.

        Abstract: alpha-Conotoxins, peptide neurotoxins from poisonous marine snails of the genus Conus that highly specifically block nicotinic acetylcholine receptors (AChRs) of various types, are reviewed. Preliminarily, the structural organization of AChRs of the muscular and neuronal types, their involvement in physiological processes, and their role in various diseases are briefly discussed. In this connection, the necessity of quantitative determination of AChR subtypes using neurotoxins and other approaches is substantiated. The chemical structure, spatial organization, and specificity of alpha-conotoxins are mainly discussed, taking into consideration the recent results on the ability of alpha-conotoxins to interact with muscular or neuronal hetero- and homooligomeric AChRs exhibiting a high species specificity. Particular emphasis is placed upon a thorough characterization of the surfaces of interaction of alpha-conotoxins with AChRs using synthetic analogues of alpha-conotoxins, mutations in AChRs, and pairwise mutations in both alpha-conotoxins and AChRs. The discovery in 2001 of the acetylcholine-binding protein from the pond snail Lymnaea stagnalis and the determination of its crystalline structure led to rapid progress in understanding the structural organization of ligand-binding domains of AChRs with which alpha-conotoxins also interact. We discuss the interaction of various alpha-conotoxins with acetylcholine-binding proteins, the recently reported X-ray structure of the complex of the acetylcholine-binding protein from Aplysia californica with the alpha-conotoxin analogue PnIA, and the application of this structure to the modeling of complexes of alpha-conotoxins with various AChRs.

          See also :

          Mordvintsev, D.Y., Polyak, Y.L., Levtsova, O.V., Tourleigh, Y.V., Kasheverov, I.E., Shaitan, K.V., Utkin, Y.N., Tsetlin, V.I. (2005) A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins. Comput Biol Chem. 29:398-411.
          Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B. (2005) Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat Struct Mol Biol. 12:582-588.
          Vulfius, C.A., Tumina, O.B., Kasheverov, I.E., Utkin, Y.N., Tsetlin, V.I. (2005) Diversity of nicotinic receptors mediating Cl- current in Lymnaea neurons distinguished with specific agonists and antagonist. Neurosci Lett. 373: 232-236.
          Kasheverov, I.E., Zhmak, M.N., Maslennikov, I.V., Utkin, Y.N., Tsetlin, V.I. (2003) A comparative study on selectivity of alpha-conotoxins GI and ImI using their synthetic analogues and derivatives. Neurochem Res.28: 599-606.
          Kasheverov, I.E., Rozhkova, A.M., Zhmak, M.N., Utkin, Iu,N., Tsetlin, V.I. (2001) [Photoactivatable analogues of alpha-conotoxins GI and MI and their interaction with nicotinic acetylcholine receptor] Bioorg Khim. 28: 101-108. Russian.
          Kasheverov, I., Rozhkova, A., Zhmak, M., Utkin, Y., Ivanov, V., Tsetlin, V.I. (2001) Photoactivatable alpha-conotoxins reveal contacts with all subunits as well as antagonist-induced rearrangements in the Torpedo californica acetylcholine receptor. Eur J Biochem. 268: 3664-3673.
          Zhmak, M.N., Kasheverov, I.E., Utkin, Iu.N., Tsetlin, V.I., Vol'pina, O.M., Ivanov, V.T. (2001) [Efficient synthesis of natural alpha-conotoxins and their analogs] Bioorg Khim.27: 83-88. Russian.
          Zolotarev, Iu.A., Bocharov, E.V., Dadaian, A.K., Kasheverov, I.E., Zhmak, M.N., Maslennikov, I.V., Borisov, Iu.A., Arsen'ev, A.S, Miasoedov, N.F., Petlin, V.I.(2000) [Study of solid-phase catalytic isotopic exchange of hydrogen in alpha-conotoxin G1 under the effect of spillover-tritium] Bioorg Khim.26: 587-592. Russian.
          Utkin, I.uN., Kasheverov, I.E., Tsetlin, V.I. (1999) [alpha-Neurotoxins and alpha-conotoxins--nicotinic cholinoreceptor blockers] Bioorg Khim.25: 805-810. Review. Russian.
          Kasheverov, I., Zhmak, M., Chivilyov, E., Saez-Brionez, P., Utkin, Y., Hucho, F., Tsetlin, V.(1999) Benzophenone-type photoactivatable derivatives of alpha-neurotoxins and alpha-conotoxins in studies on Torpedo nicotinic acetylcholine receptor. J Recept Signal Transduct Res. 19: 559-571.

      29 May, 2006

        I-superfamily of Conotoxins

        Mondal S, Vijayan R, Shichina K, Babu RM, Ramakumar S.(2005) I-superfamily conotoxins: sequence and structure analysis. In Silico Biol. 5: 557-571.
        Department of Physics, Indian Institute of Science, Bangalore 560 012, India.

        Abstract: I-superfamily conotoxins have four-disulfide bonds with cysteine arrangement C-C-CC-CC-C-C, and they inhibit or modify ion channels of nerve cells. They have been characterized only recently and are relatively less well studied compared to other superfamily conotoxins. We have detected selective and sensitive sequence pattern for I-superfamily conotoxins. The availability of sequence pattern should be useful in protein family classification and functional annotation. We have built by homology modeling, a theoretical structural 3D model of ViTx from Conus virgo, a typical member of I-superfamily conotoxins. The modeling was based on the available 3D structure of Janus-atracotoxin-Hv1c of Janus-atracotoxin family whose members have been suggested as possible biopesticides. A study comparing the theoretically modeled structure of ViTx, with experimentally determined structures of other toxins, which share functional similarity with ViTx, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels.

        Dutertre, S. and Lewis, R.J.(2006) Toxin insights into nicotinic acetylcholine receptors. Biochem Pharmacol. 2006 Apr 22; [Epub ahead of print]
        Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.

        Abstract: Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. In the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin ImI. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR.

      20 May, 2006

        New Caledonia stamp featuring Conus geographus and omega-conotoxin MVIIA !

        Tom Walker (tom@tmwalker.co.uk) has drawn my attention to a new stamp issued 12 April 2006 by New Caledonia, 150 FF, depicting Conus geogaphus together with a chemical structure of omega-conotoxin MVIIA, titled "Recherche Contre La Douleur" ("Research on Pain")

        Although the cone shell depicted on this stamp is Conus geographus, the peptide sequence depicted is from Conus magus (the "Cone of Maggi", or the Magician's cone). This cone shell venom peptide component is a powerful analgesic in humans. This 3-loop conotoxin is from Conus magus, NOT from Conus geographus! Conotoxin MVIIA (also known as Ziconotide, Prialt), is a powerful analgesic available in the US and Europe for treatment of neuropathic pain (Refs. "A toxin against pain", Gary Stix, Scientific American, April 2005, pp. 70-75 ; and "Toward Better Pain Control", Allan Bausbaum and David Julius, Scientific American, June 2006, pp. 60-67). The 6-6-0-3-4 loop structure is typical of omega conotoxins (but also found in other classes - King Kong conopeptide and delta-conotoxins for example.

        See Cone Shells on Stamps (Scroll down to Conus geographus).

      18 May, 2006

        Novel mu-conotoxins (CnIIIA & CnIIIB from C. consors, CIIIA from C. catus, and MIIIA from C. magus) that target TTX-resistant Sodium channels

        Min-Min Zhang, Brian Fiedler, Brad R. Green, Phillip Catlin, Maren Watkins, James E. Garrett, Brian J. Smith, Doju Yoshikami, Baldomero M. Olivera, and Grzegorz Bulaj (2006) Structural and Functional Diversities among mu-Conotoxins Targeting TTX-resistant Sodium Channels.Biochemistry; 45: 3723 - 3732. (click here)
        Departments of Biology, University of Utah, Salt Lake City 84112, USA.

        Abstract: mu-Conotoxins are peptides that block sodium channels. Molecular cloning was used to identify four novel mu-conotoxins: CnIIIA, CnIIIB, CIIIA, and MIIIA from Conus consors, C. catus and C. magus. A comparison of their sequences with those of previously characterized mu-conotoxins suggested that the new mu-conotoxins were likely to target tetrodotoxin-resistant (TTX-r) sodium channels. The four peptides were chemically synthesized, and their biological activities were characterized. The new conotoxins all blocked, albeit with varying potencies, TTX-r sodium currents in frog dorsal-root-ganglion (DRG) neurons. The more potent of the four new mu-conotoxins, CnIIIA and CIIIA, exhibited a strikingly different selectivity profile in blocking TTX-r versus TTX-sensitive channels, as determined by their ability to block extracellularly recorded action potentials in three preparations from frog: skeletal muscle, cardiac muscle and TTX-treated C-fibers. CnIIIA was highly specific for TTX-r sodium channels, whereas CIIIA was nonselective. Both peptides appeared significantly less potent in blocking TTX-r sodium currents in rat and mouse DRG neurons. When CnIIIA and CIIIA were injected intracranially into mice, both induced seizures, but only CIIIA caused paralysis. This is the most comprehensive characterization to date of the structural and functional diversities of an emerging group of mu-conotoxins targeting TTX-r sodium channels.

      24 April, 2006

        Conus vexillum: Two novel O-superfamily conotoxins

        Jiang, H., Xu, C.Q., Wang, C.Z., Fan, C.X., Zhao, T.Y., Chen, J.S., Chi, C.W. (2006) Two novel O-superfamily conotoxins from Conus vexillum.Toxicon. 47 (4): 425-436.
        Key Laboratory of Proteomics, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.

        Abstract: O-superfamily conotoxins include several families that have diverse pharmacological activity on Na+, K+ or Ca2+ channels. These superfamily toxins have been mainly found in fish-hunting and mollusk-hunting Conus species. Here, we reported two novel O-superfamily conotoxins, vx6a and vx6b, purified from a worm-hunting cone snail, Conus vexillum. Though their cysteine framework and signal peptides share high similarity with those of other members of O-superfamily, the mature vx6a and vx6b both have a low sequence homology with others. To test the biological function of vx6a, the toxin was chemically synthesized and then tested on the locust dorsal unpaired median (DUM) neuron system which containing various ion channels. Although no any activity on ion channels was found on the DUM neuron system, vx6a could clearly elicit a series of symptoms in mouse via intracranial injection, such as quivering, climbing, scratching, barrel rolling and paralysis of limbs at different dose.

      23 April, 2006

      31 March, 2006

        Cone Snail Movies and Envenomation

        Anderson, T. and Kohn, A. (2006) IMAGES: Mollusks With Attitude See, Science: 311, 1079 -.available on the web at http://biology.burke.washington.edu/conus
        Burke Museum of Natural History and Culture in Seattle, Washington, USA.

        Abstract: One of the newest painkillers on the market, ziconitide, comes from the sting of a snail--not the ones that demolish your cucumbers but their marine cousins, the cone snails of the genus Conus. The rapacious creatures subdue fish and other animals with their poison-tipped mouthparts. To help taxonomists tidy up this complicated group, Trevor Anderson and Alan Kohn of the Burke Museum of Natural History and Culture in Seattle, Washington, launched this catalog of the more than 3000 known Conus species. Mollusk mavens can also peruse photos and drawings of more than 600 type specimens, the original samples researchers used to delineate a species or other taxonomic group. Pioneering classifier Carolus Linnaeus consulted the Conus marmoreus specimen above when he named the genus in 1758. A dozen or so video clips show the predatory snails ambushing and gobbling their victims. Anderson and Kohn plan to add species accounts with range maps and other data.

        Envenomation by a cone snail. Web link: https://www.prialt.com/eprofessionalpharma_enu/html/conus_wm_high.html

      30 March, 2006

        Conotoxins: Therapeutic Potential and Application: REVIEW

        Layer, R.T. and McIntosh, J.M. (2006) Conotoxins: Therapeutic Potential and Application. Mar. Drugs 4: 119-142.
        Cognetix, Inc. 421 Wakara Way, Suite 201, Salt Lake City, UT 84108, USA. E-mail: rlayer@cognetix.com, and Department of Psychiatry and Dept. of Biology, University of Utah, 257 S. 1400 E., Salt Lake City, UT 84112, USA. E-mail: mcintosh.mike@gmail.com

        Abstract: The pharmacological variety of conotoxins, diverse petides found in the venoms of marine cone snails, is well recognized. Venoms from each of the estimated 500 species of cone snails contain 50 to 200 distinct biologically active peptides. Most conotoxins characterized to date target receptors and ion channels of excitable tissues, such as ligand-gated nicotinic acetylcholine, N-methyl-D-aspartate, and type 3 serotonin receptors, as well as voltage-gated calcium, sodium, and potassium channels, and G-protein-coupled recetpors including alpha-adrenergic, neurotensin, and vaospressin receptors, and the norepinephrine transporter. Several conotoxins have shown promise in preclinical models of pain, convulsive disorters, stroke, neuromuscular block. and cardioprotection. The pharmacological selectivity of the conotoxins, coupled with the safety and efficacy demonstrated in preclinical models, has led to their investigation as human therapeutic agents. In the following review, we will survey the pharmacology and therapeutic rationale of those conotoxins with potential clinical application, and discuss the unique challenges that each will face in the course of their transition from venom component to human therapeutic.

        Mechanisms of neuropathic pain: REVIEW

        Moalem, G. and Tracey, D.J. (2005) Immune and inflammatory mechanisms in neuropathic pain. Brain Res Brain Res Rev. 2005 Dec 30; [Epub ahead of print]
        School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia.

        Abstract: Tissue damage, inflammation or injury of the nervous system may result in chronic neuropathic pain characterised by increased sensitivity to painful stimuli (hyperalgesia), the perception of innocuous stimuli as painful (allodynia) and spontaneous pain. Neuropathic pain has been described in about 1% of the US population, is often severely debilitating and largely resistant to treatment. Animal models of peripheral neuropathic pain are now available in which the mechanisms underlying hyperalgesia and allodynia due to nerve injury or nerve inflammation can be analysed. Recently, it has become clear that inflammatory and immune mechanisms both in the periphery and the central nervous system play an important role in neuropathic pain. Infiltration of inflammatory cells, as well as activation of resident immune cells in response to nervous system damage, leads to subsequent production and secretion of various inflammatory mediators. These mediators promote neuroimmune activation and can sensitise primary afferent neurones and contribute to pain hypersensitivity. Inflammatory cells such as mast cells, neutrophils, macrophages and T lymphocytes have all been implicated, as have immune-like glial cells such as microglia and astrocytes. In addition, the immune response plays an important role in demyelinating neuropathies such as multiple sclerosis (MS), in which pain is a common symptom, and an animal model of MS-related pain has recently been demonstrated. Here, we will briefly review some of the milestones in research that have led to an increased awareness of the contribution of immune and inflammatory systems to neuropathic pain and then review in more detail the role of immune cells and inflammatory mediators.

      22 March, 2006

        Ziconotide (Prialt) reviewed

        Hussar DA. (2006) New drugs 06, part I. Nursing. 36:54-61; quiz 62-63.
        Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA, USA.

        Abstract: Summaries for 12 new drugs are offered. The drugs include the cone shell derived analgesic ziconotide (Prialt).

        Clinical rationale for using Prialt

        Snutch TP. Targeting chronic and neuropathic pain: the N-type calcium channel comes of age. NeuroRx. 2: 662-670.
        Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4. snutch@msl.ubc.ca

        Abstract: The rapid entry of calcium into cells through activation of voltage-gated calcium channels directly affects membrane potential and contributes to electrical excitability, repetitive firing patterns, excitation-contraction coupling, and gene expression. At presynaptic nerve terminals, calcium entry is the initial trigger mediating the release of neurotransmitters via the calcium-dependent fusion of synaptic vesicles and involves interactions with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex of synaptic release proteins. Physiological factors or drugs that affect either presynaptic calcium channel activity or the efficacy of calcium-dependent vesicle fusion have dramatic consequences on synaptic transmission, including that mediating pain signaling. The N-type calcium channel exhibits a number of characteristics that make it an attractive target for therapeutic intervention concerning chronic and neuropathic pain conditions. Within the past year, both U.S. and European regulatory agencies have approved the use of the cationic peptide Prialt for the treatment of intractable pain. Prialt is the first N-type calcium channel blocker approved for clinical use and represents the first new proven mechanism of action for chronic pain intervention in many years. The present review discusses the rationale behind targeting the N-type calcium channel, some of the limitations confronting the widespread clinical application of Prialt, and outlines possible strategies to improve upon Prialt's relatively narrow therapeutic window.

        Kohlmeier, K.A. and Leonard, C.S. (2005) Transmitter modulation of spike-evoked calcium transients in arousal related neurons: muscarinic inhibition of SNX-482-sensitive calcium influx. Eur J Neurosci. 23:1151-1162.
        Department of Physiology, New York Medical College, Valhalla, NY 10595, USA.

        Abstract: Nitric oxide synthase (NOS)-containing cholinergic neurons in the laterodorsal tegmentum (LDT) influence behavioral and motivational states through their projections to the thalamus, ventral tegmental area and a brainstem 'rapid eye movement (REM)-induction' site. Action potential-evoked intracellular calcium transients dampen excitability and stimulate NO production in these neurons. In this study, we investigated the action of several arousal-related neurotransmitters and the role of specific calcium channels in these LDT Ca(2+)-transients by simultaneous whole-cell recording and calcium imaging in mouse (P14-P30) brain slices. Carbachol, noradrenaline and adenosine inhibited spike-evoked Ca(2+)-transients, while histamine, t-ACPD, a metabotropic glutamate receptor agonist, and orexin-A did not. Carbachol inhibition was blocked by atropine, was insensitive to blockade of G-protein-coupled inward rectifier (GIRK) channels and was not inhibited by nifedipine, omega-conotoxin GVIA or omega-agatoxin IVA, which block L-, N- and P/Q-type calcium channels, respectively. In contrast, SNX-482 (100 nm), a selective antagonist of R-type calcium channels containing the alpha1E (Cav2.3) subunit, attenuated carbachol inhibition of the somatic spike-evoked calcium transient. To our knowledge, this is the first demonstration of muscarinic inhibition of native SNX-482-sensitive R-channels. Our findings indicate that muscarinic modulation of these channels plays an important role in the feedback control of cholinergic LDT neurons and that inhibition of spike-evoked Ca(2+)-transients is a common action of neurotransmitters that also activate GIRK channels in these neurons. Because spike-evoked calcium influx dampens excitability, our findings suggest that these 'inhibitory' transmitters could boost firing rate and enhance responsiveness to excitatory inputs during states of high firing, such as waking and REM sleep.

      14 February, 2006

        Some conotoxin references

        Zorn S, Leipold E, Hansel A, Bulaj G, Olivera BM, Terlau H, Heinemann SH. (2006)
        The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3. FEBS Lett. 2006 Jan 26; [Epub ahead of print] Institute of Molecular Cell Biology, Research Unit "Molecular and Cellular Biophysics", Medical Faculty of the Friedrich Schiller University Jena, Drackendorfer Str. 1, D-07747 Jena, Germany.

        Abstract: Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

        Jiang H, Xu CQ, Wang CZ, Fan CX, Zhao TY, Chen JS, Chi CW. (2006)
        Two novel O-superfamily conotoxins from Conus vexillum. Toxicon. 2006 Jan 31; [Epub ahead of print]
        Key Laboratory of Proteomics, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China; Institute of Protein Research, Shanghai Tong-Ji University, Shanghai 200092, China; Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China.

        Abstract: O-superfamily conotoxins include several families that have diverse pharmacological activity on Na(+), K(+) or Ca(2+) channels. These superfamily toxins have been mainly found in fish-hunting and mollusk-hunting Conus species. Here, we reported two novel O-superfamily conotoxins, vx6a and vx6b, purified from a worm-hunting cone snail, Conus vexillum. Though their cysteine framework and signal peptides share high similarity with those of other members of O-superfamily, the mature vx6a and vx6b both have a low sequence homology with others. To test the biological function of vx6a, the toxin was chemically synthesized and then tested on the locust dorsal unpaired median (DUM) neuron system which containing various ion channels. Although no any activity on ion channels was found on the DUM neuron system, vx6a could clearly elicit a series of symptoms in mouse via intracranial injection, such as quivering, climbing, scratching, barrel rolling and paralysis of limbs at different dose.

        Green BR, Bulaj G.(2006)
        Oxidative folding of conotoxins in immobilized systems. Protein Pept Lett. 2006 Jan;13(1):67-70.
        Cognetix, Inc., 421 Wakara Way, Suite 201, Salt Lake City, Utah 84108, USA.

        Abstract: We tested two alternative oxidation strategies to produce conotoxins alpha-GI and micro-PIIIA. The peptides were either reversibly immobilized on a solid support and then oxidized, or the immobilized disulfide reagent (CLEAR-OX(trade mark)) was used to oxidize the peptides. Both strategies appeared more efficient at higher peptide concentrations, consistent with pseudo-dilution effects.

        Ellison M, Haberlandt C, Gomez-Casati ME, Watkins M, Elgoyhen AB, McIntosh JM, Olivera BM.(2006)
        alpha-RgIA: A Novel Conotoxin That Specifically and Potently Blocks the alpha9alpha10 nAChR(,). Biochemistry. 2006 Feb 7;45(5):1511-1517.
        Departments of Biology, Pathology, and Psychiatry, University of Utah, Salt Lake City, Utah 84112, and Instituto Investigaciones en Ingenieria Genetica y Biologia Molecular, Consejo Nacional de Investigaciones Cientificas y Tecnicas-Universidad de Buenos Aires, Buenos Aires 1428, Argentina

        Abstract: The alpha9 and alpha10 nicotinic acetylcholine receptor (nAChR) subunits assemble to form the alpha9alpha10 nAChR subtype. This receptor is believed to mediate cholinergic synaptic transmission between efferent olivocochlear fibers and the hair cells of the cochlea. In addition alpha9 and/or alpha10 expression has been described in dorsal root ganglion neurons, lymphocytes, skin keratinocytes, and the pars tuberalis of the pituitary. Specific antagonists that selectively block the alpha9alpha10 channel could be valuable tools for elucidating its role in these diverse tissues. This study describes a novel alpha-conotoxin from the Western Atlantic species Conus regius, alpha-conotoxin RgIA (alpha-RgIA), that is a subtype specific blocker of the alpha9alpha10 nAChR. alpha-RgIA belongs to the alpha4/3 subfamily of the alpha-conotoxin family; sequence and subtype specificity comparisons between alpha-RgIA and previously characterized alpha4/3 toxins indicate that the amino acids in the C-terminal half of alpha-RgIA are responsible for its preferential inhibition of the alpha9alpha10 nAChR subtype.

        Liu Z, Dai J, Dai L, Deng M, Hu Z, Hu W, Liang S. (2006)
        Function and solution structure of huwentoxin-X, a specific blocker of N-type calcium channels, from the chinese bird spider ornithoctonus huwena. J Biol Chem. 2006 Jan 26; [Epub ahead of print]
        College of Life Sciences, Changsha, Hunan 410081.

        Abstract: Huwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I-IV, II-V and III-VI. Its cDNA, determined by rapid amplification of 3' and 5' cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with -conotoxins GVIA and MVIIA, two well-known blockers of N-type Ca2+ channels. Nevertheless, whole-cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40nM) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA HWTX-X>MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low-frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N -type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels.

        Teichert RW, Lopez-Vera E, Gulyas J, Watkins M, Rivier J, Olivera BM. (2006)
        Definition and Characterization of the Short alphaA-Conotoxins: A Single Residue Determines Dissociation Kinetics from the Fetal Muscle Nicotinic Acetylcholine Receptor. Biochemistry. 2006 Jan 31;45(4):1304-12.
        Departments of Biology and Pathology, University of Utah, Salt Lake City, Utah, 84112, and Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.

        Abstract: We report the definition and characterization of a conotoxin subfamily, designated the short alphaA-conotoxins (alphaA(S)) and demonstrate that all of these share the unique property of selectively antagonizing the fetal subtype of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR). We have characterized newly identified alphaA(S)-conotoxins from Conus pergrandis and have conducted a more detailed characterization of alphaA-conotoxins previously reported from additional Conus species. Among the results, the characterization of the short alphaA-conotoxins revealed diverse kinetics of a block of the fetal muscle nAChR, particularly in dissociation rates. The structure-function relationships of native alphaA(S)-conotoxins and some analogues revealed a single amino acid locus (alternatively either His or Pro in native peptides) that is a critical determinant of the dissociation kinetics. The unprecedented binding selectivity for the fetal muscle nAChR, coupled with the kinetic diversity, should make alphaA(S)-conotoxins useful ligands for a diverse set of studies. The rapidly reversible peptides may be most suitable for electrophysiological studies, while the relatively irreversible peptides should be most useful for binding and localization studies.

        Hirota K, Lambert DG. (2006)
        Measurement of [3H]PN200-110 and [125I]omega-conotoxin MVIIA binding. Methods Mol Biol. 2006;312:147-59. No abstract available.
        Department of Anesthesiology, University of Hirosaki School of Medicine, Japan.

        Tournier JM, Maouche K, Coraux C, Zahm JM, Cloez-Tayarani I, Nawrocki-Raby B, Bonnomet A, Burlet H, Lebargy F, Polette M, Birembaut P. (2006)
        {alpha}3{alpha}5{beta}2-Nicotinic Acetylcholine Receptor Contributes to the Wound Repair of the Respiratory Epithelium by Modulating Intracellular Calcium in Migrating Cells. Am J Pathol. 2006 Jan;168(1):55-68.
        INSERM UMRS-514, 45 rue Cognacq-Jay, 51092 Reims Cedex, France. jm.tournier@univ-reims.fr.

        Abstract: Nicotinic acetylcholine receptors (nAChRs), present in human bronchial epithelial cells (HBECs), have been shown in vitro to modulate cell shape. Because cell spreading and migration are important mechanisms involved in the repair of the bronchial epithelium, we investigated the potential role of nAChRs in the wound repair of the bronchial epithelium. In vivo and in vitro, alpha3alpha5beta2-nAChRs accumulated in migrating HBECs involved in repairing a wound, whereas alpha7-nAChRs were predominantly observed in stationary confluent cells. Wound repair was improved in the presence of nAChR agonists, nicotine, and acetylcholine, and delayed in the presence of alpha3beta2 neuronal nAChR antagonists, mecamylamine, alpha-conotoxin MII, and kappa-bungarotoxin; alpha-bungarotoxin, an antagonist of alpha7-nAChR, had no effect. Addition of nicotine to a repairing wound resulted in a dose-dependent transient increase of intracellular calcium in migrating cells that line the wound edge. Mecamylamine and kappa-bungarotoxin inhibited both the cell-migration speed and the nicotine-induced intracellular calcium increase in wound-repairing migrating cells in vitro. On the contrary alpha-bungarotoxin had no significant effect on migrating cells. These results suggest that alpha3alpha5beta2-nAChRs actively contribute to the wound repair process of the respiratory epithelium by modulating intracellular calcium in wound-repairing migrating cells.

        Copeland Jr RL, Leggett YA, Kanaan YM, Taylor RE, Tizabi Y.(2006)
        Neuroprotective effects of nicotine against salsolinol-induced cytotoxicity: implications for Parkinson's disease. Neurotox Res. 2005;8(3-4):289-93.
        Department of Pharmacology and Department of Microbiology, College of Medicine, Howard University, Washington, DC 20059, USA. ytizabi@howard.edu.

        Abstract: Parkinson's disease is associated with degeneration of dopaminergic cell bodies in the substantia nigra. It has been suggested that salsolinol, an endogenous metabolite of dopamine, may be involved in this process. An inverse relationship between Parkinson's disease and smoking (nicotine intake) has been observed in epidemiological studies. Moreover, neuroprotective effects of nicotine in various experimental models have been observed. In this study we sought to determine whether salsolinol-induced cytotoxicity in SH-SY5Y human neuroblastoma cells, a cloned cell line which expresses dopaminergic activity, could also be prevented by nicotine pretreatment, and if so, which nicotinic receptors may mediate the actions of nicotine. Exposure of SH-SY5Y cells to 0.8 mM salsolinol for 24 hours resulted in approximately 80% cell death as determined by 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Pretreatment of cells with 0.1 mM nicotine resulted in inhibition of salsolinol-induced cytotoxicity. The effects of nicotine were blocked by mecamylamine, a non-selective nicotinic antagonist as well as conotoxins with selective antagonism against alpha3-containing nicotinic receptor subunits. The effects of nicotine were not affected by dihydro-beta-erythroidine or methyllycaconitine, selective antagonists against alpha4-beta2 or alpha7 nicotinic receptors, respectively. It is suggested that selective nicotinic agonists may be of therapeutic potential in at least a subpopulation of Parkinsonian patients. FP Graham Publ. Co. www.NeurotoxicityResearch.com.

        Ragnarsson L, Yasuda T, Lewis RJ, Dodd PR, Adams DJ. (2006)
        NMDA receptor subunit-dependent modulation by conantokin-G and Ala7-conantokin-G. J Neurochem. 2006 Jan;96(1):283-91. Epub 2005 Nov 29.
        School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland, Australia.

        Abstract: The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage clamp technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.

        Moller C, Rahmankhah S, Lauer-Fields J, Bubis J, Fields GB, Mari F. (2006)
        A novel conotoxin framework with a helix-loop-helix (Cs alpha/alpha) fold. Biochemistry. 2005 Dec 13;44(49):15986-96.
        Department of Chemistry and Biochemistry and Center of Excellence in Biomedical and Marine Biotechnology, Florida Atlantic University, Boca Raton, Florida 33431, USA.

        Abstract: Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and cone snails, produce a variety of highly stable cystine-constrained peptide scaffolds as part of their neurochemical strategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designated framework 14). Three peptides of this family (flf14a-c) were isolated from the venom of Conus floridanus floridensis, and one (vil14a) was isolated from the venom of Conus villepinii, two worm-hunting Western Atlantic cone snail species. The primary structure for these peptides was determined using Edman degradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combination of CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of these conotoxins directly isolated from the cone snails revealed a highly helical secondary structure for the four conotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs alpha/alpha scorpion toxins kappa-hefutoxin, kappa-KTx1.3, and Om-toxins, which adopt a stable three-dimensional fold where the two alpha-helices are linked by the two disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately 6A, which is a conserved structural feature in K(+) channel blockers. The presence of this framework in scorpions and in cone snails indicates a common molecular imprint in the venom of apparently unrelated predatory animals and suggests a common ancestral genetic origin.

        [No authors listed](2006)
        Ziconotide (Prialt) for chronic pain. Med Lett Drugs Ther. 2005 Dec 5-19;47(1223-1224):103-4. No abstract available.

        Zugasti-Cruz A, Maillo M, Lopez-Vera E, Falcon A, Cotera EP, Olivera BM, Aguilar MB.(2006)
        Amino acid sequence and biological activity of a gamma-conotoxin-like peptide from the worm-hunting snail Conus austini. Peptides. 2005 Dec 1; [Epub ahead of print] Posgrado en Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, Mexico D.F. 04510, Mexico; Laboratory of Marine Neuropharmacology, Institute of Neurobiology, Universidad Nacional Autonoma de Mexico, Juriquilla, Queretaro 76230, Mexico.

        Abstract: A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVgammaCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif -gammaCCS-, which has only been found in the gamma-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other gamma-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.

        Wang CZ, Zhang H, Jiang H, Lu W, Zhao ZQ, Chi CW. (2006)
        A novel conotoxin from Conus striatus, mu-SIIIA, selectively blocking rat tetrodotoxin-resistant sodium channels. Toxicon. 2006 Jan;47(1):122-32. Epub 2005 Dec 1.
        Key Laboratory of Proteomics, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

        Abstract: mu-Conotoxin SIIIA, a novel blocker of tetrodotoxin-resistant (TTX-R) voltage-gated sodium channels (VGSCs) has been identified from the fish-hunting cone snail, Conus striatus. The deduced sequence consists of a 20-residue signal peptide, a 31-residue pro-peptide, and a 20-residue mature toxin with its N-terminal Gln cyclized and C-terminus amidated. mu-SIIIA shares the common cysteine arrangement with other mu-conotoxins. Besides, it exhibits high sequence homology with mu-SmIIIA, a toxin recently characterized from C. stercusmuscarum which potently blocks the TTX-R VGSCs in frog neurons. With whole-cell recording, mu-SIIIA potently and selectively inhibits the TTX-R VGSCs of dissociated adult rat small-diameter dorsal root ganglia (DRG) neurons with a dose- and time-dependent property and irreversibly. Homology-based modeling of mu-PIIIA, SIIIA and SmIIIA implies that they share a common backbone conformation except at the N termini. The hydroxyl-proline residue only present in mu-PIIIA is absent and substituted by an Asp residue in mu-SIIIA and SmIIIA. Similarly, one crucial basic residue (Arg(12) in mu-PIIIA) is replaced by serine in the latter two toxins. Such differences might endow them with the capacity to selectively inhibit TTX-S or TTX-R VGSCs. Considering that TTX-R VGSCs predominantly expressed in DRG neurons play pivotal roles in the nociceptive information transmission and that their specific antagonists are still lacking, mu-SIIIA might provide a useful tool for functional studies of these channels, and potentially be developed as an efficient pain killer.

        Westerlind U, Norberg T. (2006)
        Chemical synthesis of analogs of the glycopeptide contulakin-G, an analgetically active conopeptide from Conus geographus. Carbohydr Res. 2006 Jan 16;341(1):9-18. Epub 2005 Dec 1.
        Department of Chemistry, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden.

        Abstract: Cone snails are marine predators that use immobilizing venoms for catching prey. Chemical analysis of the venoms has revealed a variety of biologically active small and intermediate size peptides rich in post-translational modifications (modified amino acids, glycosylation). The glycopeptide contulakin-G (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-[beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1--> ]Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH) is a potent analgesic from Conus geographus venom. The in vivo activity of synthetic contulakin-G was previously found to be significantly higher compared to that of a peptide lacking the glycan. In order to further investigate the importance of the glycan, we have now synthesized analogs of contulakin-G where the glycan chain O-linked to threonine has been altered either to beta-D-Galp-(1-->3)-beta-D-GalpNAc-, alpha-D-Galp-(1-->3)-alpha-D-GalpNAc-, or beta-D-Galp-(1-->6)-alpha-D-GalpNAc-. The glycopeptides were assembled on a Wang resin using commercially available Fmoc amino acids and synthetically prepared Fmoc-protected threonine derivatives carrying O-acetyl protected sugar chains. The final products were thoroughly characterized by NMR and mass spectroscopy.

        Narayana AK. (2005)
        Elan: ziconotide review focused on off-label uses. Am J Hosp Palliat Care. 2005 Nov-Dec;22(6):408. No abstract available.

        Publication Types: Comment; Letter

        Buczek O, Bulaj G, Olivera BM.(2005)
        Conotoxins and the posttranslational modification of secreted gene products. Cell Mol Life Sci. 2005 Dec;62(24):3067-79.
        Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.

        Abstract: The venoms of predatory cone snails (genus Conus) have yielded a complex library of about 50-100,000 bioactive peptides, each believed to have a specific physiological target (although peptides from different species may overlap in their target specificity). Conus has evolved the equivalent of a drug development strategy that combines the accelerated evolution of toxin sequences with an unprecedented degree of posttranslational modification. Some Conus venom peptide families are the most highly posttranslationally modified classes of gene products known. We review the variety and complexity of posttranslational modifications documented in Conus peptides so far, and explore the potential of Conus venom peptides as a model system for a more general understanding of which secreted gene products may have modified amino acids. Although the database of modified conotoxins is growing rapidly, there are far more questions raised than answers provided about possible mechanisms and functions of posttranslational modifications in Conus.

        Aguilar MB, Lezama-Monfil L, Maillo M, Pedraza-Lara H, Lopez-Vera E, Heimer de la Cotera EP. (2005)
        A biologically active hydrophobic T-1-conotoxin from the venom of Conus spurius. Peptides. 2005 Nov 15; [Epub ahead of print]
        Laboratory of Marine Neuropharmacology, Institute of Neurobiology, Universidad Nacional Autonoma de Mexico, Campus UNAM-UAQ Juriquilla, Km 15 Carr. Queretaro-S.L.P., Juriquilla, Qro. 76230, Mexico

        Abstract: A major, very hydrophobic peptide, sr5a, was purified from the venom duct of Conus spurius specimens collected in the Yucatan Channel, Mexico. Its amino acid sequence (IINWCCLIFYQCC; calculated monoisotopic mass assuming two disulfide bridges 1616.68Da) was determined by automatic Edman degradation after reduction and alkylation, and confirmed by mass spectrometry (ESI monoisotopic mass, 1616.60; MALDI monoisotopic mass 1616.42Da). The primary structure of sr5a showed the pattern that characterizes the family of the T-1-conotoxins, which belong to the T-superfamily of conotoxins. The disulfide bonds were determined by partial reduction and alkylation with N-ethylmaleimide, followed by total reduction and alkylation with 4-vinylpyridine, and automatic Edman sequencing. The connectivity of the Cys residues (I-III, II-IV) is the same as that found in the T-1-conotoxin family. When injected intracranially (2.0nmol) into mice, peptide sr5a caused depressed behavioral activity.

        Hussar DA. (2005)
        New drugs: tigecycline, ziconotide, and clofarabine. J Am Pharm Assoc (Wash DC). 2005 Sep-Oct;45(5):636-9. No abstract available.
        Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, PA, USA.

        Mordvintsev DY, Polyak YL, Levtsova OV, Tourleigh YV, Kasheverov IE, Shaitan KV, Utkin YN, Tsetlin VI. (2005)
        A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins. Comput Biol Chem. 2005 Dec;29(6):398-411. Epub 2005 Nov 11.
        Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, Miklukho-Maklaya str., 16/10, GSP-7, Moscow, Russia. chlorine@yandex.ru

        Abstract: Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a 'surface-touch' manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15-25 ps according to AMBER'99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long alpha-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long alpha-neurotoxins differ in the ways of loop II entry into nAChR.

      5 January, 2006

        New issues of cone shells on stamps

        Tom Walker has notified me of the following new stamps issued during 2005 that depict cone shells: Pitcairn Islands (10-02-2005)1 species depicted; Wallis and Fatuna Islands (2005)4 species depicted.




      Continued in What's new in 2005
      See also : What's new in 2004, What's new in 2003, What's new in 2002, What's new in 2001, What's new in 2000, What's new in 1999, What's new in 1998, What's New in 1997 and What's New in 1996

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      Continued in What's new in 2005

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