What's New in 2004

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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 (1998-2004)

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31 December, 2004

    New Cone Shells on Stamps from Papua New Guinea

    The Cone Shells on Stamps site has been updated with two species of Conus from Papua New Guinea, issued November 17, 2004. The stamps are 70t Conus auratus and 4.60k Conus suratensis. In addition, two images have kindly been provided of Conus aulicus on stamps from Qatar of 5dh and 4r value.

28 December, 2004

    Elan Receives FDA Approval for PRIALT (Ziconotide Intrathecal Infusion) for Severe Chronic Pain

    PRIALT(R) [other names: Ziconotide, SNX-111, omega-Conotoxin MVIIA] was approved today by the Federal Drug Administration, FDA, for the management of severe chronic pain:

    • Novel Non-Narcotic Treatment Based on Marine Snail Peptide Blocks Pain Signals
    • PRIALT(R) is Approved for use only in the Medtronic SynchroMed(R) EL, SynchroMed(R) II Infusion System and Simms Deltec Cadd Micro(R) External Microinfusion Device and Catheter
    • Approval Based on Results From Three Pivotal Trials

    Dec. 28, 2004--Elan Corporation, plc (NYSE:ELN): Elan Corporation, plc today announced that the U.S. Food and Drug Administration (FDA) has approved PRIALT(R) (ziconotide intrathecal infusion) for the management of severe chronic pain in patients for whom intrathecal (IT) therapy is warranted, and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies or IT morphine. FDA approval of PRIALT was based on the treatment of more than 1,200 patients and three Phase III clinical trials, which evaluated the efficacy and safety of IT PRIALT in patients with severe chronic pain that was not adequately managed despite a regimen of systemic and/or IT analgesic and other drugs.

  • For further details of this long-awaited and important announcemement read the Press Release from Elan Corp.
  • A brief summary of the information is available from the Doctors Guide
  • Detailed Product Information about PRIALT(R) is also available.
  • Information about PRIALT, including prescribing information and comprehensive support services, is available through a toll-free number, 1-888-PRIALT-1, and at www.PRIALT.com.

23 December, 2004

    Seven new species of Conus described in VISAYA II

    In this second issue of Visaya: E. L. Heiman, Felix Lorenz, Manuel J. Tenorio & Carlos M. L. Afonso, Gabriella Raybaudi Massilia, Eva Pip and Guido T. Poppe contributed with 6 articles containing descriptions of several new species.

    • Lorenz, F. (2004). "Two New Species of CONIDAE from Southern Madagascar". pp.19-23.
    • Tenorio, M.J. and Alfonso, C.M. (2004). "Description of Four New Species of Conus from the Cape Verde Islands". pp. 24-37.
    • Raybaudi Massilia, G. (2004). "An 'Old' New Species of Conus from the Philippines". pp. 38-41.

    Other articles are: "Conchological Treasures from the Philippines" and "Intraspecific Variation in mauritia arabica". Visaya is printed in color from cover to cover.
    There are 7 Newly described species of Conus in this issue: Conus medoci sp. nov.; Conus chiapponorum sp. nov.; Conus vulcanus n. sp.; Conus claudiae n. sp.; Conus isabelarum n. sp.; Conus crioulus n. sp.; and Conus suduirauti n. sp.

    Another 3 new species Conus frausseni n. sp.; Conus grohi n. sp.; and Conus terryni n. sp. were described in the first issue of the journal, VASAYA I (see entry below for 9 August 2004).

    For information about VISAYA click here.

    Conus imperialis alpha-toxins ImI and ImII target separate inhibitory sites on the alpha7 nicotinic receptor

    Ellison, M., Gao, F., Wang, H.L., Sine, S.M., McIntosh, J.M. and Olivera, B.M. (2004) alpha-Conotoxins ImI and ImII Target Distinct Regions of the Human alpha7 Nicotinic Acetylcholine Receptor and Distinguish Human Nicotinic Receptor Subtypes. Biochemistry. 43:16019-16026.
    Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah 84112, and Receptor Biology Laboratory, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.

    Abstract: The Conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII. The effects of the mutations on alpha-ImI binding and functional antagonism are explained by computational docking of the NMR structure of alpha-ImI to a homology model of the ligand binding domain of the alpha7 nAChR. A distinct binding site for alpha-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the alpha7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT(3)). The two toxins also discriminate between different subtypes of human nicotinic receptors; alpha-ImII most strongly blocks the human alpha7 and alpha1beta1deltaepsilon receptor subtypes, while alpha-ImI most potently blocks the human alpha3beta2 subtype. Collectively, the data show that while alpha-ImI targets the classical competitive ligand binding site in a subtype selective manner, alpha-ImII is a probe of a novel inhibitory site in homomeric alpha7 nAChRs.

    Conotoxins from Conus magus and Conus imperialis used to distinguish different nicotinic receptor subtypes involved in dopamine release.

    Cao, Y.J., Surowy, C.S., Puttfarcken, P.S. (2005) Different nicotinic acetylcholine receptor subtypes mediating striatal and prefrontal cortical [(3)H]dopamine release. Neuropharmacology. 48:72-79.
    Neurological Diseases Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6125, USA.

    Different nicotinic acetylcholine receptor subtypes appear to modulate dopamine release from the striatum and prefrontal cortex. In this study a combination of subtype-selective antagonists and agonists were used to extensively characterize the nAChRs involved in dopamine release from slice preparations of these two brain regions. alpha-conotoxin-MII inhibited nicotine-evoked [(3)H]dopamine (DA) release from striatum by 45%, but did not affect cortical dopamine release. Neither methyllycaconitine, alpha-bungarotoxin, nor alpha-conotoxin-ImI affected nicotine-evoked [(3)H]DA release from either striatum or prefrontal cortex. MG 624, a novel selective nAChR antagonist, inhibited cortical [(3)H]DA by 53%, but had no effect on striatal release. Compared to nicotine, (+/-)-UB-165 showed less efficacy with respect to dopamine release from striatum, and had no effect on cortical dopamine release. (+/-)-UB-165-evoked striatal dopamine release was completely blocked by mecamylamine, partially blocked (up to 55%) by alpha-conotoxin-MII, and unaffected by methyllycaconitine or alpha-conotoxin-ImI. alpha4beta2* and alpha6beta2beta3* nAChRs appear to play a role in striatal dopamine release, whereas alpha4beta2* nAChRs modulate release from prefrontal cortex. alpha7* nAChRs do not appear to play a role in nAChR-mediated dopamine release from either brain region.

21 December, 2004

    Conotoxin tx5a from Conus textile: a heavily post-translationally modified glycopeptide

    Kang, J., Low, W., Norberg, T., Meisenhelder, J., Hansson, K., Stenflo, J., Zhou, G.P., Imperial, J., Olivera, B.M., Rigby, A.C. and Craig, A.G. (2004) Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-d-Gal-(1-->3)-alpha-d-GalNAc Eur J Biochem. 271:4939-4949.
    The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA, USA.

    Abstract: The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-d-Gal-(1-->3)-alpha-d-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the resulting synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-d-Gal-(1-->3)-alpha-d-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional (1)H homonuclear and (13)C-(1)H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-d-Gal-(1-->3)-alpha-d-GalNAc.

19 December, 2004

    omega-conotoxin MVIIA structure and functions

    Contoxin MVIIA from Conus magus
    Other names: SNX111, Ziconotide, Prialt. from database PubChem compound. Small molecule chemical structures.

  • View list of published pharmacological articles about omega-conotoxin MVIIA (SNX-111,Ziconotide, Prialt) on Toxline.
  • View latest Clinical Trials of Ziconotide as listed on NIH ClinicalTrials.gov.
  • 3 key articles on Conotoxin MVIIA structure and function (from TOXLINE SPECIAL).
    • Mitchell, S.S., Shon, K.J., Olivera, B., Ireland, C.M. (1996) NMR structures of conotoxins. J. Natural Toxins 5: 191-208
      Abstract: This review discusses the methodology, structural details, and biological implications regarding NMR structures of conotoxins. NMR and molecular modeling techniques have improved to the point that three-dimensional structures of conotoxins can now be determined with a significant degree of confidence. At the same time, biochemical techniques have made important progress in disseminating critical areas of the toxin receptors. As the two areas of research converge, they can begin to explain the extraordinary selectivity of conotoxin binding on a molecular level. An understanding of how molecular interactions between the toxins and their receptors leads to binding specificity should have broad applications in many fields. NMR structures of conotoxins have now been published for each of the major toxin classes. This review includes a brief discussion on the NMR and modeling techniques used for each of the published conotoxin structures to date.
    • Norton, R.S. (1996) Structure and function of peptide and protein toxins from marine organisms. J. Toxicology Toxin Reviews 17: 99-130.
      Abstract: This literature review discusses sea anemones, snakes, cone snails and other marine organisms with respect to the toxicology and biophysics of their protein toxins. It includes a discussion of analytical methods, amino acid sequence, three-dimensional structure of the peptide toxins and their related ion channel proteins, receptor binding sites, X-ray crystallography, NMR spectroscopy of alpha-conotoxins, and neurotoxins that act as sodium channel modulators and potassioum channel blockers. there is also discussion of cytolysins.
    • West, D.J., Andrews, E.B., Bowman, D., McVean, A.R., Thorndyke, M.C.(1998) Toxins from some poisonous and venomous marine snails. J. Comp Biochem Physiol C. Pharmacol. Toxicol and Endocrinol. 113: 1-10. Abstract: This literature review discusses conotoxins from Conus venom that act as calcium channel blockers.

17 December, 2004

    Chinese bird spider toxin, a more potent analgesic than omega-conotoxin MVIIA

    Chen, J.Q., Zhang, Y.Q., Dai, J., Luo, Z.M. and Liang, S.P. (2005) Antinociceptive effects of intrathecally administered huwentoxin-I, a selective N-type calcium channel blocker, in the formalin test in conscious rats. Toxicon. 45:15-20.
    Biochemistry Department, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, People's Republic of China.

    Abstract: The present study was undertaken to elucidate the antinociceptive effect of intrathecal administration of huwentoxin-I (HWTX-I), a N-type calcium channel blocker from the venom of the Chinese bird spider Ornithoctonus huwena (Wang) [=Selenocosmia huwena wang], by comparison with omega-Conotoxin-MVIIA (omega-CTX-MVIIA) and morphine hydrochloride in the formalin test in conscious rats. Similar to omega-CTX-MVIIA and morphine, intrathecal pre-treatment with HWTX-I resulted in suppression of nociceptive behavior in a dose-dependent manner. The ED(50) values of HWTX-I and omega-CTX-MVIIA were 0.28 and 0.19mug/kg, respectively. It was also found that, at lower doses (0.1 and 0.5mug/kg), the antinociceptive effect of HWTX-I was identical to that of omega-CTX-MVIIA, while omega-CTX-MVIIA acted more remarkably than HWTX-I at higher dose (1.0mug/kg). However, the antinociception induced by omega-CTX-MVIIA were companied with motor dysfunction, and these side-effects became more evident with the doses of omega-CTX-MVIIA increasing. In contrast, HWTX-I did not show these side-effects at the doses of 0.5-1.0mug/kg. Compared with HWTX-I and omega-CTX-MVIIA, the analgesic effect of intrathecal morphine hydrochloride was initiated faster, but lasted for a shorter time (about 2-3h at 1.0mug/kg) than that of HWTX-I and omega-CTX-MVIIA (about 4-5h at 1.0mug/kg). Therefore, the present results show that, like omega-CTX-MVIIA, the intrathecal administration of HWTX-I is effective in antinociception in the rat model of the formalin test.

15 December, 2004

    Conotoxins as tools for invetigating sodium channels

    French, R.J. and Terlau, H. (2004) Sodium channel toxins--receptor targeting and therapeutic potential. Curr Med Chem. 11: 3053-3064.
    Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, T2N 4N1, Canada. french@ucalgary.ca

    Abstract: Sodium channels underlie propagated electrical signalling in most excitable cells, including neurons and the myocytes of skeletal muscle and heart. These proteins are targeted by a variety of current therapeutic drugs to combat such maladies as pain, myotonias, epilepsies and cardiac arrhythmias. Typically, these problems are associated with overactivity of sodium channels leading to hyperexcitability in the relevant tissue. More than ten distinct but closely related molecular isoforms of mammalian sodium channel are now known to be specifically expressed in different cell types and tissues. Therapeutic attenuation of sodium channel activity must be effected with great precision in both targeting and the degree of reduction in channel activity if a malfunction is to be corrected without introducing deleterious or even catastrophic side effects. Numerous natural toxins have evolved to target sodium channels, either by blocking current through the pore or by modifying channel gating. Among the well studied toxins, the peptide conotoxins from cone snail venoms show a remarkable ability to discriminate among closely related forms of sodium channel, as well as exhibiting a variety of modes of action. Here, we examine the molecular basis of action of different Na channel targeted conotoxins and explore their potential as models for the future design of more specifically targeted drugs.

9 December, 2004

    Conantokins - a REVIEW

    Layer RT, Wagstaff JD, White HS. (2004) Conantokins: peptide antagonists of NMDA receptors. Curr Med Chem. 11: 3073-3084.
    Cognetix, Inc. 421 Wakara Way, Suite 201, Salt Lake City, UT 84108, USA. rlayer@cognetix.com

    Abstract: Conantokins are small peptides (17-27 amino acids) found in the venoms of cone snails (Conus sp.) that inhibit the activity of N-methyl-D-aspartate (NMDA) receptors. Unlike most of the peptides characterized from cone snail venom that contain multiple disulfide bridges, conantokins are linear peptides that possess a high degree of alpha-helicity in the presence of divalent cations, and contain gamma-carboxyglutamic acid residues. Four naturally occurring conantokins have been identified and characterized to date, conantokin-G, conantokin-T, conantokin-R, and conantokin-L. The most extensively characterized, conantokin-G, is selective for subtypes of NMDA receptors containing the NR2B subunit. The conantokins have been synthesized and characterized in a number of animal models of human pathologies including pain, convulsive disorders, stroke, and Parkinson's disease. The potential pharmacological selectivity of the conantokins, coupled with their efficacy in preclinical models of disease and favorable safety profiles indicate that these peptides represent both novel probes for NMDA receptor function as well as an important class of compounds for continued investigation as human therapeutics.

8 December, 2004

    Novel conotoxin rg11a from Conus regius

    Vianna Braga, M.C., Konno, K., Portaro, F.C., Carlos de Freitas, J., Yamane, T., Olivera, B.M. and Pimenta, D.C. (2005) Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin. Toxicon. 45 :113-122
    Department of Physiology, Bioscience Institute, University of Sao Paulo, Sao Paulo, SP, Brazil.

    Abstract: Carnivorous mollusks belonging to the genus Conus paralyze their prey by injecting a rich mixture of biologically active peptides. Conus regius is a vermivorous member of this genus that inhabits Brazilian tropical waters. Inter-, intra-species and individual variations of cone snail venom have been previously reported. In order to investigate intra-specific differences in C. regius venom, its feeding behavior and the correlation between these two factors, animals were pooled according to gender, size and season of collection, and their venom composition was compared by high performance liquid chromatography (HPLC). Both the whole venom and one specific peak were monitored by HPLC. Chromatographic profiles revealed no significant differences in their peak areas, indicating that the venom composition, based solely in the presence or absence of the major peaks, is stable regardless of season, gender and size. Therefore, analysis of one given toxin, eluting in one of the major peaks, is representative among the population. Moreover, this work presents the identification of one novel conotoxin (rg11a), which amino acid sequence was deduced by mass spectrometry.

    Live Conus regius with a fire-worm (from a series by David Touitou from Martinique).
    Note: the above paper by Braga and colleagues includes images showing the envenomation of a fire-worm E. complanata by the vermivorous carnivor, Conus regius. It was observed that the prey's body changed colour after venom inoculation, from reddish to purple. The authors speculate that besides paralysis, "there might be some secondary effect on the fire-worm's clotting cascade". They also note that when provided with a variety of prey (fish, marine worms, other molluscs), the cone snail extended its proboscis only towards the correct prey, the fire-worm. None of the 30 specimens of Conus regius observed extented their proboscis towards fish or molluscs. Paralysis of the fire-worms took longer than did paralysis of fish by piscivorous cones such as Conus ermineus, studied in parallel. Statistical analysis comparison of the chromatographic profiles of pooled venom from different specimens taken at different locations, of different size groups, male or female, and from different locations, did not reveal a significant pattern of variation, even though visual interpretation of the chromatographic profiles indicate some differences.

    The deduced amino acid sequence of peak 54, the novel conopeptide rg11a,


    reveals a peptide with a new characterized pattern (C-C-CC-CC-C-C-) from the I-superfamily of conotoxins. This peptide has some sequence homology to other conotoxins that are known to interact with vertebrate K+ channels. The pre-peptide sequence, cysteine bridging pattern, and functional properties are under investigation to confirm the assignment of rg11a to a superfamily. The authors comment that "without proper profiling and normalization of any possible intraspecific variation in the venom composition, one cannot state that a novel toxin is a constitutive component of the venom rather than the product of gender, season or size variation".

28 November, 2004

27 November, 2004

    Conotoxin r11a from Conus radiatus exhibits L-D amino acid isomerization

    Buczek, O., Yoshikami, D., Bulaj, G., Jimenez, E.C. and Olivera, B.M. (2004) Posttranslational amino acid isomerization: A functionally important D-amino acid in an excitatory peptide. J Biol Chem.280 : 4247-4253.
    Biology, University of Utah, Salt Lake City, UT 84112-0840.

    Abstract: The posttranslational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 AA in length. In this report, we demonstrate that a 46-amino-acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, while the unmodified polypeptide had no biological activity, demonstrating that the chirality of the posttranslationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide crosslinks. The D-Phe in r11a is at the third amino acid from the C-terminus, the same relative position from the C-terminal end as the D-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although posttranslational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N- or C-terminus that are preferential sites for isomerization to a D-amino acid.

    See also Jimenez et al (2003) J. Neurochem 85: 610-621.

23 November 2004

    Conus victoriae duct venom yields over 40 novel conotoxins

    Jakubowski, J.A. and Sweedler, J.V. (2004) Sequencing and mass profiling highly modified conotoxins using global reduction/alkylation followed by mass spectrometry. Anal Chem. 76: 6541-6547.
    Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801.

    Abstract: A novel high-throughput method for characterizing heavily modified peptides from cone snail venom is described. Unpurified cone snail duct venom, consisting primarily of multiply disulfide-bonded peptides, is reduced and alkylated using a global procedure in order to simultaneously reduce and derivatize dozens of disulfide-bonded peptides. Samples of Conus victoriae venom are analyzed by online liquid chromatography-electrospray ionization-ion trap-mass spectrometry (LC-ESI-MS) with collisionally induced dissociation (CID). Comparison of the mass profiles of peptides and CID spectra before and after the global reduction and alkylation enables cysteine-containing conopeptides to be ascertained. In this case, over 40 conotoxins are characterized based on only two LC-ESI-MS experiments in terms of mass, number of disulfide-linked cysteine residues (and hence, potential toxin superfamilies), relative hydrophobicity, and other posttranslational modifications. Using this technique, over half of the amino acids (by mass) of several peptides are defined prior to any detailed sequencing studies. Further comparison of the mass data with previously published genetic information allows sequence verification of three novel peptides, termed vc5b, vc6b and vc6c, based on both LC-ESI-MS CID and nanoelectrospray ionization-ion trap-mass spectrometry (nanoESI-MS) experiments. This global method is ideally suited to the use of larger genetic databases in order to efficiently sequence peptides in Conus venoms and is also applicable to analysis of other disulfide-rich classes of peptides such as defensins, chemokines, and snake, spider, or other venoms.

    Conus toxins and their targets: A Review

    Heading, C.E. (2004) Conus toxins: Targets and properties. IDrugs. 7: 1011-1016.

    The Open University, Department of Biological Sciences, Walton Hall, Milton Keynes, MK7 6AA UK. moore11@globalnet.co.uk.

    Abstract: The venoms from Conus snails are rich in peptides with potent specificity for mammalian receptor sites. Each venom typically contains up to 100 conopeptides, and with approximately 500 species of Conus snail, the number of active peptides is considerable. The receptor sites targeted appear to be mostly linked to ion channels, with voltage-gated, ligand-gated and G-protein linked sites identified. Both the central and peripheral nervous system present possible physiological targets for therapeutic products derived from the venoms, although the molecules in the most advanced development target the central nervous system. In turn, this presents problems of bioavailability, while the potency is a potential source of toxicity.

    The I-superfamily of conopeptides is widespread

    Kauferstein, S., Huys, I., Kuch, U., Melaun, C., Tytgat, J. and Mebs, D. (2004) Novel conopeptides of the I-superfamily occur in several clades of cone snails. Toxicon 44: 539 - 548.
    Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt am Main, Germany. kauferstein@em.uni-frankfurt.de

    Abstract: The I-superfamily of conotoxins represents a new class of peptides in the venom of some Conus species. These toxins are characterized by four disulfide bridges and inhibit or modify ion channels of nerve cells. When testing venoms from 11 Conus species for a functional characterization, blocking activity on potassium channels (like Kv1.1 and Kv1.3 channels, but not Kv1.2 channels) was detected in the venom of Conus capitaneus, Conus miles, Conus vexillum and Conus virgo. Analysis at the cDNA level of these venoms using primers designed according to the amino acid sequence of a potassium channel blocking toxin (ViTx) from C. virgo confirmed the presence of structurally homologous peptides in these venoms. Moreover, peptides belonging to the I-superfamily, but with divergent amino acid sequences, were found in Conus striatus and Conus imperialis. In all cases, the sequences of the precursors' prepro-regions exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between species. We then performed phylogenetic analyses of new and published mitochondrial 16S rDNA sequences representing 104 haplotypes from these and numerous other Conus species, using Bayesian, maximum-likelihood, maximum-parsimony and neighbor-joining methods of inference. Cone snails known to possess I-superfamily toxins were assigned to five different major clades in all of the resulting gene trees. Moreover, I-superfamily conopeptides were detected both in vermivorous and piscivorous species of Conus, thus demonstrating the widespread presence of such toxins in this speciose genus beyond evolutionary and ecological groups.

22 November, 2004

    Conus peptides - a REVIEW

    Wang, C.Z. and Chi, C.W. (2004) Conus peptides - a rich pharmaceutical treasure. Acta Biochim Biophys Sin (Shanghai) 36: 713-723.
    Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031 China; Institute of Neuroscience, the Chinese Academy of Sciences, Shanghai 200031, China; Institute of Protein Research, Tongji University, Shanghai 200092, China.

    Abstract Marine predatory cone snails (genus Conus) with over 500 species represent what is arguably the largest single genus of marine animals alive today. All Conus are venomous and utilize a complex mixture of Conus peptides to capture their preys and for other biological purposes. Each component of Conus peptides selectively targets a specific subtype of ion channels, neurotransmitter receptors or transporters. Owing to their diversity, more than 50,000 distinct active peptides are theoretically estimated in Conus venoms. These diversified toxins are generally categorized into several superfamilies and/or families based on their characteristic arrangements of cysteine residues and pharmacological actions. Some mechanisms underlying the remarkable diversity of Conus peptides have been postulated: the distinctive gene structure, gene duplication and/or allelic selection, genus speciation, and sophisticated expression pattern and post-translational modification of these peptides. Due to their highly pharmacological potency and target selectivity, Conus peptides have attracted extensive attention with their potentials to be developed as new research tools in neuroscience field and as novel medications in clinic for pain, epilepsy and other neuropathic disorders. Several instructive lessons for our drug development could be also learnt from these neuropharmacological "expertises". Conus peptides comprise a rich resource for neuropharmacologists, and most of them await to be explored.
    Click here for the full text of this REVIEW.

18 November, 2004

    Prialt (Ziconotide, SNX-111, omega-conotoxin MVIIA) given European "approval" for human use.

    PRIALT -Ziconotide- Receives Positive CHMP Recommendation for Intrathecal Treatment of Severe Chronic Pain Media Announcement: Elan Corporation, plc announced that PRIALT(TM) (ziconotide) has received a positive recommendation from the European Committee for Medicinal Products for Human Use (CHMP), the human medicines scientific body of the European Medicines Agency. The CHMP has recommended that Prialt be indicated for the treatment of severe, chronic pain in patients who require intrathecal (IT) analgesia. The CHMP's positive recommendation will now be proposed for final marketing approval by the European Commission. Final approval customarily follows the CHMP's recommendation in approximately three months.

13 November, 2004

    Decreased [(125)I]-alpha-Conotoxin MII binding to thalamic nuclei in dementia with impaired consciousness

    Ray, M., Bohr, I., McIntosh, J.M., Ballard, C., McKeith, I., Chalon, S., Guilloteau, D., Perry, R., Perry, E., Court, J.A. and Piggott, M. (2004) Involvement of alpha6/alpha3 neuronal nicotinic acetylcholine receptors in neuropsychiatric features of Dementia with Lewy bodies: [(125)I]-alpha-conotoxin MII binding in the thalamus and striatum.Neurosci Lett. 372: 220-225.
    Institute for Ageing and Health, University of Newcastle upon Tyne, MRC Building, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, UK
    Abstract: Dementia with Lewy bodies (DLB) is a neurodegenerative disease associated with a range of neuropsychiatric symptoms and reduced expression of neuronal nicotinic acetylcholine receptors (nAChRs) in neocortex, hippocampus, thalamus and basal ganglia. To determine whether there are selective associations between alterations in alpha6/alpha3 neuronal nicotinic acetylcholine receptors (nAChRs) and the two key neuropsychiatric features of DLB, impaired consciousness (IC) and visual hallucinations (VH), quantitative [(125)I]-alpha-conotoxin MII ([(125)I]-alpha-Ctx MII) autoradiography was undertaken on 28 people with DLB and 15 control cases from the Newcastle Brain Bank. There was a highly significant overall trend for reduced thalamic [(125)I]-alpha-Ctx MII binding in DLB (p<0.001), with significant deficits in the centromedian, ventral lateral and ventroposterior medial thalamic nuclei (p<0.05), together with caudate and putamen (p<0.001). [(125)I]-alpha-Ctx MII binding was significantly lower in DLB cases with IC than without IC in the putamen (p<0.05), however there was no significant association between [(125)I]-alpha-Ctx MII binding and VH. Reductions in [(125)I]-alpha-Ctx MII binding in caudate and putamen were paralleled by similar reductions in [(125)I]PE2I binding. [(125)I]PE2I binding was also significantly lower in DLB cases with IC than without IC in the caudate (p<0.05) and putamen (p<0.001). These results demonstrate that deficits in alpha6/alpha3 nAChRs occur in specific brain regions in DLB, may in part be related to the loss of dopaminergic neurons and may contribute to the development of impaired consciousness in the disorder.

    Conotoxins and nicotinic ligands distinguish dopamine and noradrenaline release in the hippocampus

    Cao, Y.J., Surowy, C. and Puttfarcken, P.S. (2004) Nicotinic Acetylcholine Receptor-Mediated [3H]-Dopamine Release from Hippocampus.J. Pharmacol. Exp. Ther. (in press). 2004 Nov 12; [Epub ahead of print]
    Abbott Laboratories.
    Abstract: The mechanism of nicotinic acetylcholine receptor (nAChR)-induced hippocampal dopamine (DA) release was investigated using rat hippocampal slices. nAChRs involved in hippocampal DA and norepinephrine (NE) release were investigated using prototypical agonists and antagonists, as well as several relatively novel compounds, ABT-594, +/-UB-165, and MG 624. +/--Epibatidine (EB), +/--UB-165, anatoxin-a, ABT-594, (-)-nicotine, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), and (-)-cytisine (in decreasing order of potency) evoked [(3)H]-DA release in a mecamylamine-sensitive manner. Aside from +/--UB-165, all agonists displayed full efficacy relative to 100 microM (-)-nicotine in [(3)H]-DA release. In contrast, +/--UB-165 was a partial agonist, evoking 58% of 100 microM (-)-nicotine response. Mecamylamine (Mec), MG 624, hexamethonium (Hex), d-Tubocurare (d-TC), and dihydro-beta-erythroidine (DHbetaE) (in decreasing order of potency), but not alpha-conotoxin-MII (alpha-CtxMII), methyllycaconitine (MLA), alpha-conotoxin-ImI (alpha-CtxImI) or alpha-bungarotoxin (alpha-BTX), attenuated 100 microM (-)-nicotine -evoked [(3)H]-DA release in a concentration-dependent manner. +/--UB-165, ABT-594 and MG 624 exhibited different pharmacological profiles in the [(3)H]-NE release assay when compared to their effect on [(3)H]-DA release. ABT-594 was 4.5-fold more potent, and +/--UB-165 was a full agonist, in contrast to its partial agonism in [(3)H]-DA release. MG 624 potently and completely blocked NE release evoked by 100 microM (-)-nicotine, and 10 microM +/--UB-165, whereas it only partially inhibited (-)-nicotine -evoked [(3)H]-DA release. In conclusion, we provide evidence that [(3)H]-DA can be evoked from the hippocampus, and that the pharmacological profile for nAChR-evoked hippocampal [(3)H]-DA release suggests involvement of alpha3beta4* and at least one other nAChR subtype, thus distinguishing it from that of nAChR-evoked hippocampal [(3)H]-NE release.

    [See also Neuropharmacology. 48:72-79, listed above].

18 November, 2004

    A total of 15,589 species face extinction (including 7 Conus species), according to the 2004 IUCN Red List of Threatened Species. One in three amphibians and almost half of all freshwater turtles are threatened, on top of the one in eight birds and one in four mammals known to be in jeopardy. No new species of Conus have been added to the list since the previous listing in 1996
    Conus africanus, Kiener 1845, Angola.
    Conus cepasi,Trovao, 1975. Angola
    Conus kohni, McLean & Nybakken, 1979, Ecuador (Galápagos).
    Conus kohni
    McLean & Nybakken, 1979: The Veliger 22(2):140-142, figs. 4, 24.
    HOLOTYPE: LACM 1885 (shell dry, animal wet)
    18-37 m, Caleta Tagus, Isla Isabela (Albemarle), Islas Galapagos (0° 24.5' S, 90° 23' W)
    Leg.: Gerald Wellington, 15 JAN 1975
    Conus nobrei, Trovao, 1975, Angola.
    Conus zebroides, Kiener, 1845, Angola.

13 November, 2004

    Ziconotide (omega-conotoxin MVIIA): Clinical trials for refractory pain in Cancer and AIDS

    Doggrell, S.A. (2004) Intrathecal ziconotide for refractory pain. Expert Opin Investig Drugs13 : 875-877.
    Doggrell Biomedical Communications, 47 Caronia Crescent, Lynfield, Auckland, New Zealand. s.doggrell@xtra.co.nz
    Abstract: For cancer and AIDS patients, 10-30% of pain is refractory to strong opioids, requiring intraspinal administration for pain management. Ziconotide is a selective N-type calcium channel blocker, which inhibits neurotransmitter release, and following intrathecal administration will affect primary nociceptive afferents. In 108 patients with previously unmanaged refractory pain despite the use of systemic or intrathecal opioids, in the initial titration phase, the mean Visual Analogue Scale of Pain Intensity scores improved more in the ziconotide group (53%) than the placebo group (18%). Serious adverse effects were more common in the ziconotide group (31%) than placebo group (10%) in the initial titration phase. In the 48 patients receiving Ziconotide, who proceeded to the maintenance phase, the benefit of Ziconotide was continued. Until a new approach with a better effectiveness/adverse effects profile than ziconotide for refractory pain emerges, further optimisation of Ziconotide for use in the treatment of refractory pain should be undertaken.

7 November, 2004

    Cone shells in the Read Shell Collection 1845-1928

    For a glimpse into the past at an impressive shell collection, take a look at The Victorian Shell Collection 1845-1928 of Ella and Clara Read.

    Drawer 34 Drawer 35 Drawer 36

    The Read Family Shell Collection: Evolution of A Passion
    History (Extract): "What began as a simple gift of seashells was to evolve into an intricately organized collection of some 30,000 sea, land and freshwater shells gathered from all parts of the globe, including all coastlines of the U.S. (including Hawaii, Alaska, Puerto Rico), Mexico and South America, the Mediterranean, East and South Africa, Australia, New Zealand, Indonesia, and many islands along the Pacific Rim. Each shell, preserved in it’s original state, was individually labled and catalogued according to its family, Latin classification, place of origin preserved in its original state. The shells were then stored in small spool-and-thread boxes manufactured by Joseph Reades textile mill, which were then organized into fifty-two 16’’x24’’ mahogany drawers.Having remained completely in tact for over a century, The Read Family Shell Collection provides a rare view of the intersection of natural history and human history as seen through the fascinated eyes of two Victorian women". (Prints are available - Click here. For cone shells select Drawers 34, 35 and 36)

3 November, 2004

    alpha-conotoxin BuIA from Conus bullatus distinguishes beta2- from beta4-containing nAChRs

    Azam, L., Dowell, C., Watkins, M., Stitzel, J.A., Olivera, B.M., McIntosh, J.M. (2004) alpha-conotoxin BuIA, a novel peptide from Conus bullatus distinguishes among neuronal nicotinic acetylcholine receptors. J Biol Chem. 2004 Nov 1 [Epub ahead of print]
    Departments of Psychiatry and Biology, University of Utah, Salt Lake City, UT 84112.

    alpha 4/4 BuIA GCCSTPPCAVLYC*
    Conus bullatus (from which the sequence of BuIA, a 13 amino acid amidated alpha-conopeptide, was obtained)

    Abstract: Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. alpha subunits, together with beta2 and/or beta4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3' untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha3- and chimeric alpha6-containing nAChRs; BuIA blocks alpha6/alpha3beta2 nAChRs with a 40,000-fold lower IC50 than alpha4beta2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta4 subunit have very slow off-times, compared to the corresponding beta2 subunit-containing nAChR. In each instance, rat alphaxbeta4 may be distinguished from rat alphaxbeta2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha3beta2 to mouse alpha3beta4, and human alpha3beta2 to human alpha3beta4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta2- and beta4-containing nAChRs.

30 October, 2004

    Farming Killer Cone Snails a Risky Affair

    Associated Press Writer ALICIA CHANG has penned a feature article about Jon-Paul Bingham's cone shell milking of venoms. The article syndicated worldwide is accesible here.
    Extract: POTSDAM, N.Y. (AP) Oct 30, 10:02 PM EDT -- Jon-Paul Bingham puts his life on the line every week for the sake of science. One wrong move, and he could become the hapless victim of a snail attack that could kill him. Bingham, a biochemist at Clarkson University, is a self-described "conehead" whose livelihood depends on scuba diving for tropical marine cone snails and coaxing them to discharge their venom in his laboratory.
    "It's like snake-charming," he said with a grin.......

26 October, 2004

    Conus catus envnomates a fish ( + video)

    Schulz, J.R., Norton,A.G. and Gilly, W.F. (2004). The projectile tooth of a fish-hunting cone snail: Conus catus injects venom into fish prey using a high-speed ballistic mechanism. Biol. Bull. 207:77-79
    Abstract: The sequence of stills in this video illustrates the mechanism of prey-capture by a fish-hunting cone snail (Conus catus). The action is taking place in a chamber 1.2 mm high. The cone snail's proboscis approaches from the left, and the fish is positioned at the right side of the chamber. The times before (-ms) and after (+ms) the release of the snail's harpoon-shaped radular tooth are indicated in the upper left corner of the video frame. In the final few milliseconds before the radular tooth is released (-7 to -3 ms), the base of the tooth is propelled up against a constriction within the proboscis. The first frame after the release of the tooth occurs at 0 ms. The movie ends (at +200 ms) with the cone snail's proboscis holding the base of the radular tooth, which is impaling the fish. The video was constructed of stills from a high-speed video clip (1000 frames/s); the clip is available from the corresponding author (Email: jschulz@oxy.edu).

    VIDEO (Requires Quicktime)

    Fastest Gun in the Sea Science Now, Daily News, 19 October, 2004
    Fish-hunting cone snail uses unique mechanism to capture prey
    by David Grim
    "One of the sea's slowest creatures may also be its fastest hunter. A new study reveals that the fish-hunting cone snail immobilizes passing fish by firing a harpoonlike tooth at them. The entire process--from detecting the prey to stunning it--takes less than 300 milliseconds, making it one of the quickest captures known. Quick draw. The fish-hunting cone snail fires a harpoon-shaped tooth from its proboscis (inset) to capture prey". [CREDIT: JOSEPH SCHULZ]
    "Scientists have long been interested in the deadly toxins cone snails use to paralyze their prey, exploiting them for neuroscience research and drug development. But little was known about how the snails get this venom into their victims and how they stop fish dead in their tracks. To address these questions, a team of researchers from Stanford University's Hopkins Marine Station in Pacific Grove, California, constructed a setup that would give them a view to a kill. They attached a fish and an underwater high-speed video camera to one end of a small acrylic trough. When a Conus catus snail at the opposite end of the trough sensed the fish, it guided its translucent proboscis down the trough's furrow until it touched the fish. Then, in less than a third of a second, the camera recorded the proboscis constricting, launching a hollow harpoon-shaped tooth into the fish, and injecting venom through the tooth into the fish. The base of the tooth is gripped by the proboscis during the entire event--so it never actually leaves the snail's body--and is used as a hook to draw the fish in, the team reports in the October issue of The Biological Bulletin 207:77-79.".

    "The incredible speed of this ballistic method of prey capture explains how these snails can capture such fast prey, says lead researcher Joseph Schulz, now at Occidental College in Los Angeles. He notes that it is still unclear how the constriction of the proboscis readies the tooth for launch, but he believes it may somehow build up pressure at the base of the tooth."

    " 'This is a really good piece of work,' says Jon-Paul Bingham, a biochemist and cone snail farmer at Clarkson University in Potsdam, New York. The trick used by the fish-hunting cone snail is "extremely unique" in the animal kingdom, he says, and may have evolved to allow these creatures to expand their diet."
    The original article in Biological Bulletin by Schulz, and colleagues was commented upon above by -- DAVID GRIMM in Science Magazine, 22 October 2004, Vol. 306 No. 5696.

    Related sites: (Video of cone snail shooting its prey; Study collaborator William Gilly's site; Further information on cone snails)

    See also: Article by Jeff Hecht "Venomous snails with lightning strikes", in NewScientist.com news service, 15 October 2004.

25 October, 2004

    Conchological Iconography (Part10). Conidae
    Monteiro, A., Tenorio, M. J. & Poppe, G. T. (2004) The genus Conus of West Africa and the Mediterranean (with a foreword by Dieter Röckel and a contribution on the radulae by E. Rolan). - 2+102 pp., 10 numbered and 108 unnumbered text-figs, 98 maps, 4 b/w + 164 col. pls., loose-leaf 4to [US$136]
    Description: That 10th part of the Conchological Iconography is the first devoted to the vast family of Conidae. The West-Ethiopian and Mediterranean Provinces house nearly 100 (excactly 99) species and subspecies of which most are endemic to very restricted areas, mainly in the Cape Verde Islands (48), Angola (25) and Senegal & Guinea (12). Also the Conus-fauna of the Madeiras, Canaries and St. Helena is included in that part. One new species to science is described . The text gives a short history of the scientific approach to the fauna, but also shows nice collectibles from West African countries. The 4 pages with the Systematic List are followed by 62 pages of a commented Taxa List which gives detailed descriptions of animal and shell and information on synonymy, distribution (with detailed map) habitat and biology. The description of each species is attached to a figure showing a typical specimen in ventral and dorsal view. 11 pages focus the interest on the radulae of West African cones and a Selected Bibliography of nearly 100 titles is followed by an Alphabetical Index.
    The perfect colour-figures show roughly 1,300 shells, so covering all the high variability of the fauna. Five plates illustrate live animals and their habitats, the last four plates exhibit a very useful determination tool.
    A further part on the South-Ethiopian Province is in preparation by the same author-team.

22 October, 2004

    Biosynthesis of conotoxins: Role of propeptide and protein disulfide isomerase

    Buczek, O., Goodsell, I., Garrett, J., Buczek, P., Olivera, B.M. and Bulaj, G. (2004) Role of Propeptide and Protein Disulfide Isomerase in the Oxidative Folding of Conotoxins. Poster.
    Abstract: Conotoxins are a diverse set of peptide neurotoxins from Conus snail venoms, most of which contain multiple disulfide bonds. These peptides are initially translated as precursors, consisting of an N-terminal signal sequence, an intervening propeptide region and the C-terminal mature conotoxin. Formation of the native disulfide bonds during the oxidative folding of conotoxins is prerequisite to proper biological function, but numerous in vitro folding experiments with mature conotoxins have suggested a lack of specificity in the formation of native Cys-Cys connectivities. To explore the function of propeptide sequence in the maturation of conotoxins, we studied the oxidative folding of two conotoxin precursors, namely pro-GI and pro-PVIA, belonging to alpha- and delta-conotoxin families, respectively. The results indicate that the propeptide sequence does not strongly affect folding kinetics and thermodynamics. However, when oxidative folding is catalyzed by protein disulfide isomerase (PDI), the early steps in the folding of precursor were more efficient compared to the mature conotoxin alone. In addition, we present the cloning and recombinant expression of the Conus PDI, a major protein component of the venom duct from Conus textile. Taken together, our data are suggestive that the propeptide and protein disulfide isomerase may play an important role in the biosynthesis of conotoxins.

    REVIEW: Nicotinic ligands for the treatment of pain
    In this article the potential of nicotinic receptor ligands in the treatment of pain is discussed focussing on the perceived advantages and disadvantages of non-selective and subtype-selective agonists, peripherally acting agonists and, more speculatively, whether a disease-specific approach or nicotinic modulators or antagonists (including alpha-conotoxins such as AuI, MII, PIA and Vc1.1) might offer possibilities for treatment of pain.

    Rueter, L.E., Decker, M.W. and Bitner, R.S. (2004)Improving the therapeutic window in the treatment of pain with nicotinic ligands. Drug Discovery Today: Therapeutic Strategies - Pain and anethesia. 1:(1),89-96.
    Abbott Laboratories, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Park, IL, USA.

    Abstract: ALthough the analgesic properties of nicotine have been known for many years, the pharmaceutical exploration of nicotinic acetylcholine recetpors (nAChR) ligands for the treatment of pain has really only developed during the past 10 years. In that time, it has become apparent that the greates hurdle in the development of nAChR ligands is the therapeutic window, specifically separating analgesia from nAChR-mediated adverse effects on the gastrointestinal, cardiovascular, respiratory and central nervous systems. We discuss here five strategies designed to improve the therapeutic window. The grewatest chances for the successful development of a nAChR analgesic will probably be through combinations of these strategies, such as selective recetpor subunit agonist developed for a specific type of pain.

    See also:

    Decker, M.W., Rueter, L.E. and Bitner, R.S. (2004) Nicotinic acetylcholine receptor agonists: a potential new class of analgesics. Curr Top Med Chem. 4:369-84. Review.

    Abstract: Current analgesics, such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs), are largely refinements of approaches available for more than 100 years and have critical liabilities and limitations. A number of new molecular targets for analgesia have been proposed in recent years, including the neuronal nicotinic acetylcholine receptor (nAChR). Agonists at neuronal nAChRs have antinociceptive effects in a variety of preclinical pain models. Moreover, nicotine can decrease experimentally-induced pain in humans without disrupting normal tactile sensation. These data from both experimental animals and humans suggest that compounds targeting neuronal nAChRs may represent a new class of analgesic agents. In this paper, we provide brief overviews of the physiology of pain, the animal models used to assess potential analgesics preclinically, and the biology of nAChRs. We then provide a review of preclinical data on the antinociceptive effects of a variety of neuronal nAChR agonists and a discussion of potential mechanisms, including evidence that antinociception is mediated by activation of brainstem nuclei with descending inhibitory inputs to the spinal cord. An evaluation of the clinical potential of this approach must also consider potential side effects. Undesirable side effects of nicotine are well known, but as we will discuss in detail, these effects are not produced by all neuronal nAChR agonists and the existence of neuronal nAChR subtypes may provide a basis for separating therapeutic effects from toxicities.

    Bunelle, W.H. and Decker,, M.W. (2003) Neuronal nicotinic acetylcholine receptors ligands as potential analgesics. Expert Opin. Ther. Patents 13: 1003-1021.

13 October, 2004

    Assembly of conantokins

    Dai, Q., Castellino, F.J., Prorok, M. (2004) A single amino acid replacement results in the Ca2+-induced self-assembly of a helical conantokin-based peptide. Biochemistry 43:13225-13232.
    Department of Chemistry and Biochemistry and the W. M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, Indiana 46556.
    Abstract: Conantokins are short (17-27 amino acid residues), gamma-carboxyglutamate (Gla)-rich peptide components of the venoms of marine snails of the genus Conus. They display high apo and/or Ca2+-induced helicity and act as potent and selective inhibitors of the N-methyl-d-aspartate receptor (NMDAR). We have previously established that one of the conantokins, conantokin-G (con-G), self-associates in the presence of Ca2+ with high specificity for antiparallel chain orientation [Dai, Q., Prorok, M., and Castellino, F. J. (2004) J. Mol. Biol. 336, 731-744]. The dimerization appears to be driven by interhelical Ca2+ coordination between the following residue pairings: Gla(3)-Gla(14)('), Gla(7)-Gla(10)('), Gla(10)-Gla(7)('), and Gla(14)-Gla(3)('). A second member of the conantokin family, conantokin-T (con-T), shares sequence identity with con-G at 8 of 21 amino acids, including 4 Gla residues. These similarities notwithstanding, several primary and secondary structural differences exist between con-T and con-G. Particularly notable is that con-T contains a Lys, rather than a Gla, at position 7. Moreover, unlike con-G, con-T does not undergo Ca+ -triggered self-assembly. In the present study, sedimentation equilibrium ultracentrifugation is employed to demonstrate that a single amino acid replacement analogue of con-T, con-T[K7gamma], assumes a dimeric superstructure in the presence of Ca2+ at pH values consistent with the ionization of Gla carboxylate groups. Furthermore, HPLC-monitored thiol-disulfide folding and rearrangement assays with Cys-containing con-T variants suggest that the relative chain alignment preference in the noncovalent complex is antiparallel. Our results suggest that interchain Ca+ coordination in con-T[K7gamma] is incumbent upon an "i, i + 4, i +7, i +11" arrangement of Gla residues, as occurs in native con-G.

    Conus pappiliferus live - a small Australian cone shell.

7 October, 2004

    w-Conotoxin GVIA reveals N-type calcium channels in dorsal horn of spinal cord

    Murakami, M., Nakagawasai, O., Suzuki, T., Mobarakeh, II., Sakurada, Y., Murata, A., Yamadera, F., Miyoshi, I., Yanai, K., Tan-No, K., Sasano, H., Tadano, T. and Iijima, T. (2004) Antinociceptive effect of different types of calcium channel inhibitors and the distribution of various calcium channel alpha(1) subunits in the dorsal horn of spinal cord in mice. Brain Res.1024:122-129.
    Department of Pharmacology, Akita University School of Medicine, 1-1-1 Hondoh, Akita 010-8543, Japan.
    Abstract: To understand better which voltage-dependent calcium channels (VGCCs) are involved in nociceptive neurotransmission, we investigated the pharmacological properties and distribution of VGCCs in the mouse spinal cord. A behavioral assay revealed that intrathecal injections of omega-agatoxin TK, omega-agatoxin IVA, omega-conotoxin GVIA, and SNX-482, which block P/Q-, P/Q-, N-, and R-type calcium channels, respectively, produced analgesic effects, while an L-type channel blocker had no such effect. An electrophysiological study demonstrated the presence of various types of VGCCs within dorsal root ganglion (DRG) neurons. Immunohistochemistry revealed distinct localization of P/Q-, N-, L-, and R-type calcium channel subunits to the dorsal horn of the spinal cord. The results of this study revealed the localization and functions of several calcium channels that are involved in nociceptive neurotransmission within the dorsal horn of the mouse spinal cord.

25 September, 2004

    'Light sleeper' peptide from Conus radiatus

    Olivera and colleagues have isolated a novel
    “light sleeper peptide”, conotoxin r7a, from Conus radiatus venom.

    (W = 6-bromotryptophan; Ê = gamma-carboxyglutamate; ^ = C-terminal free acid)

    This is the third peptide from Conus radiatus venom that induces a sleep-like state. This highly posttranslationally modified peptide is distinctly different from two other sleeper peptides characterized previously from this venom:

    Jimenez, E.C., Watkins, M. and Olivera, B.M. (2004) Multiple 6-bromotryptophan residues in a sleep-inducing peptide. Biochemistry 43: 12343-12348.
    Departments of Biology and Pathology, University of Utah, Salt Lake City, Utah 84112, and Department of Physical Sciences, College of Science, University of the Philippines Baguio, Baguio City, Philippines.
    Abstract: We have characterized a novel sleep-inducing peptide comprising 33 amino acids with three residues of the unusual posttranslationally modified amino acid, 6-bromotryptophan. The peptide, termed "light sleeper" or the r7a conotoxin, was purified from the venom of the fish-hunting Conus radiatus. The light sleeper peptide has additional notable biochemical properties; it equilibrates slowly between two distinct conformers, and has four gamma-carboxyglutamate residues. The pattern of posttranslational bromination in the light sleeper peptide suggests that tryptophan residues at N- and C-termini may be preferential sites for posttranslational bromination.

15 September, 2004

    Review: Conus peptides - and video clip of envenomation
    Cruz, LJ and Jimenez, EC (2004) Conus venom neuropeptides Encyclopedia of Neuroscience 3rd Edition, Edited by George Adelman and Barry H. Smith, CD-ROM,p.291 ISBN: 0444514325, Elsevier, BV.
    Includes video clip showing envenomation of a small fish by a piscivorous cone shell.

    Conus textile envenomating a marine snail
    In issue #32 of Marine Life, Guido Poppe and Philippe Poppe have a nice photo of a Conus textile in the act of envenomating a small snail on Cabilao Island in the Philippines.

10 September, 2004

    {alpha}5 Subunit of nicotinic receptor contributes to native alpha6*-nAChR-like pharmacology with high affinity for alpha-conotoxin MII
    Grinevich, V.P., Letchworth, S.R., Lendenberger, K.A., Menager, J., Mary, V., Sadieva, K.A., Buhlman, L., Bohme, G.A., Pradier, L., Benavides, J., Lukas, R.J. and Bencherif, M. (2004) Heterologous Expression of Human {alpha}6{beta}4{beta}3{alpha}5 Nicotinic Acetylcholine Receptors: Binding Properties Consistent with Their Natural Expression Require Quaternary Subunit Assembly Including the {alpha}5 Subunit. J Pharmacol Exp Ther. 2004 Sep 8 [Epub ahead of print]
    Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, Arizona 85013, USA. rlukas@chw.edu
    Abstract: Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners for nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits (alpha6*-nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human alpha6beta2-, alpha6beta4-, alpha6beta2beta3-, alpha6beta4beta3-, or alpha6beta4beta3alpha5-nAChR. Cells expressing subunits needed to form alpha6beta4beta3alpha5-nAChR exhibited saturable [(3)H]-epibatidine binding (Kd = 95.9 +/- 8.3 pM and Bmax = 84.5 +/- 1.6 fmol/mg protein). The rank order of binding competition potency (Ki) for prototypical nicotinic compounds was alpha-conotoxin MII (6 nM) > nicotine (156 nM) approximately methyllycaconitine (200 nM) > alpha-bungarotoxin (>10 mM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that beta3 and alpha5 subunits are likely partners of the alpha6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time RT-PCR of human brain indicated that alpha6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, the human alpha5 subunit makes a critical contribution to alpha6beta4beta3alpha5-nAChR assembly into a ligand-binding form with native alpha6*-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.

3 September, 2004

    Different sensitivity of two classes of DRG neurons to contoxin GVIA
    In this study, Drs Wu and Pan found that block of N-type Ca2+ channels with omega-conotoxin GVIA produced a significantly greater reduction of Ca+ currents in IB(4)-positive than in the smaller IB(4)-negative DRG neurons. They also suggest that IB(4)-positive neurons have a higher density of N-type calcium channels than the smaller IB(4)-negative DRG neurons.These differences may contribute to the different function of these two classes of nociceptive neurons.

    Wu, Z.Z. and Pan, H.L. (2004). High voltage-activated Ca+ channel currents in isolectin B(4)-positive and -negative small dorsal root ganglion neurons of rats. Neurosci Lett. 368: 96-101.
    Department of Anesthesiology, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, PA 17033, USA.

    Abstract: Voltage-gated Ca+ channels in the primary sensory neurons are important for neurotransmitter release and regulation of nociceptive transmission. Although multiple classes of Ca channels are expressed in the dorsal root ganglion (DRG) neurons, little is known about the difference in the specific channel subtypes among the different types of DRG neurons. In this study, we determined the possible difference in high voltage-activated Ca+ channel currents between isolectin B(4) (IB(4))-positive and IB(4)-negative small-sized (15-30microm) DRG neurons. Rat DRG neurons were acutely isolated and labeled with IB(4) conjugated to a fluorescent dye. Whole-cell patch clamp recordings of barium currents flowing through calcium channels were performed on neurons with and without IB(4). The peak current density of voltage-gated Ca2+ currents was not significantly different between IB(4)-positive and IB(4)-negative neurons. Also, both nimodipine and omega-agatoxin IVA produced similar inhibitory effects on Ca2+ currents in these two types of neurons. However, block of N-type Ca+ channels with omega-conotoxin GVIA produced a significantly greater reduction of 2Ca2+currents in IB(4)-positive than IB(4)-negative neurons. Furthermore, the IB(4)-positive neurons had a significantly smaller residual Ca2+ currents than IB(4)-negative neurons. These data suggest that a higher density of N-type Ca2+ channels is present in IB(4)-positive than IB(4)-negative small-sized DRG neurons. This differential expression of the subtypes of high voltage-activated Ca+ channels may contribute to the different function of these two classes of nociceptive neurons.

29 August, 2004

    MOLLUSK - a Conotoxin sequence database

    MOLLUSK is an integrated database of conotoxin sequence information, focusing on the molecular components of conotoxin sequences. MOLLUSK is meant to provide a unique source of information, data retrieval and analysis tools on conotoxin sequences. All records are fully referenced.
    MOLLUSK facilitates:

  • Keyword search - enables search for entries from the entire database using keywords, ion channel, superfamily and family
  • Alignments - contains alignments of protein sequences and superfamilies
  • BLAST Mollusk - a query amino acid or nucleotide sequence can be compared against all known sequences available in the database using BLAST.
  • Download FASTA - enables the downloading of fasta-formatted files of amino acids and nucleotide sequences.
  • Annotate Mollusk - analyse and generates putative functional annotation for the query sequence
  • Mollusk Structures - display 3-D structures of conotoxin from PDB and the homology models of toxins whose structures have not been solved as yet.
    (Source: The Institute for Infocomm Research (I²R), Singapore. The Institute for Infocomm Research (I²R) integrates R&D in communications and information technology to develop holistic solutions across the ICT value chain. The Institute's research capabilities are in wireless and optical communications, and information technology and science. I²R seeks to enable technologies and processes that will drive new and enhanced services for Singapore's knowledge-based economy. Dr. Vladimir Brusic and Dr. Rekha Pillai, coordinators, last updated this database on October 10, 2003).

27 August, 2004

    The Shell Collector: A tale of a blind professor who collects cones
    THE SHELL COLLECTOR by Anthony Doerr, Paperback: 224 pages ; Dimensions (in inches): 0.59 x 7.94 x 5.10 Publisher: Penguin Books; Reissue edition (February 1, 2003) ISBN: 0142002968

    The Shell Collector, Anthony Doerr's first collection of stories, ranges from Liberia and Tanzania to Montana and Maine. This short story collection was named winner of bookseller Barnes and Noble's 10th Annual "Discover Great New Writers Awards" in the fiction category on March 5, 2003. The Shell Collector is Doerr's first work of fiction, and it is the first short-story collection to receive the Discover Award (Doerr received a cash prize of $10,000 and a full year of additional marketing and advertising support). Traversing the vast terrain of the world, Doerr shows an extraordinary empathy in stories that most often concern themselves with the interaction between humans and nature. In the title story he applies his empathy to a retired professor ("The Shell Collector"), a lyrical and somber tale constructed of achingly beautiful, precise language. Synopsis: The shell collector is a blind former professor who at age 63 is attempting a retreat from the human world by retiring to the coast of Tanzania to live out his days wading in a quiet lagoon and collecting shells with fingers that seem to see. His world is simple and empirical: "Ignorance was, in the end, and in so many ways, a privilege: to find a shell, to feel it, to understand only on some unspeakable level why it bothered to be so lovely. What joy he found in that, what utter mystery." Yet the world intrudes on the shell collector, asking him to be a father, a guide, and a savior; reluctantly, and with devastating and then surprisingly hopeful consequences, he is drawn in. Doerr's greatest gift is to see an equal humanity in people belonging to all ages, cultures, and countries. "Doerr's proze dazzles, his sinewy sentences blending the naturalist's unswerving gaze with the poet's gift for metaphore." - New York Times Book Review. Stories include a winner of the O. Henry Award and two finalists for the National Magazine Award.

    Dextral or Sinistral ?
    Wandelt, J. and Nagy, L.M. (2004) Left-right asymmetry: more than one way to coil a shell. Curr Biol. 14: R654-656.
    Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721, USA.
    Abstract:Snail shells can be left-handed or right-handed, sometimes within one species. For over a century, it has commonly been assumed that mirror-image shell coiling in snails is correlated with a mirror- image reversal of early spindle orientation and cleavage. The results of an exciting and elegant new study refute this model, showing that right doesn't have to be the mirror image of left.

26 August, 2004

    Synthesis of cis-Pro isomer analogs of delta-Conotoxin EVIA from Conus erminius
    Chierici, S., Jourdan, M., Figuet, M. and Dumy, P. (2004) A case study of 2,2-dimethylthiazolidine as locked cis proline amide bond: synthesis, NMR and molecular modeling studies of a [small delta]-conotoxin EVIA peptide analog. Org Biomol Chem. 2: 2437-2441. Epub 2004 Aug 05.
    LEDSS UMR 5616 & ICMG-FR2607, Universite Joseph Fourier, BP 53, 38041, Grenoble cedex 9, France. pascal.dumy@ujf-grenoble.fr
    Abstract:The delta-conotoxin EVIA from the Conus ermineus venom, a recently characterized toxin, exhibits cis-trans isomerism of the Leu(12)-Pro(13) bond associated with the triggering of its biological activity. In this paper we use the pseudoproline concept to target the presumed bioactive cis conformation. We report the design and the synthesis of loop 2 analogs from residue 8 to 18 containing either the cis-inducing Cys(Psi(Me,Me)Pro)(13) unit or the natural proline residue. NMR studies in water and molecular modeling allowed us to identify the amide bond "locked" in a cis conformation for as in the suggested bioactive form of the natural toxin.

24 August, 2004

    Conotoxin-receptor interactions aid drug discovery
    Grant, M.A., Morelli, X.J. and Rigby, A.C. (2004) Conotoxins and structural biology: a prospective paradigm for drug discovery. Curr Protein Pept Sci. 5: 235-248.
    Division of Hemostasis and Thrombosis, Department of Medicine, Beth Israel Deaconess Medical Center, 41 Avenue Louis Pasteur, Boston, MA 02115, USA. arigby@bidmc.harvard.edu

    Abstract: Understanding the interactions between activating or antagonizing ligands and their cognate receptors at a molecular level offers promise for the development of pharmacological therapeutics for CNS disorders. The discovery of novel molecules that are capable of discriminating between the varied molecular subunits or isoforms of ion channels should provide a more detailed understanding of the pathophysiology of many CNS disorders. Abundant natural sources of pharmacologically active agents that demonstrate this refined selectivity and specificity are found in the animal toxins of venomous species including: snakes, spiders and the marine snail of the genus Conus. The uniquely fascinating combinatorial ability of the marine snail, genus Conus to modify the pharmacological properties of these neurotoxins or conopeptides within its venom is depicted throughout this review. The myriad of posttranslational modifications and disulfide bonded architectures that have been identified in the conopeptides, are described with an emphasis on the unique pharmacological properties and receptor target specificities that have been ascribed to each of these modifications. The ability of NMR spectroscopy to provide three-dimensional structural information within the interaction interface for both the ligand and target protein following complex formation and its application to conopeptide drug discovery are discussed. Similarly, the strength of merging NMR spectroscopy data with ab initio "restrained soft-docking" for rational pharmacophore design and the identification of lead compounds from in silico library screens will also be discussed. The initial phases of this stratagem are illustrated using two toxin antagonists and the recently determined structure of the KcsA potassium channel. These data exemplify the utility of this approach in elucidating important molecular interfaces of specific toxin-receptor/ion channel complexes, which can be further exploited in drug discovery initiatives.

    Cone snail venom toxins and neuroprotection
    Rajendra W, Armugam A, Jeyaseelan K.(2004) Neuroprotection and peptide toxins. Brain Res Brain Res Rev. 45: 125-141. Review.
    Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore.
    Abstract: Neurodegeneration induced by excitatory neurotransmitter glutamate is considered to be of particular relevance in several types of acute and chronic neurological impairments ranging from cerebral ischaemia to neuropathological conditions such as motor neuron disease, Alzheimer's, Parkinson's disease and epilepsy. The hyperexcitation of glutamate receptors coupled with calcium overload can be prevented or modulated by using well-established competitive and non-competitive antagonists targeting ion/receptor channels. The exponentially increasing body of pharmacological evidence over the years indicates potential applications of peptide toxins, due to their exquisite subtype selectivity on ion channels and receptors, as lead structures for the development of drugs for the treatment of wide variety of neurological disorders. This review comprehensively highlights the overview of the diversity in the molecular as well as neurobiological mechanisms of different peptide toxins derived from venomous animals [for conotoxins see pp. 130-135] with particular reference to neuroprotection. In addition, the potential applications of peptide toxins in the diagnosis and treatment of neurological disorders such as neuromuscular disorders, epilepsy, Alzheimer's and Parkinson's diseases, gliomas and ischaemic stroke and their future prospects in the diagnosis as well as in the therapy are addressed.

    Clinical applications of conotoxins in pain and inflammation
    Rajendra, W., Armugam, A. and Jeyaseelan, K. (2004) Toxins in anti-nociception and anti-inflammation. Toxicon 44: 1-17.
    Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8, Medical Drive, Singapore, Singapore 117597.
    Abstract: The use of toxins as novel molecular probes to study the structure-function relationship of ion-channels and receptors as well as potential therapeutics in the treatment of wide variety of diseases is well documented. The high specificity and selectivity of these toxins have attracted a great deal of interest as candidates for drug development. This review highlights the involvement of the proteins and peptide toxins as well as non-proteinaceous compounds derived from both venomous and non-venomous animals, in anti-nociception and anti-inflammation. The possible mechanisms of these potential therapeutic agents and possible clinical applications in the treatment of pain and inflammation are also summarized.

20 August, 2004

    Biotinylated mu-Conotoxin GIIIA probes sodium channels
    Nakamura M, Ishida Y, Kohno T, Sato K, Oba Y, Nakamura H. (2004) Effects of modification at the fifth residue of mu-conotoxin GIIIA with bulky tags on the electrically stimulated contraction of the rat diaphragm. J Pept Res. 64: 110-117.
    Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601, Japan.
    Abstract: mu-conotoxin GIIIA, a peptide toxin from the cone snail, blocks muscle-type sodium channels. Thr-5 of mu-conotoxin GIIIA, located on the opposite side of the active site in the globular molecule, was replaced by Cys to which the bulky tags were attached. The tagged mu-conotoxin GIIIA derivatives, except for the phospholipid-tagged one, exerted the biological activity with a potency slightly weaker than natural mu-conotoxin GIIIA. When the biotinylated tags of various lengths were added, the presence of avidin suppressed the action of the biotinylated toxins of <4 nm, but not with 5 nm. The bulky biotinylated tags are useful as a caliper to measure the depth of receptor sites in the channels.

19 August, 2004

    alpha6-containing nicotinic receptors mediate dopamine release
    Azam, L. and McIntosh, J.M. (2004) Effect of novel {alpha}-conotoxins on nicotine-stimulated [3H]dopamine release from rat striatal synaptosomes. J Pharmacol Exp Ther. 2004 Aug 17 [Epub ahead of print]
    Abstract:Nicotine's action on the midbrain dopaminergic neurons is mediated by nicotinic acetylcholine receptors (nAChRs) that are present on the cell bodies and the terminals of these neurons. Previously, it was suggested that one of the nAChR subtypes located on striatal dopaminergic terminals may be an alpha3beta2 subtype, based on partial inhibition of nicotine-stimulated [(3)H]dopamine release by alpha-conotoxin MII, a potent inhibitor of heterologously expressed alpha3beta2 nAChRs. More recent studies indicated that alpha-conotoxin MII also potently blocks alpha6-containing nAChRs. In the present study, we have examined the nAChR subtype(s) modulating [(3)H]dopamine release from striatal terminals by using novel alpha-conotoxins that have 37-78 fold higher selectivity for alpha6- vs. alpha3-containing nAChRs. All of the peptides partially (20-35%) inhibit nicotine-stimulated [(3)H]dopamine release with IC50s consistent with those obtained with heterologously expressed rat alpha6-containing nicotinic acetylcholine receptors. These results, together with previous studies by others, further support the idea that alpha6-containing nicotinic receptors modulate nicotine-stimulated dopamine release from rat striatal synaptosomes.

13 August, 2004

    Evolution of Conus feeding modes
    Duda Jr TF, Palumbi SR. (2004) Gene expression and feeding ecology: evolution of piscivory in the venomous gastropod genus Conus. Proc R Soc Lond B Biol Sci. 271: 1165-1174.
    Naos Marine Laboratory, Smithsonian Tropical Research Institute, Box 2072, Balboa, Ancon, Republic of Panama.
    Abstract:Differential expression of gene-family members is typically associated with the specific development of certain tissues and organs, but its importance in the ecological adaptation of organisms has rarely been investigated. Several specialized feeding modes have evolved within the predatory marine gastropod genus Conus, including molluscivory and piscivory. Based on phylogenetic investigations of Conus species, it has been concluded that piscivory arose at least twice in this genus. Moreover, molecular analyses of conotoxin mRNA transcripts reveal that piscivores from independent evolutionary lineages express the same subset of four-loop conotoxins, contrary to phylogenetic expectations. These results demonstrate that differential expression of gene-family members can play a key role in adaptive evolution, particularly during shifts to new ecological niches.

11 August, 2004

9 August, 2004

    3 New species of Conus described in VISAYA

    VISAYA is a new malacological journal with taxonomical and nomenclatoral articles. In the first issue Guido T. Poppe, Manuel J. Tenorio, Carlos M. L. Afonso and Koen Fraussen have contributed with 5 articles containing 9 descriptions of new species of molluscs including 3 new species of Conus (Conus frausseni, Conus grohi and Conus terryni). Conus cuneolus and its relatives of Sal has been revised and documented. VISAYA, in A4, is printed in colour from cover to cover, the number 1 has 52 pages and 16 full page coloured plates. Numerious in text figures and maps.VISAYA is an occasional publication and will be published as manuscripts become available. Authors are invited to submit articles for publication.

9 August, 2004

    Review: mu-Conotoxins, sodium channel antagonists
    Li, R.A. and Tomaselli, G.F. (2004) Using the deadly micro-conotoxins as probes of voltage-gated sodium channels. Toxicon 44: 117-122.
    The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 871, Baltimore, MD 21205, USA.
    Abstract:micro-Conotoxins (micro-CTX) are potent Na channel inhibitory peptides isolated from the venom of the predatory marine snail Conus geographus. micro-CTXs exert their biological action by physically occluding the ion-conducting pore of voltage-gated Na (Na(v)) channels with a 1:1 stoichiometry in an all-or-none fashion. This article reviews our current knowledge of the mechanism of micro-CTX and the associated structural and functional insights into its molecular target-Na(v) channels.

3 August, 2004

    Review: Conopeptides as potential therapeutics
    Livett, B.G., Gayler, K.R., Khalil, Z. (2004) Drugs from the sea: conopeptides as potential therapeutics. Curr Med Chem. 11: 1715-1723.
    Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3010, Australia.
    Abstract:Marine cone snails from the genus Conus are estimated to consist of up to 700 species. These predatory molluscs have devised an efficient venom apparatus that allows them to successfully capture polychaete worms, other molluscs or in some cases fish as their primary food sources. The toxic venom used by the cone shells contains up to 50 different peptides that selectively inhibit the function of ion channels involved in the transmission of nerve signals in animals. Each of the 700 Conus species contains a unique set of peptides in their venom. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing a high degree of selectivity for particular types of channels. We have undertaken a study of the conotoxins from Australian species of Conus that have the capacity to inhibit specifically the nicotinic acetylcholine receptors in higher animals. These conotoxins have been identified by mass spectroscopy and their peptide sequences in some cases deduced by the application of modern molecular biology to the RNA extracted from venom ducts. The molecular biological approach has proven more powerful than earlier protein/peptide based technique tor the detection of novel conotoxins [1,2]. Novel conotoxins detected in this way have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. This review describes the progress made by several research groups to characterise the properties of conopeptides and to use them as drug leads for the development of novel therapeutics for the treatment of a range of neurological conditions.

    Peptide "turritoxins" from Turrids
    Lopez-Vera, E., Heimer de la Cotera, E.P., Maillo, M., Riesgo-Escovar, J.R., Olivera B.M. and Aguilar, M.B. (2004) A novel structural class of toxins: the methionine-rich peptides from the venoms of turrid marine snails (Mollusca, Conoidea). Toxicon.43: 365-374.
    Laboratory of Marine Neuropharmacology, Institute for Neurobiology, Universidad Nacional Autonoma de Mexico, Queretaro 76230, Mexico.

    Abstract:The objective of this investigation was to purify and characterize polypeptides from the venom ducts of the turrid snails Polystira albida and Gemmula periscelida (superfamily: Conoidea, family: Turridae), collected in Mexican waters. Venoms of other groups in the superfamily (family: Conidae, genus: Conus) have peptide toxins ('conotoxins'), but no venom components have been characterized from any turrid species. Crude venoms were fractionated using reversed-phase high performance liquid chromatography, and one major component from each venom was characterized. In contrast to most conotoxins, the polypeptides characterized contain a high proportion of Met, Tyr and Arg residues, and few, if any, Cys residues. The two peptides had some regions of homology, but were not significantly similar to other peptides. Both peptides are predicted to contain alpha-helical structures, and the peptide from P. albida is predicted to form a coiled-coil motif. This structural motif could provide conformational stability for these turrid venom components ("turritoxins"), which in the case of conotoxins is primarily achieved by disulfide bonds. Thus, the first turritoxins characterized are strikingly different from the conotoxins, suggesting divergent biochemical strategies in the venoms of different major groups included in the superfamily Conoidea.

28 July, 2004

    D'Alessandro, M., Paci, M. and Amadei, A. (2004) Characterization of the conformational behavior of peptide Contryphan Vn: a theoretical study. Biopolymers 74: 448-456.
    Dipartimento di Scienze e Tecnologie Chimiche, Universita di Roma Tor Vergata, via della Ricerca Scientifica 1, 00133 Roma, Italy.
    Abstract: In this work we report the study of a peptide, the Contryphan Vn produced by Conus ventricosus, a vermivorous cone snail living in the temperate Mediterranean sea. This cyclic peptide of nine residues is a ring closed by a Cys-Cys (Cys: cysteine) disulfide bond containing two proline (Pro) residues and two tryptophans (Trp), one of them being a D-Trp. We present a statistical mechanical characterization of the peptide, simulated in water for about 200 ns with classical molecular dynamics (MD). In recent years there has been a growing interest in the study of the mechanics and dynamics of biological molecules, and in particular for proteins and peptides, about the relationship between collective motions and the active conformations which exert the biological function. To this aim we used the essential dynamics analysis on the MD trajectory and extracted, from the total fluctuations of the molecule, the dominant dynamical modes responsible of the principal conformational transitions. The Contryphan Vn small size allowed us to investigate in details the all-atoms dynamics and the corresponding thermodynamics in conformational space defined by the most significant intramolecular motions.

    Conotoxin GVIA- and CVID-sensitive N-type calcium channels in terminals of sacral preganglionic neurons.
    Jobling, P., Gibbins, I.L., Lewis, R.J. and Morris, J.L. (2004) Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs. Neuroscience 127: 455-466.
    Department of Anatomy and Histology, Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia.

    Abstract: Neurons in pelvic ganglia receive nicotinic excitatory post-synaptic potentials (EPSPs) from sacral preganglionic neurons via the pelvic nerve, lumbar preganglionic neurons via the hypogastric nerve or both. We tested the effect of a range of calcium channel antagonists on EPSPs evoked in paracervical ganglia of female guinea-pigs after pelvic or hypogastric nerve stimulation. omega-conotoxin GVIA (CTX GVIA, 100 nM) or the novel N-type calcium channel antagonist, CTX CVID (100 nM) reduced the amplitude of EPSPs evoked after pelvic nerve stimulation by 50-75% but had no effect on EPSPs evoked by hypogastric nerve stimulation. Combined addition of CTX GVIA and CTX CVID was no more effective than either antagonist alone. EPSPs evoked by stimulating either nerve trunk were not inhibited by the P/Q calcium channel antagonist, omega-agatoxin IVA (100 nM), nor the L-type calcium channel antagonist, nifedipine (30 microM). SNX 482 (300 nM), an antagonist at some R-type calcium channels, inhibited EPSPs after hypogastric nerve stimulation by 20% but had little effect on EPSPs after pelvic nerve stimulation. Amiloride (100 microM) inhibited EPSPs after stimulation of either trunk by 40%, while nickel (100 microM) was ineffective. CTX GVIA or CTX CVID (100 nM) also slowed the rate of action potential repolarization and reduced afterhyperpolarization amplitude in paracervical neurons. Thus, release of transmitter from the terminals of sacral preganglionic neurons is largely dependent on calcium influx through N-type calcium channels, although an unknown calcium channel which is resistant to selective antagonists also contributes to release. Release of transmitter from lumbar preganglionic neurons does not require calcium entry through either conventional N-type calcium channels or the variant CTX CVID-sensitive N-type calcium channel and seems to be mediated largely by a novel calcium channel.

    Xenome announce Phase I clinical trial for Xen2174
    In a Media Release, 20 July 2004, Xenome announced commencement of a Phase I clinical trial on its lead analgesic compound, Xen2174 (a chi-conotoxin that acts as a noradrenaline reuptake inhibitor). This Phase I trial is a randomised, placebo-controlled, double blind, dose-escalating study involving up to 20 healthy male volunteers. The primary purpose of this trial is to evaluate the safety and tolerability of Xen2174 following intravenous administration. "Additional information on the pharmacokinetics and potential anti-nociceptive effects of Xen2174 will also be examined during the trial", commented Xenome's Head of Drug Development, Dr Michael Thurn.

13 July, 2004

    A novel alphaA((1-3))-conotoxin OIVA from milked venom of Conus obscurus
    Teichert, R.W., Rivier, J., Dykert, J., Cervini, L., Gulyas, J., Bulaj, G., Ellison, M. and Olivera, B.M. (2004) alphaA-Conotoxin OIVA defines a new alphaA-conotoxin subfamily of nicotinic acetylcholine receptor inhibitors. Toxicon 44: 207-214.
    Department of Biology, University of Utah, 254 South 1400 East, Salt Lake City, UT 84112, USA.

    Abstract:The venoms of cone snails are rich in multiply disulfide-crosslinked peptides, the conotoxins. Conotoxins are grouped into families on the basis of shared cysteine patterns and homologous molecular targets. For example, both the kappaA- and alphaA-conotoxin families share the same Class IV Cys pattern (-CC-C-C-C-C-), but differ in their molecular targets. The kappaA-conotoxins are excitatory toxins that purportedly block potassium channels, while the alphaA-conotoxins are paralytic conotoxins that inhibit nicotinic acetylcholine receptors (nAChRs). In this work, we describe the isolation and characterization of a novel Conus peptide from venom milked from Hawaiian specimens of Conus obscurus. This peptide shares the Class IV Cys pattern but differs from both previously characterized alphaA- and kappaA-conotoxins in the spacing of amino acids between Cys resides. However, the peptide is similar to previously characterized alphaA-conotoxins in its paralytic effects on fish and its antagonist activity on the neuromuscular nAChR. Unexpectedly, the peptide differs in its disulfide bonding from alphaA-conotoxin PIVA. We have named this unique peptide alphaA-conotoxin OIVA, and we consider it the defining member of a subfamily of alphaA-conotoxins that we designate the alphaA((1-3))-conotoxins to identify them by their unique disulfide bonding framework. These results indicate that the alphaA-conotoxin family is both more structurally diverse and broadly distributed than previously believed.

    A benzothiazole-derived mimetic of omega-conotoxin GVIA
    Baell, J.B., Duggan, P.J., Forsyth, S.A., Lewis, R.J., Phei Lok, Y. and Schroeder, C.I. (2004) Synthesis and biological evaluation of nonpeptide mimetics of omega-conotoxin GVIA. Bioorg Med Chem.12: 4025-4037.
    School of Chemistry, Monash University, Clayton, Vic 3800, Australia.

    Abstract: A benzothiazole-derived compound (4a) designed to mimic the C(alpha)-C(beta) bond vectors and terminal functionalities of Lys2, Tyr13 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low microM binding affinity to N-type VGCCs (IC(50)=1.9microM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC(50)= 4.1microM) showed a greater than 25-fold selectivity for the N-type channel.

12 July, 2004

    kappaM-Conotoxin RIIIK from Conus radiatus blocks voltage-activated K(+) channels with a novel pharmacophore
    Al-Sabi, A., Lennartz, D., Ferber, M., Gulyas, J., Rivier, J.E., Olivera, B.M., Carlomagno, T. and Terlau, H.(2004) kappaM-Conotoxin RIIIK, Structural and Functional Novelty in a K Channel Antagonist. Biochemistry 43: 8625-8635.
    Molecular and Cellular Neuropharmacology Group, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, D-37075 Gottingen, Germany, Department of NMR Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Gottingen, Germany, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037, and Department of Biology, University of Utah, Salt Lake City, Utah 84112.

    Abstract: Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. kappaM-conotoxin RIIIK (kappaM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K channels. The functional and structural study presented here reveals that kappaM-conotoxin RIIIK blocks voltage-activated K channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of kappaM-RIIIK in the C-terminal half is highly similar to that of micro-conotoxin GIIIA, a specific blocker of the skeletal muscle Na+ channel Na(v)1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of kappaM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of kappaM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K channel-targeted toxins, kappaM-RIIIK blocks voltage-gated K channels with a pharmacophore that is not organized around a lysine-hydrophobic amino acid dyad motif.

1 July, 2004

    Conus cancellatus from the Northern Gulf of Mexico
    Kirsten Larsen and Harriet Perry from the Gulf Coast Research Laboratory, Ocean Springs, MS, USA, have created a web page entitled A Picture Guide to Shelf Invertebrates from the Northern Gulf of Mexico. This guide was assembled to assist NOAA/NMFS personnel with at-sea identification of invertebrates caught during their Groundfish and Pelagic cruises. The guide of over 300 pages includes listings of Molluscs (snails, clams, squid); Crustaceans (shrimp, crabs, lobsters, hermit crabs); Echinoderms, (starfish, sea urchins, sea cucumbers) and others (jellyfish, ascidians, polychaetes). The plates were designed for identification of fresh specimens at sea. Among the molluscs is just one cone shell Conus cancellatus (= C. austini) Cancellate Cone, Hwass, 1792. Habitat: 20 fathoms, common off Yucatan, rare off Florida. Range: Southeast Florida to Yucatan and West Indies.

    Receptor selectivity of conopeptide rho-TIA from Conus textile
    Chen, Z., Rogge, G., Hague, C., Alewood, D., Colless, B., Lewis, R.J. and Minneman, K.P. (2004) Subtype-selective noncompetitive or competitive inhibition of human alpha 1-adrenergic receptors by rho-TIA. J Biol Chem. 279: 35326-35333.
    Pharmacology, Emory University, Atlanta, GA 30322.

    Abstract: The 19 amino acid conopeptide (rho-TIA) was shown previously to noncompetitively antagonize alpha(1B)-adrenergic receptors (ARs). Since this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)- over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B(max)) for human alpha(1B)-ARs without changing affinity (K(D)), and this inhibition was unaffected by length of incubation but reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing K(D) without changing B(max), suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated (3)H-inositol phosphate formation in HEK 293 cells expressing human alpha(1B)-ARs, but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs, and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA non-competitively inhibits alpha(1B)-ARs, but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini, are not due to the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.

    Binding sites of alpha-conotoxins
    Dutertre, S., Nicke, A., Tyndall, J.D. and Lewis, R.J. (2004) Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors. J Mol Recognit. 17: 339-347.
    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

    Abstract: alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that ImI and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists.

    Cyclic pentapeptides that mimic omega-conotoxins
    Schroeder, C.I., Smythe, M.L. and Lewis, R.J. (2004) Development of small molecules that mimic the binding of omega-conotoxins at the N-type voltage-gated calcium channel. Mol Divers. 8: 127-134.

    Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia.

    Abstract: Cone snails (Conidae) are marine predators with some extraordinary features. Their venom contains a hundred or more peptides that target numerous ion channels and receptors in mammals, including several that are involved in disease. omega-Conotoxins from fish hunting snails are 24-27 residue peptides with a rigid 4-loop cysteine framework that target the N-type voltage-gated calcium channel (VGCC). Two omega-conotoxins, MVIIA and CVID are currently in clinical development for chronic pain management (Ziconotide or Prialt, and AM336, respectively). In an attempt to develop small molecule equivalents of CVID, we defined the Calpha-Cbeta vectors of the residues believed to be important for binding to the N-type VGCC. Using these vectors, we undertook a virtual screening of virtual libraries approach to identify compounds that matched the pharmacophore. Cyclic pentapeptides containing residues of loop 2 of CVID, with one or more being a D-amino acid were designed and synthesised and were found to be active at the N-type VGCC (IC50 approximately 20 microM). Agreeing with the specificity profile of CVID, molecules were inactive at the P/Q-type VGCC.

    Mini-Review series on alpha-conotoxins
    Nicke A. (2004) Learning about structure and function of neuronal nicotinic acetylcholine receptors. Lessons from snails. Eur J Biochem. 271: 2293.

    Extract: This minireview series gives an account of alpha-conotoxins and their interaction with neuronal nAChR subtypes covering chemical, pharmacological and structural aspects.The first review in this series by Loughnan & Alewood, provides a brief introduction into the biology of cone snails before covering analytical aspects of alpha-conotoxin isolation and characterization and strategies for their synthesis, a key requirement for their use as research tools. As small peptides, alpha-conotoxins show a superior selectivity to small ligands which often can not differentiate between highly conserved nAChR binding sites. The second review by Nicke, Wonnacott and Lewis, provides an overview of the diversity of neuronal nAChRs and summarizes the pharmacological characterization of selective alpha-conotoxins with a focus on their application in deciphering neuronal nAChR subtypes in native tissues. The crystal and/or NMR solution structures of most alpha-conotoxins have been determined. Thus information about common structural elements and three dimensional orientation of the amino acid residues that contribute to binding is available. In the third review by Millard, Daly and Craik. these structures are compared, and features that contribute to binding and underlie subtype selectivity are discussed. The high resolution structure of AChBP serves as a template for homology modelling and docking studies which are described in the fourth review by Dutertre & Lewis. This paper summarizes data from mutagenesis studies and integrates the available structural information to develop first models of the alpha-conotoxin–nAChR interaction.

    Structural studies on alpha-conotoxins
    Millard, E.L., Daly, N.L., Craik, D.J. (2004) Structure-activity relationships of alpha-conotoxins targeting neuronal nicotinic acetylcholine receptors. Eur J Biochem. 271: 2320-2326.
    Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia.

    Abstract: alpha-Conotoxins that target the neuronal nicotinic acetylcholine receptor have a range of potential therapeutic applications and are valuable probes for examining receptor subtype selectivity. The three-dimensional structures of about half of the known neuronal specific alpha-conotoxins have now been determined and have a consensus fold containing a helical region braced by two conserved disulfide bonds. These disulfide bonds define the two-loop framework characteristic for alpha-conotoxins, CCX(m)CX(n)C, where loop 1 comprises four residues (m = 4) and loop 2 between three and seven residues (n = 3, 6 or 7). Structural studies, particularly using NMR spectroscopy have provided an insight into the role and spatial location of residues implicated in receptor binding and biological activity

    Characterization and chemical synthesis of alpha-conotoxins
    Loughnan, M.L. and Alewood, P.F. (2004) Physico-chemical characterization and synthesis of neuronally active alpha-conotoxins. Eur J Biochem. 271: 2294-2304.
    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.

    Abstract: The high specificity of alpha-conotoxins for different neuronal nicotinic acetylcholine receptors makes them important probes for dissecting receptor subtype selectivity. New sequences continue to expand the diversity and utility of the pool of available alpha-conotoxins. Their identification and characterization depend on a suite of techniques with increasing emphasis on mass spectrometry and microscale chromatography, which have benefited from recent advances in resolution and capability. Rigorous physico-chemical analysis together with synthetic peptide chemistry is a prerequisite for detailed conformational analysis and to provide sufficient quantities of alpha-conotoxins for activity assessment and structure-activity relationship studies.

    Binding of {kappa}-Conotoxin PVIIA to Shaker K Channels
    Boccaccio, A., Conti, F., Olivera, B.M. and Terlau, H. (2004) Binding of {kappa}-Conotoxin PVIIA to Shaker K Channels Reveals Different K+ and Rb+ Occupancies within the Ion Channel Pore. J. Gen. Physiol. 124: 71-81.
    Max-Planck-Institute for Experimental Medicine, Molecular and Cellular Neuropharmacology Group, Hermann-Rein-Str.3, D-37075 Gottingen, Germany. Fax: 49-551-3899-475. email: hterlau@gwdg.de

    Abstract: The x-ray structure of the KcsA channel at different [K] and [Rb] provided insight into how K channels might achieve high selectivity and high K+ transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K channel in the presence of K and Rb was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K channel differs in the closed and open state. When K is the only permeant ion, increasing extracellular [K] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K]. In contrast, extracellular [Rb] affects both closed- and open-channel binding. As extracellular [Rb] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb. These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.

24 June, 2004

    alpha-Conotoxins: probes for neuronal nicotinic receptors. A Review
    Nicke, A., Wonnacott, S., Lewis, R.J. (2004) alpha-Conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes. Eur J Biochem. 271:2305 -2319.

    Max Planck-Institute for Brain Research, Frankfurt, Germany.

    Abstract: Cone snails comprise approximately 500 species of venomous molluscs, which have evolved the ability to generate multiple toxins with varied and often exquisite selectivity. One class, the alpha-conotoxins, is proving to be a powerful tool for the differentiation of nicotinic acetylcholine receptors (nAChRs). These comprise a large family of complex subtypes, whose significance in physiological functions and pathological conditions is increasingly becoming apparent. After a short introduction into the structure and diversity of nAChRs, this overview summarizes the identification and characterization of alpha-conotoxins with selectivity for neuronal nAChR subtypes and provides examples of their use in defining the compositions and function of neuronal nAChR subtypes in native vertebrate tissues.

    Milking venomous cone snails
    Laura Nelson reports in a Nature News Feature today about the work of Dr. Jon-Paul Bingham from Clarkson University, upstate New York who's research involves keeping live cones in an aquarium and milking them for their venom to obtain novel bioactive compounds. This cone shell farming is aimed at conserving wild populations of cone shells while yielding a wider range of useful conotoxins. The News Feature "Venomous snails: one slip and your dead..." is published in Nature 429: 798-799 (2004). Or you can download the .pdf version of the News Feature here.

    Treatments for marine envenomations
    Watters, M.R. and Stommel, E.W. (2004) Marine Neurotoxins: Envenomations and Contact Toxins. Curr Treat Options Neurol. 6: 115-123.

    Department of Medicine, Division of Neurology, University of Hawaii, 1356 Lusitana Street, 7th Floor, Honolulu, HI 96813, USA. mwatters@hawaii.edu

    Abstract: Familiarity with the appearance and habitat of venomous sea creatures, the location of their stinging apparatus, and surveillance of population concentrations within recreational waters are essential in avoiding envenomations. Compared with the thermo-stable low molecular weighted ingestible seafood toxins, venomous toxins are often large molecular weight proteins and many are heat labile, which provides opportunity for therapeutic intervention. Heat therapy may denature the toxins, and provide immediate relief of pain in coelenterate and venomous fish envenomations. Injections of local anesthetic agents may also be used. First aid measures at the seashore may limit the spread of venom, and include immobilization of the affected sites, compression bandaging, and venous-lymphatic occlusive bandages. Measures to limit continued envenomation by attached stinging cells include topical vinegar for jellyfish tentacles and irrigation with debridment for spines of venomous fish. Antivenins are of limited availability and may be used for envenomations with sea snakes, Chironex box jellyfish, and some venomous fish. Sea snakes bites may be treated with antivenin from land snakes or with hemodialysis when antivenin is not available. Neuromuscular paralysis occurs with bites by sea snakes, cone snails, blue octopuses, and some jellyfish. Supportive treatment includes attention to cardiopulmonary resuscitation and intubation. Exposure to Pfeisteria may result in cognitive and behavioral abnormalities. Treatment with cholestyramine may be helpful in binding the toxin and improve recovery.

    See also Nimorakiotakisn B. and Winkel, K.D. (2003) Marine envenomations. Part 2--Other marine envenomations. Aust Fam Physician.32: 975-979.

23 June, 2004

    kappaM-conotoxin RIIIK blocks human Kv1.2 potassium channels
    Ferber, M., Al-Sabi, A., Stocker, M., Olivera, B.M., Terlau, H. (2004) Identification of a mammalian target of kappaM-conotoxin RIIIK. Toxicon 43:915-921.
    AG Molekulare und Zellulare Neuropharmakologie, Max-Planck-Institut fur Experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075 Gottingen, Germany.

    Abstract: Despite the great variability of the Conus peptides characterized until now only relatively few have been identified that interact with K+ channels. kappaM-conotoxin RIIIK (kappaM-RIIIK) is a 24 amino acid peptide from Conus radiatus, which is structurally similar to micro-conotoxin GIIIA, a peptide known to block specifically skeletal muscle Na+ channels. Recently, it has been shown that kappaM-RIIIK does not interact with Na+ channels, but inhibits Shaker potassium channels expressed in Xenopus oocytes. It was demonstrated that kappaM-RIIIK binds to the pore region of Shaker channels and a teleost homologue of the Shaker channel TSha1 was identified as a high affinity target of the toxin. In contrast the mammalian Shaker-homologues Kv1.1, Kv1.3, Kv1.4 are not affected by the toxin. In this study the activity of kappaM-RIIIK on other mammalian Kv1 K+ channels expressed in Xenopus oocytes was investigated. We demonstrate that kappaM-conotoxin RIIIK up to 5 microM exhibits no significant effect on Kv1.5 and Kv1.6 mediated currents, but the human Kv1.2 K channel is blocked by this peptide. The binding of kappaM-RIIIK to Kv1.2 channels is state dependent with an IC(50) for the closed state of about 200nM and for the open state of about 400nM at a test potential of 0 mV. kappaM-conotoxin RIIIK is the first conotoxin described to block human Kv1.2 potassium channels.

    Biographical and bibliographical catalogue of malacologists
    Eugene V. Coan, Alan R. Kabat & Richard E. Petit (2004), "2,400 Years of Malacology," is online at http://erato.acnatsci.org/ams/publications/epubs.html

    This publication is a comprehensive catalog of biographical and bibliographical papers on malacologists, conchologists, paleontologists, and others with an interest in mollusks. At present, the catalog is over 600 pages and indexes over 5,000 individuals. This catalog is a work in progress, and we will be posting updated versions periodically. Readers are encouraged to explore and use this catalog, and to forward to their comments to the authors.

21 June, 2004

    Cone shell gallery
    Alfonso Pina, from Málaga, Spain has updated his personal shell gallery with 53 new images. The cone shell collection can be seen here. Particlularly nice is his specimen of Conus decoratus.

18 June, 2004

    Conotoxins Gla-MrII, Gla-MrIII and Gla-MrIV from Conus marmoreus
    Hansson, K., Furie, B., Furie, B.C. and Stenflo, J. (2004) Isolation and characterization of three novel Gla-containing Conus marmoreus venom peptides, one with a novel cysteine pattern. Biochem Biophys Res Commun. 319: 1081-1087.
    Department of Clinical Chemistry, Lund University, University Hospital, Malmo, S-20502 Malmo, Sweden.

    Abstract: One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel gamma-carboxyglutamic acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five gamma-carboxyglutamic acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two gamma-carboxyglutamic acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal acid.

    The acyclic peptide Mo1659 from Conus monile targets K channels
    Sudarslal S, Singaravadivelan G, Ramasamy P, Ananda K, Sarma SP, Sikdar SK, Krishnan KS, Balaram P. (2004) A novel 13 residue acyclic peptide from the marine snail, Conus monile, targets potassium channels. Biochem Biophys Res Commun. 317:682-688.
    Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

    Abstract: A novel 13-residue peptide Mo1659 has been isolated from the venom of a vermivorous cone snail, Conus monile. HPLC fractions of the venom extract yielded an intense UV absorbing fraction with a mass of 1659Da. De novo sequencing using both matrix assisted laser desorption and ionization and electrospray MS/MS methods together with analysis of proteolytic fragments successfully yielded the amino acid sequence, FHGGSWYRFPWGY-NH(2). This was further confirmed by comparison with the chemically synthesized peptide and by conventional Edman sequencing. Mo1659 has an unusual sequence with a preponderance of aromatic residues and the absence of apolar, aliphatic residues like Ala, Val, Leu, and Ile. Mo1659 has no disulfide bridges distinguishing it from the conotoxins and bears no sequence similarity with any of the acyclic peptides isolated thus far from the venom of cone snails. Electrophysiological studies on the effect of Mo1659 on measured currents in dorsal root ganglion neurons suggest that the peptide targets non-inactivating voltage-dependent potassium channels.

15 June, 2004

    Coneshell update
    Giancarlo Paganelli has updated his Cone Shells collection (currently more than 1100 images). Conus adamsoni BRODERIP, 1836 French Polynesia, Tuamotu Is. - 36.8 mm; Conus episcopatus DA MOTTA, 1982 New Ireland, Kavieng - 60.9 mm ; Conus floccatus, SOWERBY II, 1841 Philippines, Olango Is. - 49.7 mm; Conus scalarisVALENCIENNES, 1832 Panama, Bay of Chiriqui - 51.6 mm; Conus voluminalisREEVE, 1843 Thailand, off Racha Is. - 65.6 mm, and Conus zeylanicusGMELIN, 1791 Mozambique, Nacala Bay - 53.4 mm.

    Cones with periostracum
    Included here are 3 new images of cones with periostracum Conus aureus, Conus circumcisus and Conus kinoshitai and some old ones. Conus aureus aureus HWASS in BRUGUIÈRE, 1792, Philippines, Cebu, Olango Is., off Caw-Oy - 58.1 mm; Conus circumcisus BORN, 1778, Philippines, Cebu, Olango Is., off Caw-Oy - 61.1 mm and Conus kinoshitai, KURODA, 1956, Philippines, Bohol, Panglao Is. - 45.4 mm; - and more.

9 June, 2004

    alpha-conotoxin interactions with neuronal nicotinic receptor
    Dutertre, S. and Lewis, R.J. (2004). Computational approaches to understand alpha-conotoxin interactions at neuronal nicotinic receptors. Eur J Biochem. 271:2327-2334.
    Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.

    Abstract: Recent and increasing use of computational tools in the field of nicotinic receptors has led to the publication of several models of ligand-receptor interactions. These models are all based on the crystal structure at 2.7 A resolution of a protein related to the extracellular N-terminus of nicotinic acetylcholine receptors (nAChRs), the acetylcholine binding protein. In the absence of any X-ray or NMR information on nAChRs, this new structure has provided a reliable alternative to study the nAChR structure. We are now able to build homology models of the binding domain of any nAChR subtype and fit in different ligands using docking programs. This strategy has already been performed successfully for the docking of several nAChR agonists and antagonists. This minireview focuses on the interaction of alpha-conotoxins with neuronal nicotinic receptors in light of our new understanding of the receptor structure. Computational tools are expected to reveal the molecular recognition mechanisms that govern the interaction between alpha-conotoxins and neuronal nAChRs at the molecular level. An accurate determination of their binding modes on the neuronal nAChR may allow the rational design of alpha-conotoxin-based ligands with novel nAChR selectivity.

3 June, 2004

    Contryphan-Vn a cyclic peptide from Conus ventricosus
    Eliseo, T., Cicero, D.O., Romeo, C., Schinina, M.E., Massilia, G.R., Polticelli, F., Ascenzi, P. and Paci, M. (2004) Solution structure of the cyclic peptide contryphan-Vn, a Ca2+-dependent K channel modulator. Biopolymers 74: 189-198.
    Department of Chemistry, University of Rome "La Sapienza," Piazzale Aldo Moro 5, 00198 Rome, Italy.

    Abstract: The solution structure of contryphan-Vn, a cyclic peptide with a double cysteine S-S bridge and containing a D-tryptophan extracted from the venom of the cone snail Conus ventricosus, has been determined by NMR spectroscopy using a variety of homonuclear and heteronuclear NMR methods and restrained molecular dynamics simulations. The main conformational features of backbone contryphan-Vn are a type IV beta-turn from Gly 1 to Lys 6 and a type I beta-turn from Lys 6 to Cys 9. As already found in other contryphans, one of the two prolines-the Pro4-is mainly in the cis conformation while Pro7 is trans. A small hydrophobic region probably partly shielded from solvent constituted from the close proximity of side chains of Pro7 and Trp8 was observed together with a persistent salt bridge between Asp2 and Lys6, which has been revealed by the diagnostic observation of specific nuclear Overhauser effects. The salt bridge was used as a restraint in the molecular dynamics in vacuum but without inserting explicit electrostatic contribution in the calculations. The backbone of the unique conformational family found of contryphan-Vn superimposes well with those of contryphan-Sm and contryphan-R. This result indicates that the contryphan structural motif represents a robust and conserved molecular scaffold whose main structural determinants are the size of the intercysteine loop and the presence and location in the sequence of the D-Trp and the two Pro residues.

    Structure of Glacontryphan-M from Conus marmoreus
    Grant, M.A., Hansson, K., Furie, B.C., Furie, B., Stenflo, J., Rigby, A.C. (2004) The Metal-free and Calcium-bound Structures of a gamma-carboxyglutamic acid-containing Contryphan from Conus marmoreus, Glacontryphan-M. J Biol Chem. 279: 32464-32473.
    Department of Medicine, BIDMC/ Harvard Medical School/ MBL, Boston, MA 02115.

    Abstract: Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca+ -channels expressed in mouse pancreatic beta-cells, was recently isolated from the venom of the cone-snail Conus marmoreus (Hansson et al., accompanying manuscript). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a D-Trp is present; however, unique to Glacontryphan-M is a histidine within the intercysteine-loop and two gamma-carboxyglutamic acid (Gla) residues, formed by posttranslational modification of glutamic acid. The two calcium-binding Gla residues are located in a 4-residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of Glacontryphan-M using two-dimensional (1)H NMR spectroscopy in the absence and presence of calcium. Comparisons of the Glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N-terminus and the Cys11-Cys5-Pro6 region of the intercysteine-loop. The backbone of N-terminal residues perturbed by calcium, Gla2 and Ser3, moves away from the His8 and Trp10 aromatic rings and the alignment of the D-Trp7 and His8 aromatic rings with respect to the Trp10 rings is altered. The blockage of L-type voltage-gated Ca+ -channel currents by Glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone Calpha conformation of the intercysteine-loop of calcium-bound Glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66, respectively). Taken together these data identify that Glacontryphan-M possesses the canonical contryphan intercysteine-loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.

    Glacontryphan-M from Conus marmoreus
    Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B.C., Rorsman, P., Stenflo, J. (2004) The first Gla-containing contryphan: A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus. J Biol Chem. 2004 May 20 [Epub ahead of print] PMID: 15155730 [PubMed - as supplied by publisher]
    Department of Clinical Chemistry, Lund University, Malmo, Malmo S-205 02, Sweden.

    Abstract: Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, Glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to gamma-carboxyglutamyl (Gla) residues. The amino acid sequence of Glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, Glacontryphan-M is amidated at the C-terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a KD of 0.63 mM. Cloning of the full-length cDNA encoding Glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. The non-homologous propeptide region of Glacontryphan-M contains an unusually high density of basic amino acids that might be part of a Conus gammacarboxylation recognition sequence. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that Glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.

    Conotoxins in the venom duct of Conus victoriae
    Jakubowski, J.A., Keays, D.A., Kelley, W.P., Sandall, D.W., Bingham, J.P., Livett, B.G., Gayler, K.R., Sweedler, J.V. (2004) Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry. J Mass Spectrom. 39: 548-557.
    Department of Chemistry and the Beckman Institute, University of Illinois, Urbana-Champaign, Illinois 61801, USA.

    Abstract: A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.

    See also entry for 23 November 2004, Jakubowski and Sweedler (2004)).

1 June, 2004

    Affinity of omega-conotoxins for N-type calcium channels
    J Mould J, Yasuda T, Schroeder CI, Beedle AM, Doering CJ, Zamponi GW, Adams DJ, Lewis RJ. (2004) The alpha 2delta auxiliary subunit reduces affinity of omega -conotoxins for recombinant N-type calcium channels. J. Biol Chem. 279: 34705-34714.
    Institute for Molecular Bioscience, Brisbane, Queensland 4072.
    Abstract: The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential (-120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K]MVIIA and [O10K]GVIA had greater recovery from the block, whereas [K10R]CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

28 May, 2004

    Oxidative folding of alpha-contoxin ImI from Conus imperialis
    Nielsen, J.S., Buczek, P. and Bulaj, G. (2004) Cosolvent-assisted oxidative folding of a bicyclic alpha-conotoxin ImI. J Pept Sci. 10:249-256.
    Cognetix, Inc., 421 Wakara Way, Suite 201, Salt Lake City, Utah 84108, USA.
    Abstract: alpha-conotoxin ImI is a 12-amino acid peptide, found in the venom of the marine snail Conus imperialis. This conotoxin is a selective antagonist of alpha7 nicotinic acetylcholine receptors. To produce biologically active alpha-ImI, disulfide bonds must be formed between Cys2-Cys8 and Cys3-Cys12. Oxidative folding of bicyclic conotoxins, such as alpha-ImI, has been traditionally achieved using two-step oxidation protocols with orthogonal protection on two native pairs of cysteines. In this work, two alternative oxidation protocols were explored: (1) the recently described one-pot oxidation of t-butyl/4-methylbenzyl protected Cys pairs and (2) direct oxidative folding. In contrast to the first method, the latter one resulted in high yields of correctly folded alpha-ImI. The addition of organic cosolvents, such as methanol, ethanol or isopropanol into the folding mixture significantly increased the accumulation of the native peptide. This effect was also observed for another conotoxin, alpha-PnIA. It is suggested that cosolvent-assisted direct oxidation might be of general use for other bicyclic alpha-conotoxins, but efficiency should be assessed on a case-by-case basis.

26 May, 2004

    Synthetic Iodo-conotoxin MI has 20-times higher potency for the alpha/delta interface of the muscle nicotinic receptor
    Drs Luo and McIntosh from the Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah 84112 have synthesised a potent selective inhibitor of the alpha/delta interface of the mouse muscle nicotinic acetylcholine receptor by iodinating the tyrosine residue on conotoxin-MI from Conus magus. Unlike 125I-alpha-bungarotoxin which binds irreversibly to the receptor, 125I-CTX-MI binds reversibly and is displaceable by d-tubocurarine.

    Luo, S. and McIntosh, J.M. (2004) Iodo-alpha-Conotoxin MI Selectively Binds the alpha/delta Subunit Interface of Muscle Nicotinic Acetylcholine Receptors. Biochemistry 43: 6656-6662.
    Abstract: The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [(3)H]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the alpha/delta interface. An analogue of alpha-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the alpha/delta subunit interface. alpha-conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin MI binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [(125)I]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-)(1)); k(on) is 8.5 x 10(7) min(-)(1) M(-)(1). The calculated k(d) is 0.98 nM. This potency is approximately 20-fold higher than the unmodified alpha-MI peptide. Unlike [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.

20 May, 2004

    Conus kintoki and Conus monile
    Giancarlo Paganelli has updated his Cone Shells collection (currently more than 1100 images). Included are 4 images of Conus kintoki from the Philippines and one new image of Conus monile from the Mergui Archipelago. Conus kintoki HABE & KOSUGE, 1970, Philippines; Conus monile HWASS in BRUGUIÈRE, 1792, Myanmar, Mergui Archipelago.

14 May, 2004

    mu O-Conotoxins (MrVIA, MrVIB) from Conus marmoreus inhibit TTX-resistant sodium channels
    Daly NL, Ekberg JA, Thomas L, Adams DJ, Lewis RJ, Craik DJ. (2004) Structures of mu O-conotoxins from Conus marmoreus: Inhibitors of TTX-sensitive and TTX-resistant sodium channels in mammalian sensory neurons. J Biol Chem. 259: 2774-2782.

    Centre for Drug Design and Development, Institute for Molecular Bioscience, Brisbane, QLD 4072.
    Abstract: The muO-conotoxins are an intriguing class of conotoxins targeting various voltage-dependent sodium channels and molluscan calcium channels. In the current study, we have shown MrVIA and MrVIB to be the first known peptidic inhibitors of the transient tetrodotoxin-resistant (TTX-R) Na+ current in rat dorsal root ganglion neurons, in addition to inhibiting tetrodotoxin-sensitive Na+ currents. Human TTX-R sodium channels are a therapeutic target for indications such as pain, highlighting the importance of the muO-conotoxins as potential leads for drug development. Furthermore, we have used NMR spectroscopy to provide the first structural information on this class of conotoxins. MrVIA and MrVIB are hydrophobic peptides that aggregate in aqueous solution but were solubilized in 50% acetonitrile/water. The three-dimensional structure of MrVIB consists of a small betasheet and a cystine knot arrangement of the three-disulfide bonds. It contains four backbone loops between successive cysteine residues that are exposed to the solvent to varying degrees. The largest of these, loop 2, is the most disordered part of the molecule, most likely due to flexibility in solution. This disorder is the most striking difference between the structures of MrVIB and the known delta- and omega-conotoxins, which along with the muO-conotoxins are members of the O superfamily. Loop 2 of omega-conotoxins has previously been shown to contain residues critical for binding to voltage-gated calcium channels and it is interesting to speculate that the flexibility observed in MrVIB may accommodate binding to both sodium and molluscan calcium channels.

12 May, 2004

    Conantokin-G modulation of NMDA receptors
    Drs Barton, White and Wilcox from the Anticonvulsant Drug Development Program, University of Utah, Salt Lake City, UT, USA compared the actions of two anticonvulsants (CI-1041 and CGX-1007) thought to be selective for the NR2B containing NMDA receptor. Both compounds blocked CA1 pyramidal cell, NMDA receptor-mediated excitatory postsynaptic currents (N-EPSCs). CI-1041 was the more selective of the two for the NR2B subunit.

    Barton ME, Steve White H, Wilcox KS.(2004) The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on NMDA receptor-mediated EPSCs. Epilepsy Res. 2004 59:13-24..
    Abstract: The N-methyl-d-aspartate (NMDA) receptor-gated ion channel is comprised of at least one NR1 subunit and any of four NR2 subunits (NR2A-D). The NR2 subunit confers different pharmacological and kinetic properties to the receptor. CGX-1007 (Conantokin G), a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is thought to be selective for the NR2B subunit. CGX-1007 has been reported to have highly potent, broad-spectrum anticonvulsant activity in animal seizure models. CI-1041 is an investigational compound, which also possesses anticonvulsant activity and has been shown to be highly selective for NR2B containing NMDA receptors. Although both CI-1041 and CGX-1007 are reportedly NR2B specific antagonists, they differ in their ability to block amygdala-kindled seizures, suggesting that the mechanism of action of these compounds differs. The present study was designed to test the hypothesis that CI-1041 and CGX-1007 would differentially modulate the function of NMDA receptors at excitatory synapses. Using the whole cell patch clamp technique, CGX-1007 and CI-1041 were found to block CA1 pyramidal cell, NMDA receptor-mediated excitatory postsynaptic currents (N-EPSCs) in a concentration-dependent manner in hippocampal slices from P4-P6 animals. In contrast, only CGX-1007 decreased NMDA receptor-mediated EPSC peak amplitude in slices from adult animals. The CGX-1007 block of peak amplitude was accompanied by a similar concentration-dependent decrease in decay kinetics of NMDA receptor-mediated EPSCs. These results suggest that while CI-1041 may be selective for NMDA receptors containing the NR2B subunit, CGX-1007 appears to be less selective than previously reported.

    Barton ME and Steve White H. (2004) The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on kindling acquisition and expression. Epilepsy Res. 59:1-12.
    Abstract: CGX-1007, a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is selective for the NR2B subunit. CI-1041 (PD 196860; Co 200461) is a novel, orally available NR2B selective antagonist. Both compounds possess anticonvulsant activity in a variety of well-established animal seizure models. The present study was designed to assess the effects of CGX-1007 and CI-1041 on the acquisition and expression of kindled seizures. In the corneal kindled rat, CGX-1007 [Epilepsia 36 (1998) 39] and CI-1041, administered p.o., 2h prior to the kindling stimulation displayed time- and dose-dependent block of fully expressed corneal kindled seizures ( [Formula: see text] pmol and 2.5mg/kg for CGX-1007 and CI-1041, respectively). In amygdala kindled rats, acute treatment with CGX-1007 blocked the secondarily generalized kindled seizure in a dose-dependent manner. Complete protection against the secondarily generalized seizure was only observed at a dose that produced behavioral impairment (4nmol). Acute treatment with CI-1041 did not provide any notable protection against secondarily generalized seizures. Neither compound provided protection against the focal kindled seizure. Chronic i.c.v. infusion of CGX-1007 or chronic oral administration of CI-1041 did not delay the acquisition of amygdala kindling. The results from these studies suggest that NMDA receptors containing the NR2B subunit may contribute to the expression of fully kindled secondarily generalized seizures; however, they appear less important for the development of kindling. The differential results obtained with CGX-1007 and CI-1041 suggest that several classes of mechanistically distinct NR2B antagonists may exist and that CGX-1007 may be less specific as a NR2B receptor antagonist than initially reported.

2 May, 2004

    Conus generalis
    Giancarlo Paganelli has updated his Cone Shells collection (currently more than 1100 images). Included are 3 images of Conus generalis from the Philippines and Thailand and one of Conus maldivus from Tanzania. Conus generalis LINNAEUS, 1767, Philippines, Bohol - 58.1 mm; Conus generalis krabiensis DA MOTTA, 1982 Thailand, Andaman Sea, Phuket - 48.2 mm; Conus maldivus HWASS in BRUGUIÈRE, 1792 Tanzania, Zanzibar Is., Nungwi - 51.2 mm

25 April, 2004

    Conus straitellus
    Giancarlo Paganelli has updated his Cone Shells collection (currently more than 1000 images). Included are 8 images of Conus striatellus Link 1807, from Admiralty Is., Madagascar, the Madives and the Philippines. You can view them here.

23 April, 2004

    Xenome developing painkiller from Conus marmoreus
    Biotechnology company, Xenome Limited, have filed an Investigational New Drug (IND) application with the US Food and Drug Administration's (FDA) Center for Drug Evaluation and Research (CDER) for their painkiller Xen2174 ( see Press Release). This compound is based on chi-CTX MrIA from Conus marmoreus (Sharpe et al J. Biol. Chem 2003, 278, 40317, 2003). It acts as a reversible noncompetitive inhibitor of the neuronal noradrenaline transporter, a known drug target in the central nervous system. Delivery of Xen2174 directly into the space around the spinal cord (intrathecally) deposits the drug adjacent to the noradrenaline transporter so that during episodes of pain, inhibition of the transporter by Xen2174 elevates the levels of noradrenaline. This causes activation of descending inhibitory pathways so preventing pain signals from reaching the brain.

    As reported by Renate Krelle, Australian Biotechnology News on 22/04/2004 :
    Xenome -- 25 per cent owned by Medica Holdings (ASXMCA) -- has filed an investigational new drug (IND) application with the US Food and Drug Administration for its Xen2174 cancer painkiller, which is derived from cone shell venom. In animal models, Xen2174 has proved a more potent painkiller than morphine. It is a synthetic peptide which inhibits the transport of noradrenaline,­ a nerve transmitter which amplifies messages between nerve cells, blocking pain transmission. The cone shell venom on which Xen2174 is based is native to Queensland's Great Barrier Reef. If the IND application is successful, Xenome plans to begin Phase I clinical trials in both Australia and the US in June to test the safety and tolerability of Xen2174. Xenome's CEO Dr Tony Evans said that the IND filing would trigger a second milestone payment of $2 million from the Queensland BioCapital Fund, bringing the total funds QBF has invested in Xenome to $6 million. Other Xenome investors include listed investment company Biotech Capital (ASXBTC) and University of Queensland commercialisation vehicle, UniQuest.

21 April, 2004

    Conantokin-G self assembly
    Dr. Dai and colleagues from the University of Notre Dame, Indianapolis, provide evidence that conantokin-G (con-G), a gamma-carboxyglutamate (Gla)-rich neuroactive peptide from Conus geographus can dimerize in the presence of calcium ions. A particular arrangement of Gla residues is essential for this self-assembly. The results suggest a new role for Gla residues and calcium in the controlled assembly of con-G.

    Dai Q, Prorok M, Castellino FJ. (2004)A new mechanism for metal ion-assisted interchain helix assembly in a naturally occurring peptide mediated by optimally spaced gamma-carboxyglutamic acid residues. J Mol Biol. 336:731-744.
    Helix-helix interactions, such as those that occur in coiled-coil domains, four-helix bundles, or membrane-spanning helical bundles, are important to the structural organization and function of numerous proteins. However, tractable peptide models for studying such structural elements have been limited to synthetic analogs of coiled-coil protein domains and de novo designed peptides. The present study provides evidence that conantokin-G (con-G), a gamma-carboxyglutamate (Gla)-rich neuroactive peptide from a venomous marine snail, can self-associate in the presence of certain divalent metal cations. Sedimentation equilibrium analyses of con-G show that Ca+ binding promotes peptide dimerization, while the addition of the tighter binding divalent cations, Mg+, Zn+, and Mn+, does not result in intermolecular association. The effects of specific residue replacements indicate that an i, i + 4, i + 7, i + 11 arrangement of Gla residues is essential for con-G self-assembly. To determine the relative chain orientation of the dimeric assembly, distributions of Cys-containing con-G variants were examined in thiol-disulfide rearrangement assays and the results were consistent with an antiparallel alignment. Our data suggest that the driving force for con-G dimerization stems from the appropriate balance of interchain and intrachain metal ion coordination by Gla residues in similar locations. These findings suggest a new role for Gla residues and accompanying cation binding in the stabilization of interstrand helix association in a natural product and provide a model for controlled assembly of peptide chains or segments of larger proteins.

    Killing Pain with Snail's Venom
    Linda Alvarez in a CBS 2 Special Assignment Report "Snail's Venom" that aired Tuesday, April 13, at 11 p.m. told of an unusual treatment that may help chronic pain sufferers. Chronic pain affects one out of every three people in the U.S., and for some of those people, medications provide little or no relief. In this interview with chronic pain patient, Jennifer Wissner, research scientist, Tony Yaksh and pain doctor Mark Darrow, she tells how administration of Ziconotide, a novel analgesic peptide from the venom of a marine cone snail, provided in the words of Wissner, "Immediate relief, it was just amazing."

11 April, 2004

1 April, 2004

    Can a dead cone sting you ?
    In the April 2004 issue of Hawaiian Shell News (HSN), on pages 11-12, Month Section, there is a reprint of an article from March, 1972 HSN Volume 20, page 5, by Virginia Dennis captioned "Can a dead cone sting you ?".
    She relates how in her hurry to clean a dead C. rattus and a large C. ebraeus she forgot to wear her vinyl gloves. She describes how "After working for perhaps five minutes, two fingers on my right hand began to tingle at the tips....The prickling in my fingers continued and some mild numbness set in. After about 10 minutes I felt so rocky that I lay down, with pencil and paper handy to record reactions....Hard headaches started 24 hours after the initial contact, but they responded to aspirin. In the meantime, I had consulted a doctor, who confirmed my symptoms as reaction to poison. Anyhow, the headaches continued for 16 days, then fitfully for another eight days. It was nearly three months before the tingling completely left my finger tips." She beleives she came in contact during cleaning with an unseen venomous barb (harpoon, radula) and/or venom smeared on the outside of the shell, which she handled with possibly scratched fingers. One thing is certain, now when she cleans cones she wears gloves.

    Click here to download a .pdf version of this March 1972 issue of HSN, which contains in addition:

  • a report on Conus kermadecensis, the "First Live Cone Shell Found in New Zealand",
  • an article by Mme Denise Valero on "The Discovery of Sinistral Conus mediterraneus (Hwass in B.) From the Cote D'Azur, France",
  • an article by William E. Old, Jr. on "A Remarkable Malformed Conus" (Conus quercinus, Lightfoot, from East New Britain Island), and
  • an extensive article by E.R. Cross "Conus gloriamaris - Follow-Up".

    A reminder that you can SEARCH past issues of HSN by Keyword/Text, Topic, Title, Author, Year released, or Volume at Guido T. Poppe's Concholgy Inc. site.

29 March, 2004

    alpha-conotoxin MII analogues selective for alpha6 nicotinic receptors
    McIntosh, J.M., Azam, L., Staheli, S., Dowell, C., Lindstrom, J.M., Kuryatov, A., Garrett, J.E., Marks, M.J. and Whiteaker, P. (2004) Analogs of {alpha}-Conotoxin MII Are Selective for {alpha}6-Containing Nicotinic Acetylcholine Receptors. Mol Pharmacol. 65: 944-952.
    Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. alpha-conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related alpha3 and alpha6 subunits. Structure-function analysis of alpha-conotoxin MII was performed in an attempt to generate analogs with preference for alpha6-containing [alpha6(*) (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at alpha3(*) versus alpha6(*) nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC(50) at alpha6beta4 versus alpha3beta4 nAChRs. The peptide had a 590-fold lower IC(50) for alpha6/alpha3beta2 versus alpha3beta2 and a 2020-fold lower IC(50) for alpha6/alpha3beta2beta3 versus alpha3beta2 nAChRs. MII[H9A;L15A] had little or no activity at alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nAChRs. Functional block by MII[H9A;L15A] of rat alpha6/alpha3beta2beta3 nAChRs (IC(50) = 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [(125)I]alpha-conotoxin MII binding to putative alpha6beta2(*) nAChRs in mouse brain homogenates (K(i) = 3.3 nM). Thus, structure-function analysis of alpha-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing alpha3 and alpha6 subunits.

24 March, 2004

    Fake cones
    Fake cone shells have fooled many a novice collector. You must be aware of the possibility when you buy shells. They are very hard to discern. What to look out for ? Guido Poppe of Conchology Inc. has posted images on his web site of 19 fake cones and another 5 fake cones to alert you to this problem. Species illustrated include Conus bocki, Conus capitaneus, Conus consors, Conus distans, Conus generalis, Conus magus, Conus marmoreus, Conus pergrandis, Conus praecellens, Conus pulicarius, Conus samial, Conus striatus, Conus textile and Conus virgo.

22 March, 2004

    Can cones grow a new operculum?
    Interested and curious Mrs. "Ibby" (short for Elizabeth) Harrison decided to find out. Two medium sized Conus pennaceus collected at Maile Point on June 26th. 1961 were used in the experiment. Before being placed in the tank, the operculum was removed from each animal. That it did not seriously interfere with the regular routine is proven by the fact that within an hour or two after being placed in the tank without his operculum, one of them made a meal on a Cypraea caputserpentis of which there were several in the tank. Both were examined every few days for signs of a new operculum, but none could be noticed. One of them died within a month unnoticed and the new operculum was lost in the sand. The other died a few days later and upon hosing out the shell as Mrs. Harrison expressed it, "I recovered a small, pale yellow, almost transparent operculum. My curiosity was satisified." (Source: Hawaii Shell News X, (1) 2, 1961 ).
    Download this issue in Adobe .pdf format Hawaii Shell News, Research Dept., Volume X, No. 1, New Series No. 21, November 1961, page 2).

    Hawaii Shell News, the Official Publication of the Hawaiian Malacological Society, is now available on the web. 44 years of articles in an online archive of 8989 searchable articles .

    Conotoxins inhibit ACh and catecholamine release in rat adrenals in vivo
    Drs Akiyama, Yamazaki, Mori and Sunagawa from the Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Osaka, Japan have studied the differential involvement of L, N, and P/Q calcium channels in the release of acetylcholine from splanchnic nerve terminals innervating the adrenal medulla of rats in vivo, and of the release of catecholamines from the innervated adrenal medulla.

    Akiyama T, Yamazaki T, Mori H, and Sunagawa K. (2004) Effects of Ca+ channel antagonists on acetylcholine and catecholamine releases in the in vivo rat adrenal medulla. Am J Physiol Regul Integr Comp Physiol. 2004 Mar 18 [Epub ahead of print]
    Abstract: To elucidate the types of voltage-dependent Ca+ channels controlling acetylcholine and catecholamine releases in the in vivo adrenal medulla, we implanted microdialysis probes in the left adrenal medulla of anesthetized rats and investigated the effects of Ca+ channel antagonists on acetylcholine, norepinephrine, and epinephrine releases induced by nerve stimulation. The dialysis probes were perfused with Ringer's solution containing a cholinesterase inhibitor, neostigmine. The left splanchnic nerves were electrically stimulated at 2 and 4 Hz before and after intravenous administration of Ca+ channel antagonists. omega-conotoxin GVIA (an N-type Ca+ channel antagonist, 10 micro g/kg) inhibited acetylcholine release at 2 and 4 Hz by about 40%, norepinephrine release at 4 Hz by about 50%, and epinephrine release at 2 and 4 Hz by about 45%. A 5-fold higher dose of omega-conotoxin GVIA (50 micro g/kg) did not further inhibit these releases. omega-conotoxin MVIIC (a P/Q-type Ca+ channel antagonist, 50 micro g/kg) inhibited acetylcholine and epinephrine releases at 4 Hz by about 30%. Combined omega-conotoxin GVIA (50 micro g/kg) and MVIIC (250 micro g/kg) inhibited acetylcholine release at 2 and 4 Hz by about 70%, norepinephrine and epinephrine releases at 2 and 4 Hz by about 80%. Nifedipine (an L-type Ca+ channel antagonist, 300 and 900 micro g/kg) did not change acetylcholine release at 2 and 4 Hz, but nifedipine (300 micro g/kg) inhibited epinephrine release at 4 Hz by 20% and nifedipine (900 micro g/kg) inhibited norepinephrine and epinephrine releases at 4 Hz by 30%. In conclusion, both N- and P/Q-type Ca+ channels control acetylcholine release on pre-ganglionic splanchnic nerve endings while L-type Ca+ channels do not. L-type Ca+ channels are involved in norepinephrine and epinephrine releases on chromaffin cells.

12 March, 2004

9 March, 2004

    ACV1 Progress Report
    Metabolic Pharmaceuticals, Melbourne (www.metabolic.com.au) are proceding with pre-clinical toxicity testing of ACV1, a novel and promising analgesic alpha-conotoxin from an Australian cone shell, Conus victoriae, preliminary to planned Stage I Clinical Trials for diabetic neuropathy early in 2005. (See ASX Announcement, March 9, 2004)

    Potency determinants of Conotoxin MII from Conus magus
    Everhart, D., Cartier, G.E., Malhotra, A., Gomes, A.V., McIntosh, J.M. and Luetje, C.W. (2004) Determinants of Potency on alpha-Conotoxin MII, a Peptide Antagonist of Neuronal Nicotinic Receptors. Biochemistry 43: 2732-2737.
    Abstract:alpha-conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat alpha3beta2 receptors, expressed in Xenopus oocytes, are blocked with an IC(50) of 3.7 +/- 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of alpha3beta2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and approximately 2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the alpha3beta2 receptor.

7 March, 2004

    Cone Shells from Brazil
    Giancarlo Paganelli has updated his Cone Shells collection (currently more than 1000 images), with 6 images of Cone shells from Brazil. The specimens depicted here are : Conus beddomei SOWERBY III, 1911 Brazil, Touros - 22.9 mm; Conus clenchi MARTINS, 1943 Brazil, Marataìzes - 53.4 mm; Conus mindanus f. agassizii DALL, 1889 Brazil, Vitoria - 36.8 mm; Conus pusillus LAMARCK, 1810 Brazil, Cabo Frio - 19.5 mm; Conus sanderi WILLS & MOOLENBECK 1979 Brazil, Vitoria - 37.7 mm, and Conus villepini FISCHER & BERNARDI, 1857 Brazil, Rio de Janeiro - 54.4 mm;

    NMR Structure of alpha-conotoxin GIC from Conus geographus
    Chi, S.W., Kim, D.H., Olivera, B.M., McIntosh, J.M., Han, K.H. (2004) Solution conformation of alpha-conotoxin GIC, a novel potent antagonist of alpha3beta2 nicotinic acetylcholine receptors. Biochem J. 380: 347-352
    Abstract:alpha-conotoxin GIC is a 16-residue peptide isolated from the venom of the cone snail Conus geographus. alpha-conotoxin GIC potently blocks the alpha3beta2 subtype of human nicotinic acetylcholine receptor, showing a high selectivity for neuronal versus muscle subtype [McIntosh, J. M., Dowell, C., Watkins, M., Garrett, J. E., Yoshikami, D., and Olivera, B. M. (2002) J. Biol. Chem. 277, 33610-33615]. We have determined the three-dimensional solution structure of alpha-conotoxin GIC by nuclear magnetic resonance spectroscopy. The structure of alpha-conotoxin GIC is well-defined with backbone and heavy atom root mean square deviations (residues 2-16) of 0.53 A and 0.96 A, respectively. Structure and surface comparison of alpha-conotoxin GIC with the other alpha4/7 subfamily conotoxins reveals unique structural aspects of alpha-conotoxin GIC. In particular, the structural comparison between alpha-conotoxins GIC and MII indicates molecular features that may confer their similar receptor specificity profile as well as those that provide the unique binding characteristics of alpha-conotoxin GIC.

    [125]I-alpha-conotoxin MII detects increase in alpha6 subunit of nAChR
    Parker, S.L., Fu, Y., McAllen, K., Luo, J., McIntosh, J.M., Lindstrom, J.M. and Sharp, B.M. (2004) Up-Regulation of Brain Nicotinic Acetylcholine Receptors in the Rat during Long-Term Self-Administration of Nicotine: Disproportionate Increase of the {alpha}6 Subunit. Mol Pharmacol. 65 :611-622.
    Abstract:In male rats continually self-administering nicotine (approximately 1.5 mg free base/kg/day), we found a significant increase of nicotinic acetylcholine receptors (nAChRs) labeled by epibatidine (Epb) in 11 brain areas. A large increase of high-affinity Epb binding sites was apparent in the ventral tegmentum/substantia nigra, nucleus tractus solitarii, nucleus accumbens, thalamus/subthalamus, parietal cortex, hypothalamus, and amygdala. A smaller but significant up-regulation of high-affinity Epb sites was seen in the piriform cortex, hippocampus, caudate/putamen, and cerebellar cortex. The up-regulation of nAChRs, shown by immunoadsorption and Western blotting, involved alpha4, alpha6, and beta2 subunits. As a consequence of long-term self-administration of nicotine, the alpha6 immunoreactive (IR) binding of either labeled Epb or (125)I-alpha-conotoxin MII increased to a much greater extent than did alpha4 or beta2 IR binding of Epb. In addition, the beta2 IR binding of Epb was consistently enhanced to a greater extent than was alpha4. These findings may reflect a larger surface membrane retention of alpha6-containing and, to some degree, beta2-containing nAChRs compared with alpha4-containing nAChRs during long-term self-administration of nicotine.

    Conotoxins GVIA, CVIB and CVID distinguish calcium channel subtypes involved in transmitter release
    Morris, J.L., Ozols, D.I., Lewis, R.J., Gibbins, I.L. and Jobling, P. (2004) Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons. Br. J. Pharmacol. 2004 Mar 1 [Epub ahead of print]
    Abstract:The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nM. Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nM CTX GVIA. Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced <50% by CTX GVIA 10-100 nM. Capsaicin (3 micro M) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. The novel N-type blocker CTX CVID (100-300 nM), P/Q-type blockers agatoxin IVA (10-100 nM) or CTX CVIB (100 nM), the L-type blocker nifedipine (10 micro M) or the 'R-type' blocker SNX-482 (100 nM), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl2 (100-300 micro M) reduced but did not abolish the remaining neurogenic dilations. Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

27 February, 2004

    Some conotoxin structures to view
    New Additions to conotoxin entries in Protein Data Bank (PDB) listings: 1q2j Mu-conotoxin Smiiia Structural Basis For Tetrodotoxin-Resistant Sodium Channel Binding By Mu-conotoxin Smiiia. Keizer, D.W., West, P.J., Lee, E.F., Olivera, B.M., Bulaj, G., Yoshikami, D. and Norton, R.S. (2004)
    1sbu Delta Conotoxin EVIA1 Peptide Analog Containing a Dimethylthiazolidine NMR Structure of a Delta Conotoxin EVIA1 Peptide Analog Containing a Dimethylthiazolidine, Figuet, M., Chierici, S., Jourdan, M. and Dumy, P.(2004)


21 February, 2004

    3-D structure of Conotoxin EVIA1 from Conus ermineus
    Dr. Andres Menez and colleagues from Biomolecular NMR, Universite Claude Bernard - Lyon, France have studied the three-dimensional structure of delta-conotoxin EVIA1 from Conus ermineus which inhibits sodium channel inactivation in neuronal membranes (without affecting cardiac or skeletal muscle sodium channel subtypes).

    Volpon, L., Lamthanh, H., Barbier, J., Gilles, N., Molgo, J., Menez, A. and Lancelin, J.M. (2004) NMR solution structures of delta-conotoxin EVIA1 from Conus ermineus that selectively acts on vertebrate neuronal Na+ channels. J Biol Chem. 279: 21356-21366.
    Abstract:Delta-conotoxin EVIA, isolated from the venom of Conus ermineus, is a 32 residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. Delta-conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNav1.2a, rNav1.3, rNav1.6), without affecting rat skeletal muscle (subtype rNav1.4) and human cardiac muscle (subtype hNav1.5) sodium channel subtypes [Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S.F., Gordon, D., Mnez, A. and Molg, J. (2004) preceding article submitted jointly to J. Biol. Chem.]. Its three-dimensional structure was solved in aqueous solution by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu12-Pro13 peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be separately calculated on the basis of their respective NMR-derived structural restraints. The isomerism occurs within a specific long disordered loop 2, including residues 11 to 19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the inhibitor cystine-knot motif of conotoxins from the "O-superfamily" with a high structural order. To probe a possible functional role of the Leu12-Pro13 cis/trans isomerism, a Pro13Ala delta-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A delta-conotoxin EVIA was estimated only two times less active than the wild type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice.

20 February, 2004

    Conus anemone Credit: Leon Altoff, Melbourne, Victoria, Australia.
    Griffith Point San Remo, Victoria 3 Jan. 1999; Right Image: Breamlea, Victoria 31 Dec. 2001

    Sulfated Conotoxins from Conus anemone
    Richard Lewis and colleagues from The University of Queensland, Australia have identified three novel sulfated alpha-conotoxins in the venom of the Australian marine snail Conus anemone. The importance of specific posttranslational modifications and amino acid deletions on the binding and selectivity of these 4/7-alpha conotoxins towards alpha3beta2 and alpha-7 nicotinic acetylcholine receptor subunit combinations is described.

    Loughnan, M.L., Nicke, A., Jones, A., Adams, D.J., Alewood, P.F. and Lewis, R.J. (2004) Chemical and Functional Identification and Characterization of Novel Sulfated alpha-Conotoxins from the Cone Snail Conus anemone. J Med Chem. 47:1234-1241.
    Abstract: An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC(50) 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC(50) 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC(50) 0.6 nM) and alpha7 (IC(50) 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC(50) 367 nM) than at the alpha3beta2 nAChR (IC(50) 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

16 February, 2004

    How Much at Risk Are Cone Snails?
    Duda Jr TF, Jon-Paul Bingham, Bruce G. Livett, Alan J. Kohn, Gabriella Raybaudi Massilia, Joseph R. Schultz, John Down, David Sandall and Jonathan V. Sweedler; and Mike Fainzilber (2004) How Much at Risk Are Cone Snails? ;Response: Eric Chivian, Callum M. Roberts and Aaron S. Bernstein Science 303: (Feb 13 2004) 955-957.

15 February, 2004

    Conus garywilsoni: a new conus species from Western Australia

    Conus garywilsoni: This new species of Conus was discovered late in 2003 in the North West Shelf area of Western Australia by Spectrum Shells at the very limits of S.C.U.B.A. The unique habitat is mostly sand that is covered by a film of algae with sea pens all around.
    According to Dr Felix Lorenz, who researched and named it (Lorenz and Morrison, 2003), Conus garywilsoni has three distinct conchological differences from other species, including the two similar species C. lischkeanus and C.articulatus.
    This new Conus has a mean average size of 16-21mm. Spectrum Shells retains some of the paratypes : the holotypes are in the repository of the Western Australian Museum. Spectrum Shells, Albany, Western Australia are the sole distributors and source of C. garywilsoni. (I thank Paul Harrison for bringing this information to my attention).

14 February, 2004

    Cones from Italy
    Giancarlo Paganelli has updated his Cone Shells collection (currently more than 1000 images), with 14 images of Conus ventricosus, GMELIN, 1781, from Italy. This small cone shell is also found at other sites around the Mediterranean (eg. Djerba, Tunisia; and Malaga, Spain) and is commonly, though incorrectly known as "Conus mediterraneus".
    Conus ventricosus, GMELIN, 1781.
    The different sizes, and regions of Italy from which the Conus ventricosus specimens depicted here were collected are : 56.0 mm Marina di Pulsano, TA; 55.3 mm Marina di Pulsano, TA; 54.1 mm Marina di Pulsano, TA; 49.5 mm Marina di Pulsano, TA; 46.9 mm Marina di Pulsano, TA; 41.6 mm Marina di Pulsano, TA; 42.2 Marina di Pulsano, TA; 39.3 mmMarina di Pulsano, TA; 35.9 mm Marina di Pulsano, TA; 33.1 mm Marina di Pulsano, TA; 28.6 mm Marina di Pulsano, TA; 44.6 mm Lampedusa Is.; 40.6 mm Gallipoli, LE, and 36.6 mm Gallipoli, LE;

    See also Color patterns in Conus ventricosus GMELIN, 1791 : flecks

    Conus terebra - image of live specimen
    George Sangiouloglou has some nice images of Conus terebra from Thailand on his web page "Georges Sea-shells and Underwater World". To view the shells click here. To view the live specimen photographed in its native environment, click here.

13 February, 2004

    Cone shells: a source of new painkillers
    Cherry Farrow of The Guardian wrote on Conus, and its potential to provide a breakthrough in the fight against chronic pain.
    "Small creature, big potential", The Guardian, Thursday February 12, 2004.
    "For thousands of years, medical research has depended on plants, animals and microbes to understand and treat human disease. Now increasing attention is being paid to animals for sources of new medicines. One of the most unlikely and beautiful is a small, deadly mollusc - the cone snail. There is growing excitement among scientists that this marine snail may provide a breakthrough in the development of a series of new painkillers. Cone snails may contain more medicines than any other animal. A new synthetic drug, Prialt, is now at an advanced stage in clinical trials for the treatment of what is classed as intractable pain - unremitting, untreatable pain. Prialt, derived from the venom or conotoxin of the snail, may be 1,000 times stronger than morphine but without the tolerance or addiction usually associated with opiates. Cone snails live in shallow tropical waters, on reefs, in mangrove swamps and mud flats. There are thought to be approximately 500 species, although research has focused mainly on three. The snails have a little harpoon or fleshy foot from which they inject a lethal cocktail of toxins into their prey - other molluscs, worms and fish. They can "shake" the cocktail and create a new mix of changing proportions so their victims cannot develop immunity. Each species has evolved to produce its own set of toxins, about 100, which means there could be as many as 50,000 conotoxins in all. This is unprecedented in the biological world, says Aaron Bernstein, who is currently conducting research at Harvard Medical School. "It's the sheer diversity of the chemicals they make. The cone snail is young in evolutionary terms - only 50m years old - yet each has evolved a different number of toxins. Compare this to the 10,000 known alkaloids in plants, which we have been looking at for centuries," he said. Reseach into cone snails started 20 years ago. Experiments suggest that conotoxins could treat muscle spasticity, following spinal injury and epilepsy, that is resistant to traditional medicines. A broad spectrum anti-epileptic agent is currently in the first stage of clinical trials. For years, scientists have searched for a medicine that is very specific, effective at low doses and does not cause side effects of addiction or tolerance. Having to keep increasing doses to achieve the same results has always limited the long-term effectiveness of the opiates used for treating severe pain. Conotoxins do not behave like that -they are very potent in small concentrations, as trials carried out on patients with Aids and cancers have shown. "Most conotoxins are small peptides, 10 to 40 amino acids in length. They are exquisitely selective about their receptor binding sites," says Bernstein. "This makes them powerful tools for understanding how cells work, as well as a rich source for discovery of new medicines.What is remarkable about them is this ability to be so selective." Studies suggest that conotoxins could be used to treat muscle spasticity following spinal injuries and possibly prevent cell death during strokes or head injuries. They may also help in the treatment of small-cell lung cancer. By detecting the antibodies, contoxins can provide an early diagnostic test for some types of cancer. But it is in the treatment of "untreatable" pain that conotoxin research is furthest advanced. The first large-scale trials show just how effective the new painkiller in its synthetic form may prove to be. Trial results showed no evidence of tolerance or addictive behaviour and in feasibility studies with Aids and cancer patients with chronic pain there was relief in more than half of the patients. All this from a small marine snail that may, in the words of Eric Chivian, founder and director of the Centre for Human Health and Global Environment at Harvard Medical School, "contain the largest and most clinically important pharmacopoeia of any genus in nature." However, the reefs, mud and swamps in which they thrive are all under threat from over-fishing, pollution, mangrove clearance for coastal development, shrimp farming and "bioprospecting" - for private collections as well as the biomedical industry. Millions of cone snails are imported into the US and Europe every year. "We cannot find any country that monitors this trade," says Chivian. "Although a number of researchers are careful about moving quickly to synthesise the toxins they need, others may not be. No one knows how many and what species are being sacrificed for research and for the ornamental trade in their shells," he says. Global warming is also damaging their ocean habitat. "The loss of species deprives us of invaluable tools for biomedical research. They can give us the insight into health and illness," he adds. "So just as we are appeciating the remarkable potential of the cone snail, they are coming under intense pressure."

    See also http://www.sciencedaily.com/print.php?url=/releases/2003/10/031017073822.htm>New Medicines At Risk From Biodiversity Loss

9 February, 2004

    American cones - and New sections (Patterns, Shape and Sculpture)
    Giancarlo Paganelli has updated his Cone Shells collection (currently more than 1000 images), with 9 images of cones from America. Included are : Conus anabathrum CROSSE, 1865, USA, Florida, Gullivan Bay - 36.9 mm; Conus daucus Conus daucus HWASS in BRUGUIÈRE, 1792, Curaçao, Bullen Bay - 42.1 mm; Conus gradatus WOOD, 1828 Mexico - 40.6 mm; Conus mindanus HWASS in BRUGUIÈRE, 1792, Honduras, Roatan Is. - 24.2 mm; Conus nux BRODERIP, 1933 Mexico, Baja, Bahia Concepcion - 24.3 mm; Conus poormani BERRY, 1968, Panama, Bay of Quiriqui - 50.1 mm; Conus recurvus BRODERIP, 1833 Mexico, Acapulco - 44.4 mm; Conus spurius f. atlanticus Clench, 1942, Mexico, Yucatan Canal, Cancun - 48.9 mm and Conus ximenes f. mahogani REEVE, 1843, Mexico, Gulf of California - 32.7 mm.

    In addition, he has added three New Sections, namely, Patterns (both Color Patterns [eg. Dots, Flecks, Lines and Tents] and Relief Patterns [Grooves, Threads, Ribs, Ribbons and Granules]); Shape and Sculpture (including Body Whorl, Shoulder, Spire and Aperture).

    Clinical trial of Ziconotide in chronic pain patients
    Dr. Wermeling and colleagues from the University of Kentucky Medical Center, Pain Treatment Center, Lexington, Kentucky, USA. have studied the half-life and disposition of omega-conotoxin MVIIA (Ziconotide, Prialt) following intrathecal administration to patients with non-malignant pain. They find that in cerebral spinal fluid (CSF) the mean half-life was 4.5 hours. Dose related analgesia was observed but higher doses resulted in adverse nervous system behaviours.

    Wermeling D, Drass M, Ellis D, Mayo M, McGuire D, O'Connell D, Hale V. and Chao S. (2003) Pharmacokinetics and pharmacodynamics of intrathecal ziconotide in chronic pain patients. J Clin Pharmacol. 43:624-636. Abstract: The pharmacokinetics and pharmacodynamics of ziconotide were assessed over a 48-hour period following intrathecal (i.t.) administration (1, 5, 7.5, or 10 micrograms) to 22 patients with chronic, nonmalignant pain. Plasma and cerebrospinal fluid (CSF) samples were obtained over a 24-hour period. Analgesic efficacy was monitored using Visual Analog Scale of Pain Intensity (VASPI) and Category Pain Relief Scores (CPRS) measurements. Pharmacokinetic (PK) parameters were calculated by noncompartmental methods. Plasma ziconotide data were insufficient for PK calculations. In CSF, the median half-life of ziconotide was 4.5 hours. The median CSF clearance and volume of distribution were 0.26 mL/min and 99 mL, respectively. CSF pharmacokinetics of ziconotide were linear, based on cumulative exposure and peak CSF concentrations. A dose-related analgesia was observed. Pharmacokinetic-pharmacodynamic efficacy and safety analyses showed that higher CSF ziconotide concentrations were generally associated with analgesia and increased incidence of nervous system adverse events following a 1-hour i.t. infusion.

5 February, 2004

    The Conus Biodiversity Website
    Alan Kohn has announced the launch of "The Conus Biodiversity Website" at http://faculty.washington.edu/kohn/. The first, and as yet the only, component of the site available is a catalogue of all species-level taxa proposed in Conus starting with the 10th Edition of the Systema Naturae (Linnaeus, 1758), through December, 2002. "The present version contains 3,157 species-group names. Of these, 768 were published after 1936, an average of 12 new species-group names introduced annually. During the last three decades of the 20th Century the rate of new species introductions increased to 16 per year."

    Alan is now working on the next two components, namely, a gallery of photographs of type specimens, and descriptions of valid species.

18 January, 2004

    Conotoxin mr3a from Conus marmoreus
    McDougal, O.M. and Poulter, C.D. (2004) Three-Dimensional Structure of the Mini-M Conotoxin mr3a. Biochemistry 43: 425-429.
    Abstract: conotoxin mr3a from the venom of Conus marmoreus, a novel peptide that induces rolling seizures in mice, has the peptide sequence GCCGSFACRFGCVOCCV, where O is trans-4-hydroxyproline, and the chain is cross-linked with disulfide bonds between Cys-2 and Cys-16, Cys-3 and Cys-12, and Cys-8 and Cys-15. The tertiary structure of mr3a was determined by 2D (1)H NMR in combination with a standard distance-geometry algorithm. The final set of 22 structures for the peptide had a mean global backbone RMS deviation of 0.53 +/- 0.22 A based on 51 NOE, 6 hydrogen bond, 6 phi dihedral angle, and 3 disulfide bond constraints. conotoxin mr3a is the first example of the new mini-M branch of conopeptides in the M superfamily. Members of the maxi-M branch, whose structures are known, include the micro- and psi-conotoxins, both of which share a common disulfide bond connectivity. Although mr3a has the same arrangement of Cys residues as the micro- and psi-conotoxins, its disulfide connectivity is different. This gives mr3a a distinctive "triple-turn" backbone.

14 January, 2004

    Conus macarae from the Philippines
    Guido Poppe has posted an article entitled "Conus macarae of the masbate fishing grounds". In it he describes and illustrates the large palette of backgound colours exhibited by Conus macarae, Bernardi, 1857 (formerly known as "Conus voluminalis") from this region. Also included is a discussion of nomenclature following discussions between Guido Poppe and the Conus expert E. Wils (Belgium). It is noted that C. macarae (voluminalis) is not reported from Japan or Australia. The very closely related species Conus recluzianus (C. urashimanus) and Conus macarae are both clearly distinguishable from Conus roseorapum from the Philippines. Guido ponders whether the few spirally dotted C. macarae are a result of interbreeding with C. shikamai that he was also able to obtain from the Masbate fisherman. The article is beautifully illustrated with numerous specimens covering a range of backgound colours.

12 January, 2004

    Cone shell venom peptides and their targets - a REVIEW
    Heinrich Terlau and Baldomero M. Olivera from AG Molekulare und Zelluläre Neuropharmakologie, Max-Planck-Institut für Experimentelle Medizin, Göttingen, Germany; and Department of Biology, University of Utah, Salt Lake City, Utah have penned an impressive review on cone shell venoms and their conopeptide components and targets. In this review they focus on the targeting specificity of conotoxins and their differential binding to different states of an ion channel.

    Terlau, H. and Olivera, B.M. (2004) Conus Venoms: A Rich Source of Novel Ion Channel-Targeted Peptides. Physiol Rev 84: 41–68.
    Abstract:The cone snails (genus Conus) are venomous marine molluscs that use small, structured peptide toxins (conotoxins) for prey capture, defense, and competitor deterrence. Each of the 500 Conus can express ~100 different conotoxins, with little overlap between species. An overwhelming majority of these peptides are probably targeted selectively to a specific ion channel. Because conotoxins discriminate between closely related subtypes of ion channels, they are widely used as pharmacological agents in ion channel research, and several have direct diagnostic and therapeutic potential. Large conotoxin families can comprise hundreds or thousands of different peptides; most families have a corresponding ion channel family target (i.e., omega-conotoxins and Ca channels, alpha-conotoxins and nicotinic receptors). Different conotoxin families may have different ligand binding sites on the same ion channel target (i.e., µ-conotoxins and delta-conotoxins to sites 1 and 6 of Na channels, respectively). The individual peptides in a conotoxin family are typically each selectively targeted to a diverse set of different molecular isoforms within the same ion channel family. This review focuses on the targeting specificity of conotoxins and their differential binding to different states of an ion channel.

9 January, 2004

8 January, 2004

    Prialt (Ziconotide), is an effective and non-addictive analgesic
    Steven Reinberg writing for HealthDayNews (Jan. 6, 2004) reported on recent clinical trials of Ziconotide (also known as "Prialt"), the synthetic version of omega-conotoxin MVIIA. Read his report : Drug From Snail Venom Relieves Pain: Ziconotide effective and non-addictive, but not for everyone for further information.

    This novel, non-addictive drug derived from the venom of a marine snail Conus magus provides significant relief for cancer and AIDS patients suffering from intractable pain. Results of the new study by J. Staats and colleagues (published in the Journal of the American Medical Association, JAMA 291: 63-70, Jan. 7, 2004), indicate that more than half of the patients, who did not respond to other painkillers, found relief when they were given Ziconotide intrathecally (see Abstract, below). (Ziconotide blocks neurotransmission from primary nociceptive afferents by selectively binding to N-type voltage-sensitive calcium channels on neurons. Staats and colleagues conducted a randomized trial to test the effectiveness of intrathecal ziconotide for treatment of pain refractory to conventional treatment in patients with AIDS or end-stage cancer).

    This is welcome news for sufferers of chronic neuropathic pain as well as for Elan Corporation who plan to file a modified NDA with the FDA this year and to bring the treatment to market early in 2005 (see Press Release 7 Jan. 2004 summarized in the Business Wire below).

    • DUBLIN, Ireland--(BUSINESS WIRE)--Jan. 7, 2004--Elan Corporation, plc today announced that its recent Phase III trial for Prialt(TM) (ziconotide) met its primary endpoint in patients with severe chronic pain who had not achieved pain relief with other therapies including intrathecally delivered morphine. In the double-blind, placebo-controlled study, patients taking Prialt achieved statistically significant improvement at Week 3 in the Visual Analog Scale of Pain Intensity (VASPI) score, the most commonly used pain assessment scale for clinical trials. In the company's analysis, the treatment appears to be safe, efficacious, and well tolerated. Based on the positive results, the company expects to file an amendment to its New Drug Application (NDA) with the U.S. Food and Drug Administration (FDA) in the second quarter of 2004 and to bring the treatment to market no later than the first quarter of 2005.
    • See also Elan's Former Neurex Product, Prialt, Hits Phase III Endpoint By Cormac Sheridan, 14 January 2004. In this report David Marshall, analyst at NCB Stockbrokers in Dublin, is reported as saying "the company's most immediate target is likely to be that fraction of patients using intrathecal pumps who have become refractory to morphine or who suffer from respiratory side effects through its use".

    Staats PS, Yearwood T, Charapata SG, Presley RW, Wallace MS, Byas-Smith M, Fisher R, Bryce DA, Mangieri EA, Luther RR, Mayo M, McGuire D, Ellis D. (2004) Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. JAMA. 291: 63-70.

    Division of Pain Medicine, Johns Hopkins University School of Medicine, Baltimore, Md 21205, USA. pstaats@jhmi.edu

    Abstract:CONTEXT: Ziconotide (formerly SNX-111) selectively blocks N-type voltage-sensitive calcium channels and may be effective in patients with pain that is refractory to opioid therapy or those with intolerable opioid-related adverse effects. OBJECTIVE: To assess the safety and efficacy of intrathecal ziconotide in patients with pain that is refractory to conventional treatment. DESIGN, SETTING, AND PATIENTS: Double-blind, placebo-controlled, randomized trial conducted from March 12, 1996, to July 11, 1998, at 32 study centers in the United States, Australia, and the Netherlands. Patients were 111 individuals ages 24 to 85 years with cancer or AIDS and a mean Visual Analog Scale of Pain Intensity (VASPI) score of 50 mm or greater. Patients were randomly assigned in a 2:1 ratio to receive ziconotide or placebo treatment. INTERVENTIONS: Intrathecal ziconotide was titrated over 5 to 6 days, followed by a 5-day maintenance phase for responders and crossover of nonresponders to the opposite treatment group. MAIN OUTCOME MEASURE: Mean percentage change in VASPI score from baseline to the end of the initial titration period. RESULTS: Of the evaluable population, 67 (98.5%) of 68 patients receiving ziconotide and 38 (95%) of 40 patients receiving placebo were taking opioids at baseline (median morphine equivalent dosage of 300 mg/d for the ziconotide group and 600 mg/d for the placebo group; P =.63, based on mean values), and 36 had used intrathecal morphine. Mean (SD) VASPI scores were 73.6 (1.8) mm in the ziconotide group and 77.9 (2.3) mm in the placebo group (P =.18). Mean VASPI scores improved 53.1% (95% confidence interval [CI], 44.0%-62.2%) in the ziconotide group and 18.1% (95% CI, 4.8%-31.4%) in the placebo group (P<.001), with no loss of efficacy of ziconotide in the maintenance phase. Pain relief was moderate to complete in 52.9% of patients in the ziconotide group compared with 17.5% in the placebo group (P<.001). Five patients receiving ziconotide achieved complete pain relief, and 50.0% of patients receiving ziconotide responded to therapy compared with 17.5% of those receiving placebo (P =.001). CONCLUSION: Intrathecal ziconotide provided clinically and statistically significant analgesia in patients with pain from cancer or AIDS.

7 January, 2004

    Propeptide aids PID-catalyzed folding of conotoxins
    Olga Buczek, Baldomero M. Olivera and Grzegorz Bulaj from the Department of Biology, UniVersity of Utah, and Cognetix, Inc., Salt Lake City, Utah, have examined whether the propeptide region acts as an intramolecular chaperone in the folding of conotoxins and concluded that it does not do so directly but assists the early folding catalysed by the enzyme protein disufide isomerase, suggesting that it may play a role in the PID-catalyzed oxidative folding of conotoxin precursors.
    Buczek, O., Olivera, B.M. and Bulaj, G. (2004) Propeptide Does Not Act as an Intramolecular Chaperone but Facilitates Protein Disulfide Isomerase-Assisted Folding of a Conotoxin Precursor. Biochemistry 43: 1093-1101
    Abstract:Conotoxins comprise a large and diverse group of peptide neurotoxins derived from Conus snail venoms; most contain multiple disulfide bonds. The conotoxin precursors consist of three distinct domains: the N-terminal signal sequence, an intervening propeptide region, and the C-terminal mature conotoxin. Formation of the native disulfide bonds during the oxidative folding of conotoxins is a prerequisite for their proper biological function, but in numerous in vitro folding experiments with mature conotoxins, a lack of specificity in formation of the native Cys-Cys connectivities is observed. The mechanisms that ensure that the native disulfide bonds are formed in venom ducts during biosynthesis remain unknown. To evaluate whether the propeptide could potentially function as an intramolecular chaperone, we studied the oxidative folding of a conotoxin precursor, pro-GI, belonging to the R-conotoxin family. Our results indicate that the propeptide sequence did not directly contribute to folding kinetics and thermodynamics. However, we found that the propeptide region of pro-GI played an important role when oxidative folding was catalyzed by protein disulfide isomerase (PDI). The PDI-assisted reaction was more efficient during the early folding in the context of the propeptide sequence (pro-GI), as compared to that of the mature conotoxin (R-GI). Taken together, our results suggest for the first time that the propeptide region may play a role in the PDI-catalyzed oxidative folding of conotoxin precursors.

3 January, 2004

    Conus at The NakedScientist site
    Dr. Barry Gibb from University College London, and Guest Columnist for The Naked Scientist, UK, has provided a highly readable account of the cone shell world and their conotoxins to spark interest among young aspiring scientists. His article is entitled "Deadly Snails Harbour Painkillers of the Future". Enjoy the read !

2 January, 2004

    The "A-Superfamily" of conotoxins
    Santos AD, McIntosh JM, Hillyard DR, Cruz LJ, Olivera BM.(2004) The A-superfamily of conotoxins:Structural and functional divergence. J Biol Chem. 279: 17596-17606.
    Abstract:The generation of functional novelty in proteins encoded by a gene superfamily is seldom well-documented. In this report, we define the A-conotoxin superfamily, which is widely expressed in venoms of the predatory cone snails (Conus), and show how gene products that diverge considerably in structure and function have arisen within the same superfamily. A cDNA clone encoding alpha-conotoxin GI, the first conotoxin characterized, provided initial data that identified the A-superfamily. conotoxin precursors in the A-superfamily were identified from six Conus species: most (11/16) encoded alpha-conotoxins, but some (5/16) belong to a family of excitatory peptides, the kA-conotoxins that target voltage-gated ion channels. alpha-Conotoxins are two-disulfide-bridged nicotinic antagonists, 13-19 amino acids in length; kappaA-conotoxins are larger (31-36 AA) with three disulfide bridges. Purification and biochemical characterization of one peptide, kappaA-conotoxin MIVA is reported; five of the other predicted conotoxins were previously venom-purified. A comparative analysis of conotoxins purified from venom and their precursors reveals novel post-translational processing, as well as mutational events leading to polymorphism. Patterns of sequence divergence and Cys codon usage define the major superfamily branches, and suggest how these separate branches arose.

1 January, 2004

    Minireview on alpha- and kappa-conotoxins
    V.I. Tsetlin and F. Hucho from Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia and the Institut für Chemie-Biochemie, Freie Universität, Berlin, Germany, respectively, have written a "Minireview" on the interactions of snake alpha-neurotoxins and cone snail alpha- and kappa-conotoxins that have been shown to interact with nicotinic acetylcholine receptors. Their applications for pharmacological distinction of muscle, neuronal and neuronal-like AChR subtypes and for other medical purposes is briefly discussed.
    V.I. Tsetlin and F. Hucho (2003) Minireview - snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications. FEBS Lett. 557: 9-13. Review.
    This review covers recent data on interactions of nicotinic acetylcholine receptors (AChR) with snake venom proteins (alpha- and kappa-neurotoxins, `weak' toxins recently shown to act on AChRs), as well as with peptide alpha-conotoxins from Conus snails. Mutations of AChRs and toxins, X-ray/nuclear magnetic resonance structures of alpha-neurotoxin bound to AChR fragments, and the X-ray structure of the acetylcholine-binding protein were used by several groups to build models for the alpha-neurotoxin-AChR complexes. Application of snake toxins and alpha-conotoxins for pharmacological distinction of muscle, neuronal and neuronal-like AChR subtypes and for other medical purposes is briefly discussed.

Continued in What's new in 2003
See also : What's new in 2006, What's new in 2005, 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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