What's New in 2003

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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 http://www.nature.com/nsu/020715/020715-11.html

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

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31 December, 2003

    Binding properties of kappa-Conotoxin PVIIA from Conus purpuascens
    Dr. Koch and colleagues from Max-Planck-Institute for Experimental Medicine, Molecular and Cellular Neuropharmacology Group, Gottingen, Germany in collaboration with 'Toto' Olivera from University of Utah, Salt Lake City, USA, have examined the complicated binding kinetics of kappa-conotoxin PVIIA to Shaker K+ channels.
    Koch ED, Olivera BM, Terlau H, Conti F. (2004) The Binding of kappa-Conotoxin PVIIA and Fast C-Type Inactivation of Shaker K(+) Channels are Mutually Exclusive. Biophys J. 86: 191-209
    Abstract:kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid peptide identified from the venom of Conus purpurascens, inhibits the Shaker K(+) channel by blocking its outer pore. The toxin appears as a gating modifier because its binding affinity decreases with relatively fast kinetics upon channel opening, but there is no indication that it interferes with the gating transitions of the wild-type channels (WT), including the structural changes of the outer pore that underlie its slow C-type inactivation. In this report we demonstrate that in two outer pore mutants of Shaker-IR (M448K and T449S), that have high toxin sensitivity and fast C-type inactivation, the latter process is instead antagonized by and incompatible with kappa-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of kappa-PVIIA, whereas toxin rebinding must await recovery from inactivation causing a double-exponential relaxation of the second response to double-pulse stimulations. Compared with the lack of similar effects in WT, these results demonstrate the ability of peptide toxins like kappa-PVIIA to reveal possibly subtle differences in structural changes of the outer pore of K(+) channels; however, they also warn against a naive use of fast inactivating mutants as models for C-type inactivation. Unfolded from the antagonistic effect of inactivation, toxin binding to mutant noninactivated channels shows state- and voltage-dependencies similar to WT: slow and high affinity for closed channels; relatively fast dissociation from open channels at rate increasing with voltage. This supports the idea that these properties depend mainly on interactions with pore-permeation processes that are not affected by the mutations. In mutant channels the state-dependence also greatly enhances the protection of toxin binding against steady-state inactivation at low depolarizations while still allowing large responses to depolarizing pulses that relieve toxin block. Although not obviously applicable to any known combination of natural channel and outer-pore blocker, our biophysical characterization of such highly efficient mechanism of protection from steady-state outer-pore inactivation may be of general interest.

28 December, 2003

    Two Reviews on the Chemistry of Conotoxins.
    Paul Alewood and colleagues have reviewed the chemistry, solid phase synthesis and pharmacology of conotoxins presenting a structure-activity study on alpha-conotoxin [A10L] PnIA as a case study.

    • Alewood, P., Hopping, G. and Armishaw, C. (2003) Marine toxins as sources of drug leads. Aust J Chem 56 : 769-774.

    A. Grey Craig from the Salk Inst Biol Studies, Clayton Fdn Labs Peptide Biol, La Jolla, CA 92037 USA, has reviewed the traditional and more novel techniques for the characterization of conotoxins, focussing on sites of post-translational modifications and the increasingly important role played by mass spectrometry.

    • Craig, A.G. (2003) The characterization of conotoxins. J Toxicol-Toxin Rev. 19: 53-93. Abstract: Conotoxins are peptide toxins synthesized by marine cone snails for both prey entrapment and defense. The peptides, when injected into the prey, cause immobilization and death. Cone snails are widely distributed in tropical waters, their prey includes fish, worms and other marine snails. The peptide toxins have very high specificity and selectivity for a variety of neuro receptors and ion channels. This makes the toxins very useful in studies aimed at identifying receptors and their ligands, as well as in drug development studies. Conotoxins are notable at the level of primary amino acid sequence for their high percentage of cysteine residues and other post-translational modifications including hydroxylation of proline, gamma-carboxylation of glutamate, pyroglutamic acid formation, bromination of tryptophan and C-terminal amidation. This review describes traditional and more novel techniques for the characterization of conotoxins. In particular, the identification of the nature and the site of post-translational modifications is emphasized. Among the different techniques used to characterize the conotoxins, the important role played by mass spectrometry is emphasized.

27 December, 2003

26 December, 2003

    The anal canal in the detemination of Conus species
    Wesley M. Thorsson, editor of Internet Hawaiian Shell News (IHSN), asked on CONCH-L mail list if "there (are) any opinions as to the advantage to Conus in having different posterior canal shapes (in apical view, the outer lip rises radially to curved to having a deep slit). I don't recall seeing mention of this in references as an ID factor for Conus, but this detail seems constant with specimens that are undamaged".
    To which Giancarlo Paganelli of Cone Shells web page replied: "For an investigation on the use of the anal canal in the determination of cones species see:
    PIN, M., 1985. Etude des canaux anaux des Conidae du Sénegal et leur importance dans la détermination des espèces. Publ. Ocas. Soc. Port. Malac., 14:33-38.
    PIN, M. & TACK, K.D., 1995. I coni del Senegal. La Conchiglia, annuario, 53 pp.

    I re-learn something new every day on this web site ! (see also Cones of Senegal" by by M. Pin and K.D. Leung-Tak (1995), Supplement to issue no. 277 (October-December 1995) of "La Conchiglia" (ISSN 0394-0152).

17 December, 2003

16 December, 2003

    Conus - a case of mistaken identity
    Peter Egerton of "Peter's Seashells" (including Shells of British Columbia) reported to CONCH-L mailing list that he had "just finished watching the TV show 'Killer Instinct', which had a segment on deadly cone snails. It showed the host of the show snorkeling for cones and showing us the live animals. Nearly every one that he showed was actually a Strombus luhuanus ! He did show a few actual cones, but it was funny to see most of the 'deadly cone snails' moving along the sand with the characteristic Strombus jerking. A good reminder to take anything you see on these TV shows with a grain of salt and to question the 'experts' facts".
    PS. Peter has a new Conus Gallery. Check it out here.

14 December, 2003

    Conidae: South West Florida Species
    Check out the Bailey-Matthews Shell Museum web site (www.shellmuseum.org) for a number of new items of general interest.
    The inventory of shallow-water Southwest Florida Shells now includes 180 species from the Sanibel area, including a bunch of micromollusks seldom photographed.
    Included are images of the following members of the Family Conidae including 3 species of Conus : Conus anabathrum (Conrad, 1865); Conus spurius Gmelin, 1791; and Conus stearnsii Conrad, 1869 ; and 5 Other Conidae : Cryoturris cerinella (Dall, 1889); Pyrgocythara hemphilli Bartsch & Rehder, 1939; Pyrgocythara plicosa (C. B. Adams, 1850); Stellatoma stellata (Stearns, 1872); and Tenaturris bartletti (Dall, 1889).

    In addition, PDF versions of the chapters referenced below are available for printing under the sections "What's New" and "Projects" :

  • Leal, J. H. 2002. Bivalves. In: K. E. Carpenter (ed.) The Living Marine Resources of the Western Central Atlantic. Volume 1. Introduction, mollusks, crustaceans, hagfishes, sharks, batoid fishes and chimaeras. FAO Identification Guide for Fishery Purposes. The Food and Agriculture Organization of the United Nations, Rome, pp. 25-98
  • Leal, J. H. 2002. Gastropods. In: K. E. Carpenter (ed.) The Living Marine Resources of the Western Central Atlantic. Volume 1. Introduction, mollusks, crustaceans, hagfishes, sharks, batoid fishes and chimaeras. FAO Identification Guide for Fishery Purposes. The Food and Agriculture Organization of the United Nations, Rome, pp. 99-147

    alpha-Conotoxins from Conus episcopatus and Conus aureus react differently with native vs. recombinant receptors.
    Dr. Richard Lewis and colleagues from the Institute for Molecular Bioscience (IMB), University of Queensland, Brisbane, Qld 4072, Australia, have shown that the selectivity of alpha-conotoxins for nicotinic receptor (nAChR) subunit combinations expressed in native mammalian targets is different to their interaction with those same subunit combinations expressed in Xenopus oocytes. This indicates that factors yet to be identified influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs.
    Nicke A, Samochocki M, Loughnan ML, Bansal PS, Maelicke A, Lewis RJ. (2003) Alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors. FEBS Lett. 554:219-223.
    Abstract: The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT3 chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had 25-fold lower activity. These results indicate that as yet unidentified factors influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs.

    EF-hand structure of calmodulin is needed to inhibit binding of omega-conotoxins
    Dr. Seiji Ichida and colleagues from the Department of Biological Chemistry, School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Japan, have shown that the characteristic EF-hand structure of calmodulin (CaM) itself is needed to cause the observed inhibitory effect of calmodulin on the specific binding of 125I-omega-CTX to chick brain membranes.
    Ichida, S., Abe, J., Yu-an, Z., Minami, T., Wada, T., Yazawa, M. and Sohma, H. (2003) Structural specificity for the inhibitory effect of calmodulin on specific 125I-omega-conotoxin GVIA binding. Neurochem Res.28: 1813-1818.
    Abstract: To clear the structural specificity of calmodulin (CaM) on the specific 125I-omega-CTX binding to crude membranes from whole chick brain, the following experiments were investigated in this study: (i) the attenuating effect of semisynthetic tetrahydroisoquinoline derivatives on the inhibitory effect of Ca2+/CaM, (ii) the effects of chimeras of yeast and chicken Ca2+/CaM, and (iii) the effects of Ca2+-binding proteins (such as troponin c, S 100 a and b, and annexin I, III-V). The inhibitory effect of Ca2+/CaM was attenuated by isoquinoline derivatives (PX 28, 34, 216, 224, and CPU57) and a CaM antagonist W-7. PX 34, a typical synthesized isoquinoline derivative, showed the attenuating effect in a dose-dependent manner. The ED50 value for the attenuating effect of PX 34 was about 20 microM, which is similar to that of W-7 reported previously. Some chimeric CaMs such as YC 51-53 (which are close to the properties of vertebrate CaM) showed a significant inhibitory effect on the specific 125I-omega-CTX binding, but YC 129 and 130 (which retain the properties of yeast CaM), troponin c, S100 a, b, and annexin I, III-V had no effect on the specific 125I-omega-CTX binding. These results suggest that the characteristic structure containing the EF-hand structure of CaM itself is needed to cause the inhibitory effect on the specific 125I-omega-CTX binding

8 December, 2003

    kappa-conotoxin PVIIA (CGX-1051) from Conus purpurascens reduces infarct size in rabbits.
    Shi Jun Zhang, Xi-Ming Yang, Guang S. Liu, Michael V. Cohen, Karen Pemberton and James M. Downey from the Department of Physiology, University of South Alabama, College of Medicine, Mobile, Alabama 36688, USA have reported on the protective properties afforded by administration of conotoxin PVIIA (CGX-1051) from the venom of Conus purpurascens on myocardial infarct size in a rabbit heart model of ischemia/reperfusion.

    Zhang, S.J., Yang, X.M., Liu, G.S., Cohen, M.V., Pemberton, K. and Downey, J.M. (2003). CGX-1051, A peptide from Conus snail venom, attenuates infarction in rabbit hearts when administered at reperfusion. Journal of Cardiovascular Pharmacology 42: 764-771.
    Abstract: CGX-1051, isolated from the venom of the marine snail Conus purpurasens, was previously noted to interact with potassium channels. Since potassium channels play an important role in cardiac physiology, we assessed the effect of CGX-1051 on infarct size in a rabbit heart model of ischemia/reperfusion. A coronary branch was occluded for 30 minutes followed by 3 hours of reperfusion in in situ and 2 hours in in vitro preparations. Infarct size was measured with triphenyltetrazolium chloride staining and expressed as a percent of the risk zone. In in situ studies, a bolus intravenous injection of CGX-1051, either 10 or 100 µg/kg, administered 5 minutes before reperfusion, reduced infarct size from 40.4 ± 2.8% of the risk zone in untreated animals to 19.8 ± 3.8% and 15.0 ± 1.9%, respectively. One µg/kg CGX-1051 was not protective. To see if the salvage was sustained, two groups of rabbits underwent 72 hours of reperfusion. The dose of 10 µg/kg infused 5 minutes before reperfusion reduced infarct size from 37.0 ± 1.6% in untreated rabbits to 15.5 ± 2.0%. When administered 10 minutes after reperfusion had begun, 100 µg/kg CGX-1051 had no effect. CGX-1051 also reduced infarct size in crystalloid-perfused, isolated rabbit hearts suggesting that protection did not depend on circulating leukocytes. The mitochondrial K(ATP) inhibitors glibenclamide and 5-hydroxydecanoate and the MEK(½), ERK and hence, inhibitor PD 98059 aborted protection from CGX-1051. These data indicate that functionally active ERK and mitochondrial K(ATP) channels are necessary for protection. CGX-1051 caused no hemodynamic alterations at any dose tested. We conclude that CGX-1051 has a powerful anti-infarct effect when given just before reperfusion.

    See also : Pemberton-Goodman et al 2003 - US Patent Application US 2003/224343, Publication Date: December 4, 2003 (Filed Jan 28, 2003; Provisional Application No. 60/352,219 filed on Jan 29, 2002). "Kappa-PVIIA-related conotoxins as organ protectants". Inventors: Pemberton-Goodman, Karen (Guilford, CT); Jones, Robert M. (San Diego, CA); Temple, Davis L. JR. (Wallingford, CT); McIntosh, J. Michael (Salt Lake City, UT); Olivera, Baldomero M. (Salt Lake City, UT)
    Abstract: The invention relates to .kappa.-PVIIA-related conotoxins and their use as organ protecting agents, i.e., organ protectants. These conotoxins can be used for arresting, protecting or preserving an organ, such as a circulatory organ, a respiratory organ, a urinary organ, a digestive organ, a reproductive organ, an endocrine organ or a neurological organ. These conotoxins can also be used for arresting, protecting or preserving somatic cells.

    (It is revealed in legends to Figs. 9, 10 and 11 of the above patent application that kappa-conotoxin PVIIA is CGX-1051 [Cognetix].) Kappa-PVIIA is a 27 amino acid peptide (a kappa-conotoxin) originally purified from the venom of the purple cone snail Conus purpurascens (Terlau et al., 1996; US Pat. no. 5,672,682), and has previously been identified as a potent antagonist of the Shaker H4 postassium channel (IC.sub.50.about.60nM). In the same study, no detectable activity was detected on the voltage-gated potassium channels KV1.1 or KV1.4 (Terlau et al 1996). Chimeras constructed from the SHaker and the KV1.1 K.sup.+ channels have identified the putative pore-forming region between the fifth and sixth transmembrane region as the site of toxin sensitivity (Shon et al., 1998). It appears that .kappa.-PVIIA interacts with the external tetraethyl-ammonium binding site on the Shaker channel.

    Nicotinic receptor subunit selectiviy for alpha-conotoxin MII
    Cui C, Booker TK, Allen RS, Grady SR, Whiteaker P, Marks MJ, Salminen O, Tritto T, Butt CM, Allen WR, Stitzel JA, McIntosh JM, Boulter J, Collins AC, Heinemann SF. (2003) The {beta}3 Nicotinic Receptor Subunit: A Component of {alpha}-Conotoxin MII-Binding Nicotinic Acetylcholine Receptors that Modulate Dopamine Release and Related Behaviors. J Neurosci. 2003 Dec 3;23(35):11045-11053.
    Abstract: Salk Institute for Biological Studies, La Jolla, California 92037, Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0930, Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah 84112, Department Psychiatry and Biobehavioral Science, University of California Los Angeles, Los Angeles, California 90024, and Blanchette Rockefeller Neuroscience Institute, Rockville, Maryland 20850. Nigrostriatal dopaminergic neurons express many nicotinic acetylcholine receptor (nAChR) subunits capable of forming multiple nAChR subtypes. These subtypes are expressed differentially along the neuron and presumably mediate diverse responses. beta3 subunit mRNA has restricted expression but is abundant in the substantia nigra and ventral tegmental areas. To investigate the potential role(s) of nicotinic receptors containing the beta3 subunit in dopaminergic tracts, we generated mice with a null mutation in the beta3 gene. We were thereby able to identify a population of beta3-dependent alpha-conotoxin MII-binding nAChRs that modulate striatal dopamine release. Changes were also observed in locomotor activity and prepulse inhibition of acoustic startle, behaviors that are controlled, in part, by nigrostriatal and mesolimbic dopaminergic activity, respectively, suggesting that beta3-containing nAChRs modulate these behaviors.

2 December, 2003

    Conus ebreus on a Jardinier
    Seen on "The Antiques Road Show" Program on Foxtel "Lifestyles Channel" (19-20 November, 2003) eminating from the UK (Chichester Cathedral), a Jardinier (vase) featuring painted shells including an Conus ebreus. The vase was valued at 12,000 Pounds !

    Death, where is thy sting?
    Jane Bradbury authored the following article about venoms as a source of new drugs. This article appeared on 5 November in BioMedNet News under the title "Death, where is thy sting?".

    Getting close to venomous animals is not everyone's idea of a job with a future. But deadly venoms are proving a gold mine for molecules with interesting pharmacological properties, and the potential to treat an array of medical disorders.
    Venoms are complex cocktails of molecules produced by animals for defense and for catching prey. A single snake venom may contain several hundred neurotoxic, cardiotoxic, and cytotoxic proteins and peptides.
    Then there are scorpions, spiders, even snails - the list of venomous creatures is long. "There are about 1500 species of scorpions worldwide with some 100+ novel molecules in each venom," said Paul Alewood, professor of chemical and structural biology at the University of Queensland in Brisbane. "So there is the potential to discover tens of thousands of interesting molecules and their accompanying activities in scorpions alone."
    From these molecules, isolated by high performance liquid chromatography and mass spectrometry, state-of-the-art chemistry can be used to understand their structure-function relationships and select lead molecules for development. And yet, said Alewood, "this exciting field of research is somewhat under investigated."
    Most people who are interested in natural products look in plants, he says. "They have the benefit that they stand still and don't bite you," he said. Between 25-50% of the current pharmacopoeia, estimates Alewood, comes from plant-derived materials. By contrast, few drugs have been derived from venoms.
    The prime exception, says Alewood, is angiotensin converting enzyme (ACE) inhibitors, which are used worldwide to treat high blood pressure. ACE inhibitors were developed from molecules identified in the venom of a South American viper, he explains, after it was noticed that blood pressure plummeted in people bitten by this snake.
    Among the researchers who are mining venoms for their pharmacological treasures is Ponnampalam Gopalakrishnakone, a professor in the Faculty of Medicine at the National University of Singapore. Speaking in Singapore at the Biomedical Research Applications in Drug Discovery Technology Asia-Pacific meeting at the beginning of November, Gopalakrishnakone explained that although natural toxins have often been used in traditional remedies, it is only in the past ten years that the search for new drug leads in venoms has intensified.
    Among the 23 new toxins that his group has discovered are: hannalgesin, a strong analgesic isolated from king cobra venom; an acidic phospholipase A2 toxin with anticlotting activity also from king cobra venom; and anti-inflammatory peptides derived from python venom that may be useful in the treatment of rheumatoid arthritis and some forms of brain damage. His team has also isolated lead molecules from scorpion, spider and coneshell venoms.
    This last group of venoms - the conotoxins - is of particular interest to Roger Drinkwater, head of research at biopharmaceutical firm Xenome in Brisbane, Queensland. The peptides isolated from predatory sea snails called coneshells have "an exceptionally broad range of pharmacological activities," said Drinkwater, "and are highly potent and very specific, ideal attributes for new drug leads."

1 December, 2003

18 November, 2003

    Conus erminieus delta-EVIA toxin inhibits Na+ channel inactivation
    Drs. Jean Barbier, Andrez Menez and colleagues from the Laboratoire de Neurobiologie Cellulaire et Moleculaire, UPR 9040, Centre National de la Recherche Scientifique, Gif sur Yvette 91198, France have isolated a delta conotoxin (delta-EVIA) from the venom of Conus ermineus, that increases the duration of action potentials by inhibiting Na(+) channel inactivation. delta-EVIA inhibited inactivation of rat neuronal Na(+) channel subtypes (rNaV1.2a, rNaV1.3 and rNaV1.6), but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na(+) channel subtypes.

    Barbier J, Lamthanh H, Le Gall F, Favreau P, Benoit E, Chen H, Gilles N, Ilan N, Heinemann SH, Gordon D, Menez A, and Molgo J. (2003) A delta -conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na(+) channels, but not in skeletal and cardiac muscles. J Biol Chem. Nov 13, 2003 [Epub ahead of print].
    Abstract: We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six cysteine/four loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA, inhibits Na(+) channel inactivation with unique tissue specificity upon binding to receptor site-6 of neuronal Na(+) channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na(+) channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction, but did not affect directly-elicited muscle action potentials. The neuronal-selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na(+) channel subtypes (rNaV1.2a, rNaV1.3 and rNaV1.6), but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na(+) channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na(+) channels without acting on Na(+) channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na(+) channel subtypes, for studying the distribution and modulation mechanisms of neuronal Na(+) channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.

17 November, 2003

    Coneshells from the Philippines [ex collection Jim Cootes]
    Giancarlo Paganelli has updated his Cone Shells collection (currently currently 822 images), with some images of cone shell specimens he received by Jim Cootes, an Australian conchologist who is specialized in cones of Philippines. Conus aphrodite PETUCH, 1979 Philippines, Aliguay Is. - 14.0 mm; Conus axelrodi WALLS, 1978 Philippines - 15.9 mm and 17.8 mm ; Conus blanfordianus CROSSE, 1867 Philippines, Marinduque - 24.1 mm ; Conus capitanellus FULTON, 1938 Philippines, Cebu, Sugod - 26.8 mm ; Conus corallinus KIENER, 1845 Philippines, Mactan, Punta Engano - 18.7 mm ; Conus dayriti RöCKEL & DA MOTTA, 1983 Philippines, Palawan - 18.8 mm ; Conus ebraeus LINNAEUS, 1758 Philippines, Mactan, Punta Engano - 26.0 mm ; Conus furvus REEVE, 1843 Philippines, Sulu Is. - 43.6 mm ; Conus muriculatus SOWERBY I, 1833 Philippines, Siquijor Is. - 38.4 mm and Conus proximus f. cebuensis WILS, 1990 Philippines, Cebu, Sugod - 32.0 mm

    More coneshell images
    George Sangiouloglou from Greece has 51 new cone shell images available for viewing on the web from "A to Z", Conus ateralbus to Conus zulu. To view these 51 images go to the following pages : Page 1 and Page 2

    REVIEW on Neuropathic Pain.
    Fiona E. Munro and Tracey Dickinson from Quintiles Limited, Edinburgh, UK, together with Kevin Lee and David Spanswick from NeuroSolutions Limited, University of Warwick, Coventry , UK have reviewed basic mechanisms and actions of some of the key compounds that are currently being developed for the treatment of neuropathic pain.

    Dickinson, T., Lee, K., Spanswick, D. and Munro, F.E. (2003) Leading the charge - pioneering treatments in the fight against neuropathic pain (2003) Trends in Pharmacological Sciences 24: 555-557.
    Abstract: Neuropathic pain, arising from nerve injury, results in a chronic and debilitating form of pain that in the past has been poorly diagnosed and treated. During the past few years, intensive research has resulted in major progress towards understanding the basic mechanisms that contribute to this condition, and the renewed possibility of safe and effective medicines. Indeed, the next few years should see the first fruits of this labour reaching the market place and, with them, the opportunity to assess whether the scientific advances achieved have resulted in significant clinical improvement. In this article, some of the key compounds that will lead this charge and the clinical results obtained so far are discussed.
    For further information, contact Fiona Munro

5 November, 2003

    REVIEW on the Pharmacology of traumatic brain injury.
    Drs T. McIntosh and colleagues from the Head Injury Center, Department of Neurosurgery, University of Pennsylvania, 3320 Smith Walk, 105 C Hayden Hall, Philadelphia, PA 19104-6316, USA have reviewed the literature pertaining to the pharmacology of traumatic brain injury - a condition that was once thought it may be possible to treat using N-type calcium channel blockers such as omega-conotoxin MVIIA (also known as Prialt, Ziconotide, SNX-111 and CI-1009) and other N-type conotoxins.

    Royo, N.C,, Shimizu, S, Schouten, J.W., Stover, J.F. and McIntosh, T.K. (2003) Pharmacology of traumatic brain injury, Current Opinion in Pharmacology 3: 27-32
    Abstract: The intensity of experimental and clinical research to identify a neuroprotective drug for the treatment of traumatic brain injury is motivated by the devastating morbidity and mortality of this condition. Encouraging experimental work has led so far to disappointing clinical trials and the identification of new potential therapeutic targets is critically dependent on a better understanding of the chronic pathophysiology triggered by the initial insult. Future advances in the pharmacological treatment of traumatic brain injury are likely to include the evaluation of sequentially timed therapies combining multiple and targeted agents, and manipulation of the newly discovered neurogenic potential of the adult brain together with the refinement of traditional interventions to block specific cytotoxic cascades.

    (Abbreviations: 2-AG, 2-arachidonyl glycerol; BBB, blood brain barrier; CNS, central nervous system; COX, cyclo-oxygenase; IL, interleukin; mGlu, metabotropic glutamate receptor; MPTP, mitochondrial permeability transition pore; NMDA, N-methyl-beta-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species; TBI, traumatic brain injury)

    The section referring to the conotoxins makes for interesting reading.
    "Modulators of cation homeostasis: Intracellular calcium elevation in neurons resulting from NMDA receptor activation, a major pathophysiological event contributing to cell death, remains a potential target for pharmacotherapy after TBI. Although the L-type voltage-sensitive calcium channel blocker, nimodipine, has proven to be efficacious in experimental TBI, its beneficial effect in severely injured patients with subarachnoid haemorrhage remains unproven (Clausen and Bullock, 2001). Likewise, neuroprotective effects of the N-type calcium channel blocker, Ziconotide (SNX-111, CI-1009), in animal models (Berman et al 2000; Verweij et al 2000) have not been translated to humans because of their secondary hypotensive effects. Calcium-mediated neuronal injury is mediated in part by the activation of calpains leading to the degradation of cytoskeletal proteins. Although calpain antagonists are difficult to synthesise and poorly penetrate the blood brain barrier (BBB), we encourage a renewed interest in this class of compounds for the treatment of TBI."


    Clausen, T. and Bullock, R. (2001) Medical treatment and neuroprotection in traumatic brain injury. Curr. Pharm. Des. 7: 1517-1532.
    Berman, R.F., Verweij, B.H. and Muizelaar, J.P. (2000) Neurobehavioral protection by the neuronal calcium channel blocker ziconotide in a model of traumatic diffuse brain injury in rats. J. Neurosurg. 93: 821-828.
    Verweij, B.H., Muizelaar, J.P., Vinas, F.C. Peterson, P.L., Xiong, Y. and Lee, C.P. (2000). Improvement in mitochondrial dysfunction as a new surrogate efficiency measure for preclinical trials: dose-response and time-window profiles for administration of the calcium channel blocker Ziconotide in experimental brain injury. J. Neurosurg. 93: 829-834.

4 November, 2003

    Evolution of conopeptide folding in Conus.

    Grzegorz Bulaj, 'Toto' Olivera and colleagues from the Department of Biology, University of Utah and from Cognetix, Inc., Salt Lake City, UT, USA have addressed the mechanisms used by the cone shells in vivo for folding their mature toxin peptides to yield the biologically active disulfide configuration.They found that that the correct folding is facilitated by a posttranslationally modified amino acid, gamma-carboxyglutamate suggesting that acquisition of this capability provided a competitive advantage to Conus over other organisms.

    Bulaj G, Buczek O, Goodsell I, Jimenez EC, Kranski J, Nielsen JS, Garrett JE, Olivera BM (2003). Efficient oxidative folding of conotoxins and the radiation of venomous cone snails. Proc Natl Acad Sci U S A. 100: (suppl. 2) 14562-14568..
    Abstract: The 500 different species of venomous cone snails (genus Conus) use small, highly structured peptides (conotoxins) for interacting with prey, predators, and competitors. These peptides are produced by translating mRNA from many genes belonging to only a few gene superfamilies. Each translation product is processed to yield a great diversity of different mature toxin peptides ( approximately 50,000-100,000), most of which are 12-30 aa in length with two to three disulfide crosslinks. In vitro, forming the biologically relevant disulfide configuration is often problematic, suggesting that in vivo mechanisms for efficiently folding the diversity of conotoxins have been evolved by the cone snails. We demonstrate here that the correct folding of a Conus peptide is facilitated by a posttranslationally modified amino acid, gamma-carboxyglutamate. In addition, we show that multiple isoforms of protein disulfide isomerase are major soluble proteins in Conus venom duct extracts. The results provide evidence for the type of adaptations required before cone snails could systematically explore the specialized biochemical world of "microproteins" that other organisms have not been able to systematically access. Almost certainly, additional specialized adaptations for efficient microprotein folding are required.

    Representative Publications of Grzegorz Bulaj
  • Buczek O, Olivera BM, Bulaj G "Propeptide Does not Act as an Intramolecular Chaperone, but Facilitates Protein Disulfide Isomerase - Assisted Folding of a Conotoxin Precursor" Biochemistry - in press
  • Nielsen J, Buczek P, Bulaj G "Cosolvent-Assisted Oxidative Folding of a Bicyclic (-Conotoxin ImI" J Pept Science, in press
  • Proc Natl Acad Sci USA, 100, Suppl 2, 14562-14568. (see above)
  • Keizer DW, West PJ, Lee EF, Yoshikami D, Olivera BM, Bulaj G, Norton RS (2003) "Structural basis for tetrodotoxin-resistant sodium channel binding by (delta-conotoxin SmIIIA" J Biol Chem, 278, 46805-46813
  • Dela Cruz R, Whitby F, Buczek O, Bulaj G (2003) "Detergent-assisted oxidative folding of omega-Conotoxins" J Pept Research 61, 202-212
  • West PJ, Bulaj G, Garrett JE, Olivera BM, Yoshikami D. (2002) "(delta-Conotoxin SmIIIA, a Potent Inhibitor of Tetrodotoxin-Resistant Sodium Channels in Amphibian Sympathetic and Sensory Neurons" Biochemistry 41, 15388-15393
  • Miles LA, Dy CY, Nielsen J, Barnham KJ, Hinds MG, Olivera BM, Bulaj G, Norton RS. (2002) "Structure of a novel P-superfamily spasmodic conotoxin reveals an inhibitory cystine knot motif" J Biol Chem 277, 43033-40

3 November, 2003

    Recombinant omega-contoxin MVIIA is analgesic in mice.

    Dr Zhan and colleagues from Department of Biochemistry, Zhejiang University Medical School, 353 Yan An Road, 310006, Hangzhou, PR China have used molecular cloning to create recombinant form of omega-conotoxin MVIIA using thioredoxin and expression in E.coli. The soluble expressed form of the fusion toxin had a biological half-life of approx. 1.6h and exhibited analgesic function after intracranial injection (0.6mg/kg) into mice as detected in a mouse hot-plate assay.

    Zhan J, Chen X, Wang C, Qiu J, Ma F, Wang K, Zheng S. (2003) A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity. Biochem Biophys Res Commun. 311 495-500.
    Abstract:omega-Conotoxin MVIIA (CTX MVIIA) is a potent and selective blocker of the N-type voltage-sensitive calcium channel in neurons. Its analgesic and neuroprotective effects may prove useful in treatment of severe pains and ischemia. In this paper, we report that a fusion form of CTX MVIIA with thioredoxin (Trx) has analgesic function. The DNA fragments were chemically synthesized and ligated to form the DNA sequence encoding CTX MVIIA. The synthetic gene was then cloned into the expression vector pET-32a(+) and the fusion protein Trx-CTX MVIIA containing 6x His-tag was purified by one-step metal chelated affinity chromatography (MCAC). The purity of final product was over 95% determined by HPLC and the yield of the fusion protein was approximately 40mg/L. The analgesic function was detected by using mouse hot-plate assay. After intracranially administering fusion protein with the dose of 0.6mg/kg, marked analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent and the biological half-life of the fusion toxin was approximately 1.6h.

    cDNA cloning of kappa-A-SIVA and alpha-SII conotoxin from Conus striatus.

    Dr. Wang and colleagues from Key Laboratory of Proteomics, Shanghai Institute of Biological Sciences, The Chinese Academy of Sciences, Shanghai, China, have used RACE technology to clone two A-superfamily conotoxins, kappa-A-SIVA (a voltage-gated potassium channel blocker) and alpha-SIIa (a nicotinic acetylcholine receptor antagonist) from Conus striatus. Both share the same signal sequence and both are processed at the common signal site, -X-Arg- immediately before the mature peptide sequences.

    Wang, C.Z., Jiang, H., Ou, J-L., Chen, J-S and Chi, C-W. (2003) cDNA cloning of two A-superfamily conotoxins from Conus striatus,Toxicon, 42, 613-619.
    Abstract: The full-length cDNAs of two A-superfamily conotoxins, kappa-A-SIVA and alpha-SII, were respectively cloned and sequenced from Conus striatus using 3' RACE and 5' RACE. The cDNA of kappa-A-SIVA encodes a precursor of 68 residues, including a signal peptide of 21 residues, a pro-peptide of 17 residues, and a mature peptide of 30 residues with an additional residue Gly which is prerequisite for the amidation of the preceding C-terminal Cys. The cDNA-deduced sequence of alpha-SII is composed of a signal peptide of 21 residues, a pro-peptide of 29 residues, a mature peptide of 19 residues and three additional residues Arg-Thr-Ile at the C-terminus. This tripeptide might be cleaved off by proteolytic processing. Although these two conotoxins belong to different families and target voltage-gated potassium channel and nicotinic acetylcholine receptor, respectively, they share the same signal sequence, and both are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The length of 3' untranslational region of alpha-conotoxin SII was extraordinarily large about 10 times longer than that of kappa-A-SIVA with 770 and 75 bp, respectively. The elucidated cDNAs of these two toxins will facilitate a better understanding of the process of their post-translational modifications. (Author Keywords Conotoxin; Conus striatus; Conus peptide; cDNA cloning).

31 October, 2003

    Pain killer from cone snail to go commercial

    University of Melbourne researchers have licensed Melbourne-based biotechnology company Metabolic Pharmaceuticals Limited to commercialise a potential non-addictive pain killer extracted from an Australian marine snail. UniNews 29 October 2003.

    Abstract: The compound, known as ACV1, is the outcome of a collaborative effort between a team led by Associate Professors Bruce Livett and Ken Gayler from the University’s Department of Biochemistry and Molecular Biology, and Associate Professor Zeinab Khalil at the University’s National Ageing Research Institute.

    Metabolic Pharmaceuticals plans to progress the compound immediately into preclinical toxicity studies.

    ACV1 is likely to be initially targeted at those suffering neuropathic pain associated with diabetes, a market with billion dollar annual sales potential.

    “More than 60 per cent of the community will suffer from some form of chronic pain sometime in their life and the medical profession is crying out for alternative drug treatments,” says Associate Professor Livett.

    “Scope exists for wider applications of this compound, including treating chronic pain associated with diseases such as cancer, AIDS and arthritis. It also has potential for treating sports injuries and infections, for example shingles. In tests on rats it has been found to accelerate wound healing where nerve damage has occurred,” he says.

    The research team extracted ACV1 from a species of cone shell, a beautiful but often deadly type of mollusc found in tropical waters near Broome, Western Australia. Laboratory studies have suggested the drug could be more powerful and longer lasting than morphine, yet unlike morphine, non-addictive and lacking the side-effects, namely sedation, nausea, constipation and respiratory depression. © The University of Melbourne 1994-2003

30 October, 2003

    "Conantokins in phospholipid vesicles

    Dr. Prorok and colleagues from Department of Chemistry and Biochemistry and the W. M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, Indiana 46556, USA, have used several techniques including size-exclusion chromatography, circular dichroism, fluorescence, and NMR spectroscopies to investigate the binding, conformation, and orientation of conantokins and their variants to a variety of phospholipid (PL) vesicles and multilayers.

    Dai Q, Zajicek J, Castellino FJ and Prorok M. (2003) Binding and Orientation of Conantokins in PL Vesicles and Aligned PL Multilayers. Biochemistry. 42 : 12511-12521.
    Abstract: The association of a ligand with its cognate cell surface receptor can be facilitated by interactions between the ligand and the lipid phase of the cell membrane. With respect to the N-methyl-d-aspartate receptor (NMDAR), we have previously established a low affinity, nonreceptor-mediated interaction of the peptidic conantokins with synaptic membranes in conjunction with a high affinity binding to the NMDARs present therein [Klein, R. C., Prorok, M., and Castellino, F. J. (2003) J. Pept. Res. 61, 307-317]. In the current study, several techniques including size-exclusion chromatography, circular dichroism, fluorescence, and NMR spectroscopies were used to investigate the binding, conformation, and orientation of conantokins and their variants to a variety of phospholipid (PL) vesicles and multilayers. We have found that conantokins bind to PLs and that the effectors Ca(2+) and spermine slightly increase this binding ability. The conantokins preserve a high degree of helical conformation when bound to vesicles in the presence of Ca(2+). In the absence of Ca(2+), only conantokin-G (con-G) manifests an increase in conantokin helicity with increasing vesicle concentration. In solution, the conantokins appear to be localized at the headgroup of vesicles and do not insert into the hydrophobic core of the bilayer. On aligned PL films, the helical axis of the conantokins can either reside normal to the membrane surface or partition in a parallel orientation, depending on the nature of the conantokins and the PLs used. These orientation preferences may be conjoined with the biological activities of the conantokins.

27 October, 2003

    "REVIEW: Therapeutic Potential of Venom Peptides

    Richard Lewis from the Institute for Molecular Bioscience and School of Biomedical Sciences, The University of Queensland, St. Lucia 4072, Queensland, Australia and Xenome Ltd. has joined forces with and Maria Garcia from the Dept. of Ion Channels, Merck Research Labs, New Jersey, USA to review the potential of venom peptides as therapeutics. Included are examples from cone snails, sea anemone, Glia monster, spiders and scorpions.

    Lewis, R.J. and Garcia, M.L. (2003) Therapeutic potential of venom peptides. Nat Rev Drug Discov. 2: 790 - 802.
    Abstract:Venomous animals have evolved a vast array of peptide toxins for prey capture and defence. These peptides are directed against a wide variety of pharmacological targets, making them an invaluable source of ligands for studying the properties of these targets in different experimental paradigms. A number of these peptides have been used in vivo for proof-of-concept studies, with several having undergone preclinical or clinical development for the treatment of pain, diabetes, multiple sclerosis and cardiovascular diseases. Here we survey the pharmacology of venom peptides and assess their therapeutic prospects.

22 October, 2003

    "Venom peptide chi-MrIA from Conus marmoreus inhibits the NET

    Drs Sharpe and colleagues from the Institute for Molecular Bioscience and School of Biomedical Sciences, The University of Queensland, St. Lucia 4072, Queensland, Australia, have studied the specificity of the high affinity chi-MrIA to the norepinephrine transport and found no activity against the dopamine or serotoinin transporter.

    Sharpe IA, Palant E, Schroeder CI, Kaye DM, Adams DJ, Alewood PF, Lewis RJ. (2003) Inhibition of the norepinephrine transporter by the venom peptide chi-MrIA. Site of action, Na+ dependence, and structure-activity relationship. J Biol Chem. 278: 40317 - 40323.
    Abstract: chi-Conopeptide MrIA (chi-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether chi-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 microM) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by chi-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by chi-MrIA was competitive in nature. It has previously been demonstrated that chi-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for chi-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of chi-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, chi-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of chi-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of chi-MrIA and a His in loop 2 played a dominant role in the interaction between chi-MrIA and the NET. H alpha chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of chi-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.

    Drs. Sharpe et al have characterized the pharmacology of conotoxin rho-TIA
    Sharpe IA, Thomas L, Loughnan M, Motin L, Palant E, Croker DE, Alewood D, Chen S, Graham RM, Alewood PF, Adams DJ, Lewis RJ. (2003) Allosteric alpha 1-adrenoreceptor antagonism by the conopeptide rho-TIA J Biol Chem.278: 34451-34457
    Abstract: A peptide contained in the venom of the predatory marine snail Conus tulipa, rho-TIA, has previously been shown to possess alpha1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, rho-TIA inhibited alpha1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic alpha2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, rho-TIA displayed slightly greater potency at the alpha 1B than at the alpha 1A or alpha 1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the alpha 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by rho-TIA. N-terminally truncated analogs of rho-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of rho-TIA (Arg4). An alanine walk of rho-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of rho-TIA. The unique allosteric antagonism of rho-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive alpha1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

19 October, 2003

    "Commercial development of cone shell venom drugs

    Information on the commercial development of cone shell venom drugs has been updated and is available on the Resources page.

18 October, 2003

    "Wonder Drug" Snails Face Threats, Experts Warn

    John Pickrell in England for National Geographic News reported on October 16, 2003, that Conus, famous for yielding new wonder drugs to treat chronic pain, cancer, and many other afflictions, could rapidly become extinct if measures are not taken to protect them. The 500 known "tropical cone snails may contain the largest and most clinically important pharmacopoeia of any genus in nature," said medical scientist and study author Eric Chivian of Harvard University in Cambridge, Massachusetts. "But wild populations are being decimated by habitat destruction and overexploitation. To lose these species would be a self-destructive act of unparalleled folly." If the cone snail genus was added to a part of the CITES agreement known as Appendix II, their trade would be allowed to continue, said Roberts, but countries would be obliged to both monitor trade and limit exploitation. The study is detailed in a letter to appear on October 17 in the research journal Science.
    Reference: Chivian, E., Roberts, C.M., Bernstein, A.S. (2003) "The Threat to Cone Snails" Science 302: 391.

    See also articles by:

  • Henry Fountain in The New York Times, October 21, 2003;
  • in The BBC News and,
  • the Guest Editorial by Eric Chivian and Aaron S. Bernstein, "Embedded in Nature: Human Health and Biodiversity" in Environmental Health Perspectives 112: (1), A12-A13, January 2004, available at EHP-online, the Journal of The National Institute of Environmental Health Sciences; or [Download PDF].

17 October, 2003

13 October, 2003

    Conotoxins as potential drug leads [REVIEW].:
    Dr. Bruce Livett from the Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia, together with Dr. Zeinab Khalil and Narmatha Satkunanathan from the National Aging Research Institute, Parkville, Victoria, Australia, have combined forces with Diana Alonso from NeuroPharma, Spain, to write this current mini-review on "Drugs from the Sea", in which they focus on the development of conotoxins and other conopeptides as drug leads for the treatment of neuropathic pain and other neurological conditions. Much new information is provided in their Table 1. (Download .pdf file)

    Alonso, D., Khalil, Z., Satkunanathan, N. and Livett, B.G. (2003) Drugs from the sea: conotoxins as drug leads for neuropathic pain and other neurological conditions. Mini Rev Med Chem. 2003 Oct;3(7):785-787
    Abstract: The oceans are a source of a large group of structurally unique natural products that are mainly found in invertebrates such as sponges, tunicates, bryozoans, and molluscs. It is interesting to note that the majority of marine compounds currently in clinical trials or under preclinical evaluation are produced by these species rather than as secondary metabolites by marine algae [1]. Through the combined efforts of marine natural products chemists and pharmacologists a number of promising compounds have been identified that are either already at advanced stages of clinical trials such as the new anti-cancer drug marine alkaloid ecteinascidin 743 [2], or have been selected as promising candidates for extended preclinical evaluation [3]. This is the case for conotoxins, (Table 1) where a number of conopeptides are currently being developed as analgesics for the treatment of neuropathic pain.
    PMID: 14529519 [PubMed - in process]

12 October, 2003

    A delta conotoxin from Conus amadis inhibits inactivation of mammalian sodium channels.:
    Dr. Sudarslal and colleagues from the Molecular Biophysics Unit at the Indian Institute of Science in Bangalore, India, have isolated a 26 residue delta-conotoxin (Am 2766) that inhibits inactivation of mammalian sodium channels.

    Sudarslal S, Majumdar S, Ramasamy P, Dhawan R, Pal PP, Ramaswami M, Lala AK, Sikdar SK, Sarma SP, Krishnan KS and Balaram P. (2003) Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail.
    FEBS Lett. 553:209-212. Abstract: A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na+ channels, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins that have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.

    Omega-conotoxin GVIA potentiates morphine analgesia in mice.:
    Fukuizumi T, Ohkubo T and Kitamura K. (2003) "Spinally delivered N-, P/Q- and L-type Ca2+-channel blockers potentiate morphine analgesia in mice". Life Sci. 73: 2873-2881.
    Abstract: We studied the antinociceptive effects induced at the spinal level by N-, P/Q- and L-type voltage-dependent Ca2+-channel (VDCC) blockers given alone or in combination with morphine, the test responses being the algesic ones induced by acute thermal and mechanical stimuli. When given alone, intrathecal omega-agatoxin IVA (P/Q-type blocker) produced a potent dose-dependent inhibition in the tail-flick and tail-pressure over the dose range 0.33-33 pmol/mouse. Omega-conotoxin GVIA (N-type blocker) also produced dose-dependent inhibitions, but its antinociception against thermal stimuli was weaker than against mechanical stimuli. Calciseptine (L-type blocker) slightly reduced both nociceptive responses, but only at 33 pmol. At their subthreshold doses, intrathecal omega-agatoxin IVA, omega-conotoxin GVIA and calciseptine each significantly enhanced morphine analgesia in the tail-flick and tail-pressure tests, the rank order of potencies being N-> or =P/Q->L-type. These results indicate that combining a low-dose VDCC blocker, especially the N- or P/Q-type, with morphine may be a very useful way of minimizing the dose of morphine and may reduce side effects.

    S0-3, an N-type calcium channel blocker from Conus striatus is a potent non-adictive analgesic:
    Drs Dai and colleagues from the Institute of Biotechnology, Beijing 100071, People's Republic of China have synthesised a an N-type calcium channel blocker from Conus striatus. It proved to be a potent nonaddicitive analgesic in several mouse models of pain.

    Dai Q, Liu F, Zhou Y, Lu B, Yu F and Huang P. (2003) "The synthesis of SO-3, a conopeptide with high analgesic activity derived from Conus striatus. J Nat Prod. 2003 Sep;66(9):1276-9.
    Abstract: The synthesis and characterization of the conopeptide, SO-3, originally derived from Conus striatus is reported. It contains 25 amino acid residues and three disulfide bridges and manifests 72% sequence identity with MVIIA, an N-type Ca(2+) channel inhibitor of high analgesic activity. We evaluated SO-3 in several mouse models of pain. The results indicate that SO-3 is a potent, nonaddictive, analgesic agent.
    [see also entry for 12 March 2003]

    Substitution of charged arginine by tyrosine in mu-conotoxin P-loop reduces binding affinity 300-fold:
    Xue T, Ennis IL, Sato K, French RJ and Li RA (2003) Novel interactions identified between micro-conotoxin and the Na(+) channel domain I P-loop: Implications for toxin-pore binding geometry. Biophys J. 2003 Oct;85(4):2299-2310. [PubMed - in process]
    Abstract: micro-Conotoxins (micro-CTX) are peptides that inhibit Na(+) flux by blocking the Na(+) channel pore. Toxin residue arginine 13 is critical for both high affinity binding and for complete block of the single channel current, prompting the simple conventional view that residue 13 (R13) leads toxin docking by entering the channel along the pore axis. To date, the strongest interactions identified are between micro-CTX and domain II (DII) or DIII pore residues of the rat skeletal muscle (Na(v)1.4) Na(+) channels, but little data is available for the role of the DI P-loop in micro-CTX binding due to the lack of critical determinants identified in this domain. Despite being an essential determinant of isoform-specific tetrodotoxin sensitivity, the DI-Y401C variant had little effect on micro-CTX block. Here we report that the charge-changing substitution Y401K dramatically reduced the micro-CTX affinity (approximately 300-fold). Using mutant cycle analysis, we demonstrate that K401 couples strongly to R13 (DeltaDeltaG > 3.0 kcal/mol) but not R1, K11, or R14 (<<1 kcal/mol). Unlike K401, however, a significant coupling was detected between toxin residue 14 and DI-E403K (DeltaDeltaG = 1.4 kcal/mol for the E403K-Q14D pair). This appears to underlie the ability of DI-E403K channels to discriminate between the GIIIA and GIIIB isoforms of micro-CTX (p < 0.05), whereas Y401K, DII-E758Q, and DIII-D1241K do not. We also identify five additional, novel toxin-channel interactions (>0.75 kcal/mol) in DII (E758-K16, D762-R13, D762-K16, E765-R13, E765-K16). Considered together, these new interactions suggest that the R13 side chain and the bulk of the bound toxin micro-CTX molecule may be significantly tilted with respect to pore axis.

    Like Conus, the genus Terebra has toxins (Augertoxins):
    'Toto' Olivera and colleagues from Department of Biology, University of Utah, Salt Lake City, UT, USA have recently turned their attention from Conus to venomous components of Terebra venom. This is their first report from this genus of marine gastropod.
    Imperial JS, Watkins M, Chen P, Hillyard DR, Cruz LJ and Olivera BM (2003) The augertoxins: biochemical characterization of venom components from the toxoglossate gastropod Terebra subulata. Toxicon. 42: 391-398.
    Abstract: We describe the purification and biochemical characterization of three components from the venom of the toxoglossate gastropod Terebra subulata. The three polypeptide venom components, augertoxins s6a, s7a and s11a, are 40-41AA in length with 3-4 disulfide linkages. The arrangement of Cys residues is reminiscent of certain conopeptide superfamilies, but molecular cloning failed to show the highly conserved sequence features diagnostic of the conopeptide gene superfamily with a similar arrangement of Cys residues. One of the purified peptides, s7a, elicited an uncoordinated twisting syndrome when injected into the nematode Caenorhabditis elegans, but had no effect on mice. T. subulata belongs to the family Terebridae, one of four major groups of toxoglossate gastropods in the superfamily Conacea. The results reveal that some features of the augertoxins and conotoxins are generally similar, such as the organization of prepropeptide precursors and their proteolytic processing into mature toxins; however, Terebra may have evolved generally larger venom components that are less highly post-translationally modified. The results suggest that Conus peptide gene superfamilies probably do not extend to the Terebridae, suggesting that distinctive venom gene superfamilies may be expressed in each major division of Conacean gastropods.

    Residues in the C-terminal half of conotoxin SmIIIA from Conus stercusmuscarum, confer affinity for tetrodotoxin-resistant sodium channels.:
    David Keizer, Ray Norton and colleagues from the NMR Laboratory, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. have teamed up with 'Toto' Olivera and colleagues from Salt Lake City, UT, USA to determine the structural basis for TTX-resistant blocking by the mu-conotoxin SmIIIA.
    Keizer DW, West PJ, Lee EF, Yoshikami D, Olivera BM, Bulaj G and Norton RS. (2003) Structural basis for tetrodotoxin-resistant sodium channel binding by mu-conotoxin SmIIIA. J Biol Chem. 278: 46805-46813.
    Abstract: SmIIIA is a new mu-conotoxin isolated recently from Conus stercusmuscarum. It has several distinctive features, including the absence of hydroxyproline, and is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be characterized. It therefore represents a potentially useful tool to investigate the functional roles of these sodium channels. We have determined the three-dimensional structure of SmIIIA in aqueous solution. The spatial orientations of several conserved Arg and Lys side chains, including Arg14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other mu-conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in mu-conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other mu-conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by Trp15, which is unique to SmIIIA. Arg17, which replaces Lys in the other mu-conotoxins, may also be important. Consistent with these inferences from the structure, assays of two chimeras of SmIIIA and PIIIA in which their N- and C-terminal halves were recombined, indicated that residues in the C-terminal half of SmIIIA confer affinity for tetrodotoxin-resistant sodium channels in the cell bodies of frog sympathetic neurons. SmIIIA and the chimera possessing the C-terminal half of SmIIIA also inhibit tetrodotoxin-resistant sodium channels in the postganglionic axons of sympathetic neurons, as indicated by their inhibition of C-neuron compound action potentials that persist in the presence of tetrodotoxin.

    alpha6beta2* nAChRs are functional and sensitive to alpha-conotoxin MII inhibition.:
    Champtiaux N, Gotti C, Cordero-Erausquin M, David DJ, Przybylski C, Lena C, Clementi F, Moretti M, Rossi FM, Le Novere N, McIntosh JM, Gardier AM and Changeux JP (2003) Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. J Neurosci. 2003 Aug 27;23(21):7820-7829.
    Abstract: Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinson's disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (alpha4beta2*, alpha6beta2*, and alpha4alpha6beta2*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons fromalpha4, alpha6, alpha4alpha6, and beta2 knock-out mice. Our results establish that alpha6beta2* nAChRs are functional and sensitive to alpha-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (nonalpha6)alpha4beta2* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of alpha6beta2* and alpha4beta2* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (nonalpha6)alpha4beta2* nAChRs most likely contribute to nicotine reinforcement.

11 October, 2003

    Subunit-selective alpha-conotoxin from Conus purpurascens.:
    Dowell C, Olivera BM, Garrett JE, Staheli ST, Watkins M, Kuryatov A, Yoshikami D, Lindstrom JM, and McIntosh JM. (2003) Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. J Neurosci. 23: 8445-8452.
    Abstract: Until now, there have been no antagonists to discriminate between heteromeric nicotinic acetylcholine receptors (nAChRs) containing the very closely related alpha6 and alpha3 subunits. nAChRs containing alpha3, alpha4, or alpha6 subunits in combination with beta2, occasionally beta4, and sometimes beta3 or alpha5 subunits, are thought to play important roles in cognitive function, pain perception, and the reinforcing properties of nicotine. We cloned a novel gene from the predatory marine snail Conus purpurascens. The predicted peptide, alpha-conotoxin PIA, potently blocks the chimeric alpha6/alpha3beta2beta3 subunit combination as expressed in oocytes but neither the muscle nor the major neuronal nAChR alpha4beta2. Additionally, this toxin is the first described ligand to discriminate between nAChRs containing alpha6 and alpha3 subunits. Exploiting the unusual intron conservation of conotoxin genes may represent a more general approach for defining conotoxin ligand scaffolds to discriminate among closely related receptor populations.

    Conutulakin-G reduces DNA damage associated with delayed cell death following brain injury .:

    Dr. Williams and colleagues from Walter Reed Army Institute of Research, Silver Spring, MD, USA. have reported a novel effect of the snail peptide contulakin-G (CGX-1007) from Conus geographus upon gene expression associated with delayed cell death as related to a neuroprotective relief of cerebral ischemia.
    Williams, A.J., Ling, G., Berti, R., Moffett, J.R., Yao, C., Lu, X.M., Dave, J.R. and Tortella, F.C. (2003) Treatment with the snail peptide CGX-1007 reduces DNA damage and alters gene expression of c-fos and bcl-2 following focal ischemic brain injury in rats. Exp Brain Res. Exp Brain Res. 153 : 16-26.
    Abstract: Delayed cell death following ischemic brain injury has been linked to alterations in gene expression. In this study we have evaluated the upregulation of several genes associated with delayed cell death ( c-fos, bax, and bcl-2) during the initial 24 h of transient middle cerebral artery occlusion (MCAo) in the rat and the effects of postinjury treatment with the NR2B subunit specific NMDA receptor antagonist CGX-1007 (Conantokin-G, Con-G). C-fos mRNA levels peaked at 1 h postinjury in both cortical and subcortical ischemic brain regions (30-fold increase), remained elevated at 4 h and returned to within normal, preinjury levels 24 h postinjury. The increase in mRNA levels correlated to increased protein expression in the entire ipsilateral hemisphere at 1 h. Regions of necrosis at 4 h were void of C-Fos immunoreactivity with continued upregulation in surrounding regions. At 24 h, loss of C-Fos staining was observed in the injured hemisphere except for sustained increases along the border of the infarct and in the cingulate cortex of vehicle treated rats. CGX-1007 treatment reduced c-fos expression throughout the infarct region by up to 50%. No significant differences were measured in either bcl-2 or bax mRNA expression between treatment groups. However, at 24 h postinjury CGX-1007 treatment was associated with an increase in Bcl-2 immunoreactivity that correlated to a reduction in DNA fragmentation. In conclusion, CGX-1007 effectively attenuated gene expression associated with delayed cell death as related to a neuroprotective relief of cerebral ischemia.

10 October, 2003

    Metabolic Pharmaceuticals to develop ACV1 (Conotoxin Vc1.1) for neuropathic pain.:
    Press release: 30 September, 2003
    Metabolic acquires commercial rights to high potential analgesic compound

    In-licensed for development
    Metabolic (http://www.metabolic.com.au) today announced the in-licensing of an exciting new analgesic(1) compound called ACV1, which was discovered by Associate Professor Bruce Livett and fellow scientists associated with the University of Melbourne. Owing to the success and advanced stage of efficacy testing already performed on the compound, Metabolic will accelerate the commencement of a pre-clinical toxicity program. Metabolic has acquired from the inventors the exclusive worldwide license to commercialise ACV1, in return for milestone and royalty payments. ACV1 is a peptide(2) compound discovered in the venom of the Australian marine cone snail, Conus victoriae, which has been found to have profound analgesic properties.
    Cone snails have evolved a rich cocktail of peptides in their venom, which together act by a variety of mechanisms in the nervous system to quickly immobilize or kill their prey. The potential of cone snail venoms as a source of new therapies has been recognized for many years, and the first such compound to be commercialised is the analgesic Ziconotide being developed by Elan Pharmaceuticals(3). Ziconotide acts by blocking a component of the central nervous system called the N-type calcium channel and must be injected into the spine (directly into the central nervous system) so as to avoid adverse reductions in blood pressure which would otherwise occur.
    ACV1 acts by an entirely novel mechanism, specifically blocking a subtype of a broad class of receptors in the peripheral nervous system called neuronal nicotinic acetylcholine receptors (nAChR). Unlike other cone snail venoms, ACV1 is effective and without apparent adverse effects when administered by convenient routes such as subcutaneous injection(4), providing substantial pain relief in models of nerve pain. Nerve (neuropathic) pain is the category of pain having the greatest need for improved drugs, as discussed below.
    An additional unique feature is that ACV1 also appears from the animal data to accelerate the functional recovery of injured nerves.
    (1) An analgesic is a drug that alleviates pain
    (2) A peptide is a molecule made up of a short string of amino-acid building blocks. Proteins are long strings of amino acids.
    (3) It is uncertain whether Ziconotide will obtain marketing approval from the regulatory authorities.
    (4) A subcutaneous injection is an injection under the skin, as opposed to in the muscle.
    Current therapies for neuropathic pain
    In contrast to acute pain resulting from tissue injury, neuropathic pain results from aberrant activity of damaged nerves. Neuropathic pain typically responds poorly to conventional analgesics such as morphine or aspirin. Current therapy for neuropathic pain relies largely on the ‘off-label’ use of anticonvulsants, antidepressants and local anaesthetics, which have well-documented side-effects and only limited efficacy for this indication.
    ACV1 to be progressed immediately into preclinical toxicity study program
    The inventors of ACV1 have amassed a large amount of efficacy data on the compound in animal models, and independent tests are consistent with their findings. Metabolic has therefore decided that the results are sufficiently conclusive to progress the compound immediately into preclinical toxicity studies. This pushes ACV1 ahead of Metabolic’s other three projects, and becomes the second most advanced project in the Company's development pipeline, behind its AOD9604 obesity drug.
    Manufacturing of batches of ACV1 and preliminary testing is already underway. Formal preclinical toxicity studies are expected to commence in early-2004.
    The first clinical indication for which ACV1 is likely to be targeted is neuropathic pain associated with diabetes, a market with billion dollar annual sales potential.
    Metabolic’s expenditures on ACV1 over the next year have previously been budgeted for and the Company has sufficient cash on hand to advance all of its development projects this financial year.
    Market projections
    Analysts predict that a safe and effective therapy for neuropathic pain would gain immediate acceptance by doctors. With the market for analgesics to treat neuropathic pain estimated at several billion dollars, an effective therapy could potentially reap similar rewards to the blockbuster COX-2-selective anti-inflammatory analgesics - such as Celebrex - which are currently serving the arthritis pain market.
    A paper detailing ACV1 has recently been published:
    Sandall DW et al (2003). A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo.Biochemistry 2003 Jun 10; 42(22):6904-11.
    International Patent Application No. PCT/AU02/00411 filed in 2002 covers the ACV1 compound and the analgesic uses of a broad class of compounds blocking neuronal nAChRs, and has been exclusively licensed to Metabolic. The application has completed the international phase and is entering national phase in key markets. When granted, protection will last until at least 2022.
    About Metabolic
    Metabolic Pharmaceuticals Limited is a biotechnology company based in Melbourne, Australia, and listed on the Australian Stock Exchange (ASX: MBP). The Company’s mission is to develop a pipeline of new pharmaceuticals for world markets, and currently has active programs aimed at treating obesity, pain, type 2 diabetes, osteoporosis and iron overload. AOD9604 for obesity is the company’s most advanced project, currently in Phase 2 human clinical trials. It is a peptide analogue of a fragment of human growth hormone which Metabolic believes has the potential to provide a safer and more effective pharmaceutical treatment for obesity. The drug, discovered by scientists at Monash University, reduces body fat by mimicking natural hormonal regulation of fat metabolism.
    Further details are available from the Company's website - www.metabolic.com.au.
    Contact Information:
    Chris Belyea CEO
    Ph +61 3 9860 5700 chris.belyea@metabolic.com.au
    David Kenley VP Corporate Development
    Ph +61 3 9860 5700 david.kenley@metabolic.com.au
    The inventors
    John Grace, CEO Nextec Biosciences
    Ph 0402 033 205
    (See also article by Graeme O'Neil in Australian Biotechnology News)

    Go to this site to view a collection of 8 differently coloured Conus victoriae (Reeve 1843).

15 June, 2003

    Determinants of alpha3 subunit selectivity for alpha-Conotoxin PnIA:
    Studies by Everhart and colleagues in Salt Lake City, UT, identified three amino acids in the alpha3 subunit of the nicotinic acetylcholine receptor (proline 182, isoleucine 188 and glutamine 198) that were responsible for the 1000-fold greater sensitivity towards conotoxin PnIA by alpha3beta2 receptors over alpha2beta2 receptors.

    Everhart, D., Reiller, E., Mirzoian, A., McIntosh, J. M., Malhotra, A. and Luetje, C. W. (2003) Identification of residues that confer {alpha}-conotoxin- PnIA sensitivity on the {alpha}3 subunit of neuronal nicotinic acetylcholine receptors. J Pharmacol Exp Ther May 6 [Epub ahead of print].
    Abstract: Neuronal nicotinic receptors composed of the alpha3 and beta2 subunits are at least 1000-fold more sensitive to blockade by alpha-conotoxin-PnIA (PnIA) than are alpha2beta2 receptors. A series of chimeric subunits, formed from portions of alpha2 and alpha3, were coexpressed with beta2 in Xenopus oocytes and tested for PnIA sensitivity. We found determinants of PnIA sensitivity to be widely distributed in the extracellular domain of alpha3. Analysis of receptors formed by a series of mutant alpha3 subunits, in which residues that differ between alpha3 and alpha2 were changed from what occurs in alpha3 to what occurs in alpha2, allowed identification of three determinants of PnIA sensitivity: proline 182, isoleucine 188 and glutamine 198. Comparison with determinants of alpha-conotoxin-MII and kappa- bungarotoxin sensitivity on the alpha3 subunit revealed overlapping, but distinct, arrays of determinants for each of these three toxins. When tested against an EC50 concentration of acetylcholine, the IC50 for PnIA blockade was 25+/-4 nM for alpha3beta 2, 84+/-7 nM for alpha3P182Tbeta2, 700 +/-92 nM for alpha3I188Kbeta2 and 870+/- 61 nM for alpha3Q198Pbeta2. To examine the location of these residues within the receptor structure, we generated a homology model of the alpha3beta2 receptor extracellular domain using the structure of the acetylcholine binding protein as a template. All three determinant residues are located on the C-loop of the alpha3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.

14 June, 2003

    European Malacology Meeting, La Rochelle (France):
    Presentations on Conus at the Troisieme Congres International des Societes Europeennes de Malacologie, La Rochelle (France) 24-27 June 2003.

    Friday 27 June:
    Session: Molluscs and Health (Mollusques et sante)

    10:40 LIVETT, B., SATKUNATHAN, N., KHALIL, Z., SANDALL, D., KEAYS, D., DOWN, J. and GAYLER, K : "Alpha-Conotoxin Vc1.1 from Conus victoriae is a potent analgesic in a rat model of human neuropathic pain" - from Dept. Biochemistry and Molecular Biology, and National Ageing Research Institute (NARI), Parkville,Victoria 3010, Australia

    Session: Free communications (oral)

    14:00 RICHARD, G. et SEGUIGNES, M.. : "Statut de Conus nigropunctatus et C. catus de Mer rouge" - from Laboratoire de Biologie et Environnement Marins du Pôle Sciences et Technologies (LBEM), Université de La Rochelle, La Rochelle, France

    Session: Posters 3

    15:00 - 16:00 RICHARD, G. et SEGUIGNES, M. : "Nouvelle approche morphométrique des coquilles du genre Conus" - from Laboratoire de Biologie et Environnement Marins du Pôle Sciences et Technologies (LBEM), Université de La Rochelle, La Rochelle, France

10 June, 2003

    Novel alpha-conotoxin from Conus victoriae is a potent analgesic:
    Bruce Livett and colleagues from the University of Melbourne, have characterized a novel neuronal alpha-conotoxin derived from the venom ducts of a molluscivorous cone, Conus victoriae found off Broome, Western Australia. This 16-amino acid 4:7 loop conotoxin, alpha-Conotoxin Vc1.1, is a competitive inhibitor of the neuronal-type nicotinic acetylcholine receptor and suppresses the activation of sensory nerves. This mechanism of action could contribute to its ability to alleviate pain in an animal model of human peripheral neuropathy (Satkunanathan et al 2003 - in press).

    This study relates to International Patent Application No. PCT/AU02/00411 (WO 02/079236 A1) in the name of Bruce Livett, Zeinab Khalil, Kenwyn Gayler, John Down, David Sandall and David Keays entitled "Alpha Conotoxin Peptides with Analgesic Properties" (See entry for 10 October, 2002, in 'What's New in 2002'). The synthetic compound has been termed ACV1 for its potential commercial development as an analgesic (see report by Ingrid Holmes in Nature Science Update, "Snail toxin could ease chronic pain").

    Sandall, D.W., Satkunanathan, N., Keays, D.A., Polidano, M.A., Liping, X., Pham, V., Down, J.G., Khalil, Z., Livett, B.G. and Gayler, K.R. (2003) "A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo". Biochemistry 42 (22) 6904-6911.
    Abstract: We describe the identification of a conopeptide sequence in venom duct mRNA from Conus victoriae that suppresses a vascular response to pain in the rat. PCR-RACE was used to screen venom duct cDNAs for those transcripts that encode specific antagonists of vertebrate neuronal nicotinic acetylcholine receptors (nAChRs). One of these peptides, Vc1.1, was active as an antagonist of neuronal nAChRs in receptor binding and functional studies in bovine chromaffin cells. It also suppressed the vascular responses to unmyelinated sensory nerve C-fiber activation in rats. Such vascular responses are involved in pain transmission. Furthermore, its ability to suppress C-fiber function was greater than that of MVIIA, an omega-conotoxin with known analgesic activity in rats and humans. Vc1.1 has a high degree of sequence similarity to the alpha-conotoxin family of peptides and has the 4,7 loop structure characteristic of the subfamily of peptides that act on neuronal-type nAChRs. The results suggest that neuronal alpha-conotoxins should be further investigated with respect to their potential to suppress pain.

6 June, 2003

    Images of Live Cones:
    Bob Abela from Guam has posted a few more images of live Conus on his Guam Shell Site. Included are more live Conus aureus as well as a few live Conus magnificus. Scroll down the page about half way to see the live cones. See also links below on 4 April, 2003 to other images of live Conus aureus. Watch for further images from Bob on Conus predation & envenomation.

    Gabriella Raybaudi's contributions:
    Gabriella Massilia Raybaudi is well known in malacological and conchological circles for her collection of quality shells and has recently contributed to the scientific literature with a number of significant scientific publications on Conus ventricosus, most recently the description of a novel conopeptide, Contryphan Vn (see also entry for 3 April, 2003 below). A number of her significant papers on Conus are listed below:

    Massilia G.R., Eliseo T., Grolleau F., Lapied B., Barbier J., Bournaud R., Molgo J., Cicero D.O., Paci M., Eugenia Schinina M., Ascenzi P. and Polticelli F. (2003) "Contryphan-Vn: a modulator of Ca(2+)-dependent K(+) channels." Biochem Biophys Res Commun. 303: 238-246

    Massilia G.R., Schinina, M.E., Ascenzi, P and Polticelli, F. (2002) Conopeptides: Structure and Function. Recent Res. Devel. Biochem. 3: 113-128.
    Massilia G.R., Schinina, M.E., Ascenzi, P and Polticelli, F. (2001)Contryphan-Vn: A novel peptide from the venom of the Mediterranean snail Conus ventricosus. Biochem. Biophys. Res. Commun. 288: 908-913.
    Polticelli, F., Raybaudi Massilia G., and Ascenzi, P (2001) Biochem. Mol. Biol. Edu. 29, 16-20.
    Rolan, E. and Raybaudi Massilia, G. (2002) Evaluation of character state polarity of Conus radular tooth characters. Bolletino Malacologico, Supplemento 4, 175-194.
    Rolan, E. and Raybaudi Massilia, G. (1994a) New investigations on the radular teeth of Conus. Part I. Argonauta. 8(1-6): 6-59.
    Rolan, E. and Raybaudi Massilia, G. (1994b) New investigations on the radular teeth of Conus. Part II. Argonauta.. 8(7-9): 9-68.

    Also of interest is a paper by Polticelli, F., Pascarella, S., Bordo, D., Bolognesi, M., and Ascenzi, P (1999) The T-knot motif revisited. Biol. Chem.. 380: 1247-1250.

3 June, 2003

    alpha-Conotoxins as templates for novel drugs: Review:
    Bob Janes from the School of Biological Sciences, Queen Mary, University of London, U.K. has drawn attention to the high specificity of alpha-conotoxins for their potential to develop drugs to treat diseases involving neuronal nicotinic acetylcholine receptors.
    Janes, R.W. (2003) Nicotinic acetylcholine receptors: alpha-conotoxins as templates for rational drug design. Biochem Soc Trans 31:634-636
    Abstract: Nicotinic acetylcholine receptors (nAChRs) mediate the passage of potassium and sodium ions across synaptic membranes. Two classes of receptors exist: the neuromuscular nAChRs, which mediate signals between nerve and muscle cells, and the neuronal nAChRs, which are found throughout the nervous system. For treatment of diseases involving nAChRs, drugs must be designed with a high level of selectivity towards only one of these classes or subclasses (in the case of neuronal receptors). alpha-Conotoxins, small polypeptides isolated from the venoms of marine snails, represent molecules with just this type of selectivity, with specificity even towards certain subclasses of nAChRs. The availability of high-resolution crystal structures of alpha-conotoxins provides the opportunity to examine the structural features that orchestrate their preferential blocking action. In the present study of a neuromuscular- and a neuronal-specific alpha-conotoxin, SI and EpI respectively, important and significant differences can be seen in the shapes of the molecules, which must reflect topological features of the different types of target receptor subunits. These then provide a template for computational docking studies with the homologous acetylcholine-binding protein, whose structure is known, so drug analogues of the naturally occurring toxins can be developed with the desired specificities.

    Propeptide domain of conotoxin TxVI enhances export from the ER:
    Mike Fainzilber and colleages from the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel have shown that the pro-domain in the hydrophobic conotoxin TxVI precursor from Conus textile acts as a tag that can facilitate its export from the endoplasmic reticulum (ER) by a "hitchhiking" mechanism.
    Conticello SG, Kowalsman ND, Jacobsen C, Yudkovsky G, Sato K, Elazar Z, Petersen CM, Aronheim A, Fainzilber M. (2003) The pro-domain of a secreted hydrophobic mini-protein facilitates its export from the endoplasmic reticulum by hitchhiking on sorting receptors. J Biol Chem 278: 26311-26314.
    Abstract: Misfolded secretory proteins are retained in the endoplasmic reticulum (ER) by quality-control mechanisms targeted to exposed hydrophobic surfaces. Paradoxically, certain conotoxins expose extensive hydrophobic surfaces upon folding to their bioactive structures. How then can such secreted mini-proteins traverse the secretory pathway? Here we show that secretion of the hydrophobic conotoxin-TxVI is strongly dependent on its propeptide domain, which enhances TxVI export from the ER. The propeptide domain interacts with sorting receptors from the sortilin Vps10p domain family. The sortilin-TxVI interaction occurs in the ER, and sortilin facilitates export of TxVI from the ER to the Golgi. Thus, the pro-domain in a secreted hydrophobic protein acts as a tag that can facilitate its ER export by a hitchhiking mechanism.

    3D structure of psi-Conotoxin Piiif from Conus purpurascens:
    'Toto' Olivera and colleagues from the Departments of Medicinal Chemistry and Biology, University of Utah, Salt Lake City, Utah, USA, have compared the structure and activities of two closely related conotoxins from Conus purpurascens, psi-conotoxins Piiif and Piiie that block nicotinic acetylcholine receptors, and have shown that it is the third loop in both these conopeptides, which contains no charged residues, that is the prime determinant of potency in these psi-conotoxins.
    Van Wagoner, R.M., Jacobsen, R.B., Olivera, B.M. and Ireland C.M. (2003) Characterization and Three-Dimensional Structure Determination of psi-Conotoxin Piiif, a Novel Noncompetitive Antagonist of Nicotinic Acetylcholine receptors(,). Biochemistry 42: 6353-6362
    Abstract: A novel inhibitor of nicotinic acetylcholine receptors (nAChRs), psi-conotoxin Piiif, was isolated from the venom of Conus purpurascens and found to have the sequence GOOCCLYGSCROFOGCYNALCCRK-nh2. The sequence is highly homologous to that of psi-conotoxin Piiie, a previously identified noncompetitive inhibitor of Torpedo electroplax nAChR, also isolated from C. purpurascens. Both psi-conotoxins block Torpedo and mouse nicotinic acetylcholine receptors (nAChRs), but psi-Piiif is less potent by a factor of 10(1)-10(2). A high-resolution structure of psi-Piiif was determined by NMR and molecular modeling calculations. psi-Piiif analogues containing [(13)C]-labeled cysteine at selected positions were synthesized to resolve spectral overlap of Cys side chain proton signals. The structures are well-converged, with backbone atom position RMSDs of 0.21 A for the main body of the peptide between residues 4 and 22 and 0.47 A for all residues. The overall backbone conformation is closely similar to psi-Piiie, the main difference being in the degree of conformational disorder at the two termini. psi-Piiie and psi-Piiif have similar locations of positive charge density, although psi-Piiif has a lower overall charge. One disulfide bridge of psi-Piiif appears to undergo dynamic conformational fluctuations based on both the model and on experimental observation. Chimeras in which the three intercysteine loops were swapped between psi-Piiie and psi-Piiif were tested for inhibitory activity against Torpedo nAChRs. The third loop, which contains no charged residues in either peptide, is the prime determinant of potency in these psi-conotoxins.

    Structure of psi-Conotoxin Piiie from Conus purpurascens:
    Drs Wagoner and Ireland from the Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah, USA have used (13)C-labeling of selected cysteine residues to provide an improved solution structure for psi-Conotoxin Piie.
    Van Wagoner, R.M. and Ireland, C.M. (2003) An Improved Solution Structure for psi-Conotoxin Piiie Biochemistry 42:6347-6352.
    Abstract: A revised, high-resolution structure of psi-conotoxin Piiie (psi-Piiie), a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR), produced through the use of NMR and molecular modeling calculations is presented. The original structures of psi-Piiie had a relatively high degree of disorder, particularly in the conformation of the disulfide bridges. Our studies utilized (13)C-labeling of selected cysteine residues allowing the resolution of all problems of resonance overlap for the cysteine residues. The improved data were used to produce a new set of structures by a molecular modeling process incorporating relaxation matrix methods for the determination of interproton distance restraints and a combination of distance geometry and simulated annealing for structure generation. The structures produced are very well converged with the RMSD of backbone atom positions of the main body of the peptide improving from 0.73 to 0.13 A. Other indicators of correlation with the experimental data and quality of covalent geometry showed significant improvement in the new structures. The overall conformation of the peptide backbone is similar between the two determinations with the exception of the N-terminus. This difference leads to a significant effect on the predicted distribution of positive charge within psi-Piiie, a property likely to influence interpretation of future mutational studies.

    mu-Conotoxins: Mechanism of block of sodium channels.:
    Drs Li and colleagues from the Institute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, Maryland, USA have shown that mu-CTX block of Na+ channels depends critically on but not specifically on [Na+], contrasting with the known behavior of pore-blocking K+ channel toxins. Li, R.A., Hui, K., French, R.J., Sato, K., Henrikson, C.A., Tomaselli, G.F. and Marban, E. (2003) Dependence of {mu}-conotoxin block of sodium channels on ionic strength but not the permeant [Na+]: Implications into the distinctive mechanistic actions of Na and K channel pore-blocking toxins and their molecular targets. J Biol Chem 2003 May 21; [epub ahead of print]
    Abstract: mu-Conotoxins (mu-CTX) are Na+ channel blocking, 22-amino-acid peptides produced by the sea snail Conus geographus. Although K+ channel pore-blocking toxins show specific interactions with permeant ions and strong dependence on the ionic strength (), no such dependence has been reported for mu-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of mu-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of and permeant ion concentration ([Na+]) on mu-CTX block of rat skeletal muscle (1, Nav1.4) Na+ channels. mu-CTX sensitivity of wild-type (WT) and E758Q channels increased significantly (by ~20-fold) when was lowered by substituting external Na+ with equimolar sucrose (140 35 mM Na+); however, toxin block was unaltered (p>0.05) when was maintained by replacement of [Na+] with N-methyl-D-glucamine (NMG+), suggesting that the enhanced sensitivity at low was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, kon, as the primary determinant of the changes in affinity (kon increased 40- and 333-fold for mu-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200mM Na+ was reduced to 50mM). In contrast, dissociation rates changed <2-fold for the same derivatives under the same conditions. Experiments with additional mu-CTX derivatives identified toxin residues R1, R13 and K16 as important contributors to the sensitivity to external. Taken together, our findings indicate that mu-CTX block of Na+ channels depends critically on but not specifically on [Na+], contrasting with the known behavior of pore-blocking K+ channel toxins. These findings suggest that different degrees of ion interaction underlying the fundamental conduction mechanisms of Na+ and K+ channels, are mirrored in ion interactions with pore-blocking toxins.

    A cone snail protease shows homology to CRISP proteins:
    Richard Lewis and colleagues from the Institute for Molecular Bioscience, Brisbane, Queensland, Australia have used structural homology modelling and BLAsearches on the protease Tex31 from the venom duct of Conus textile to show it is a member of the PR protein superfamily of pathogenesis related (PR) proteins. Tex31 requires four residues including aST leucine N-terminal of the cleavage site for efficient substrate processing.
    Milne, T.J., Abbenante, G., Tyndall, J.D., Halliday, J. and Lewis, R.J. (2003) Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily. J Biol Chem 2003 May 20; [epub ahead of print]
    Abstract: The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant and fungal kingdoms, and is implicated in human brain tumour growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterisation, cloning and structural homology modelling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to > 95% purity by activity-guided fractionation using a p-nitroanilide substrate based on the putative cleavage-site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins which have a cysteine-rich C-terminal tail. An homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

    Conantokin binding to the NMDA receptor:
    Dr. Catellino and colleagues from Department of Chemistry and Biochemistry and the W.M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN, USA, have demonstrated direct binding of a radiolabeled conantokin to the NMDA receptors present in rat brain.
    Klein, R.C., Prorok, M. and Castellino, F.J. (2003) Direct binding properties of conantokins to native N-methyl-d-aspartate receptors. J Pept Res 61:307-317
    Abstract: Conantokin-G (con-G) is a small, gamma-carboxyglutamic acid (Gla)-containing peptide that functions neurophysiologically by inhibiting the N-methyl-d-aspartate receptor (NMDAR). In the current study, the receptor binding properties of an alanine-rich, Gla-deficient con-G variant, Ala-con-G, were assessed following tracer radioiodination with 125I. Direct binding experiments with [125I]Ala-con-G yielded a single site defined by a Kd value of 516 +/- 120 nm. Displacement of [125I]Ala-con-G binding by Ala-con-G resulted in 100% displacement with an IC50 value of 564 +/- 33 nm, while heterologous displacement by con-G[S16Y], con-G, con-T, and con-R[1-17] yielded IC50 values in the range of 15-45 &mgr;m. No displacement was observed with d-gamma-con-G or con-G[L5A], analogs that are inactive at NMDARs. Specific [125I]Ala-con-G binding was displaced by NMDA and 2-amino-5-phosphopentanoic acid in a dose-dependent manner, suggesting an interaction at the glutamate binding site. The direct binding of [125I]Ala-con-G to adult rat brain sections revealed an anatomical distribution of binding sites in all regions known to contain the NR2B subunit of the NMDAR. These results constitute the only known demonstration of the direct binding of a radiolabeled conantokin to the NMDARs present in rat brain membrane preparations and rat brain sections, and suggest that radiolabeled Ala-con-G, and similar conantokin derivatives, may find utility as probes of NMDARs in a variety of systems.

25 May, 2003

    Specificity determinants of a-conotoxin PnIA from Conus penaceus :
    Hogg RC, Hopping G, Alewood PF, Adams DJ, Bertrand D. (2003) alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha 7-L247T nicotinic acetylcholine receptor.
    J Biol Chem. 278: 26908-26914
    Abstract: The effects of the native alpha-conotoxin PnIA, its synthetic derivative [A10L]PnIA and alanine scan derivatives of [A10L]PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nAChRs expressed in Xenopus oocytes. PnIA and [A10L]PnIA inhibited ACh-activated currents at wtalpha7 receptors with IC50s of 349 nM and 168 nM respectively. Rates of onset of inhibition were similar for PnIA and [A10L]PnIA, however, the rate of recovery was slower for [A10L]PnIA, indicating that the increased potency of [A10L]PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [A10L]PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [A10L]PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nM. In contrast, [A10L]PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [A10L]PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas, [A10L]PnIA stabilized a state which is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

    American Malacological Society, June 2003 :
    The following presentations on cone snails: conotoxins are to be presented at the American Malacological Society meeting Thursday June 26, 2003

    Plenary Session, Symposium I – Diversification In The Sea

    Plenary Speaker, Baldomero M. Olivera
    "Venomous Fish-hunting Cone Snails From the Biochemistry and Genetics of Toxins, to Behavior and Natural History"

    Thomas F. Duda, Jr.
    "Conotoxin Diversity among Populations of C. ebraeus"

    Alan J. Kohn & Thomas F. Duda, Jr.
    "Does a Cryptic Sibling Species Lurk within C. ebraeus?"

    - see Abstracts below - American Malacological Society, Ann Arbor 2003

    Venomous Fish-hunting Cone Snails From the Biochemistry and Genetics of Toxins, to Behavior and Natural History.
    Baldomero M. Olivera

    Department of Biology, University of Utah, Salt Lake City
    Utah, 84112, USA, olivera@biology.utah.edu
    Abstract: All cone snails can potentially express 100-200 different peptidic toxins in their venoms to capture prey, defend against predators and interact with competitors. Of the ca. 500 different cone snails, approximately 70 species are primarily fish-hunting. Both molecular data and a biochemical analysis of venoms suggest that fish-hunting Conus species fall into four distinct groups. Some venom components are relatively conserved among all of the groups of fish-hunting cone snails, while other venom components are innovations characteristic of particular groups of species, or individual species. Thus, the biochemistry and genetics of venom components gives insights into both the evolution of Conus species, as well as of toxin genes. Diverse strategies for prey capture have evolved in fish-hunting cone snail species. A correlation can be made between the biochemical and pharmacologic al characteristics of toxins expressed in the venom of a snail, and the species-specific behavior associated with prey capture. Thus, understanding individual venom components at the biochemical and genetic level should provide useful insight into the natural history and phylogeny of a Conus species. An integrated overview of the present state of our understanding of the fish-hunting cone snails will be presented.

    Conotoxin Diversity among Populations of C. ebraeus.
    Thomas F. Duda, Jr.
    Calle del Campesino #14, Colonia Obrero Campesina, Xalapa, Veracruz, C.P. 91020, Mexico, tfduda@yahoo.com
    Abstract: Conus ebraeus is one of the most widely distributed species of the genus Conus, with a range encompassing tropical regions of the Indian and Pacific Oceans. Moreover, C. ebraeus shows slight differences in diet among populations. Analyses of conotoxin mRNA transcripts of C. ebraeus demonstrate that this species does not express orthologous counterparts of loci expressed by a close relative, C. abbreviatus. These studies also reveal that allelic diversity of a conotoxin locus, locus E1, is quite high and that diversifying selection has operated among these alleles. Comparisons of expressed conotoxin transcripts among populations of C. ebraeus from Okinawa, Guam, Samoa, Hawaii and Panamá show that allele frequencies of conotoxin locus E1 differ among populations and that a second locus, E2,is differentially expressed among both populations and individuals. These findings suggest that differences in conotoxin allele frequencies among populations, strong diversifying selection operating among alleles of conotoxin loci and differential expression of conotoxin loci among species, populations and individuals are three additional factors that contribute to differences in venom composition of Conus.

    Does a Cryptic Sibling Species Lurk within C. ebraeus ? Alan J. Kohn, Department of Biology, University of Washington, Seattle, WA 98195 USA, kohn@u.washington.edu, and Thomas F. Duda, Jr., Calle del Campesino #14, Colonia Obrero Campesina, Xalapa, Veracruz, C.P. 91020, Mexico
    Abstract: Conus ebraeus, the most widely distributed species in its genus, extends from the Red Sea throughout the Indo-Pacific region, and across the East Pacific Barrier to Central America. Nevertheless, current studies suggest genetic uniformity throughout its range (average 16S rDNA sequence differences 0.2%). Some specimens apparently of C. ebraeus from Okinawa were observed to diverge markedly, however (mean sequence difference 3.6%), a level commonly associated with different but congeneric species. This suggested a distinct sympatric species present among specimens with shells that may be indistinguishable from C. ebraeus. Radular tooth morphology of the putative sibling species differed markedly from C. ebraeus, The tooth of the former is much larger relative to shell size and has a barb and row of denticles lacking in C. ebraeus. These features conform to C. judaeus Bergh, 1895 from Philippines, originally distinguished from C. ebraeus solely on radular characters. We conclude that C. judaeus is a valid species. We have begun to determine the geographic distribution of C. judaeus and to quantify radular tooth and ecological differences between the two species. To date, we have found C. judaeus only in Okinawa and Seychelles, sympatrically with C. ebraeus in both places. All specimens examined genetically and morphologically from Panama, Hawaii, Samoa, Marshall Islands, Papua New Guinea, and Chagos Islands were C. ebraeus. The diets in nature of the two species differ in both Okinawa and Seychelles; both prey exclusively on polychaetes, but C. ebraeus eats mainly nereids and eunicids, while C. judaeus eats mainly capitellids.

5 May, 2003

9 April, 2003

5 April, 2003

    Conus peptides and neuroprotection:
    Christine Heading from Department of Biological Sciences, The Open University, Milton Keynes, UK, (and who previously wrote an extensive review on the clinical use of Ziconotide as an analgesic, entitled: "Ziconotide, Neurex Corp". Current Opinion in CPNS Investigational Drugs 1: 153-166 (1999), has now reviewed the action of Conus peptides in neuroprotection.

    Heading CE. (2002) "Conus peptides and neuroprotection." Curr Opin Investig Drugs 3 : 915-920.
    Abstract: Conus peptides comprise a diverse class of bioactive molecules, each of which is believed to have a biological target. From the 50,000 plus molecules thought to exist, several groups are judged to have potential neuroprotective activity. Those that block voltage-gated calcium channels were seen as most promising, but this is now in some doubt. Those that act at NMDA receptors may be able to protect against acute and chronic assault, while those acting at nicotinic receptors or at potassium channels seem mainly to have potential against chronic assault. The poor pharmacokinetic profile of the peptides remains a challenge.

4 April, 2003

3 April, 2003

    Conotoxin interaction with calcium channels:
    Dr. Feng and colleagues from the Dept. of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, CANADA, have examined the ability of omega-conotoxins GVIA from Conus geographus and MVIIA from Conus magus to interact with voltage gated calcium channels. They found that the overall architecture of the MVIIA site is conserved in all types of high voltage activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Based on their observations, they propose a model in which N-type calcium channels can form both reversible and irreversible complexes with conotoxin MVIIA.

    Feng ZP, Doering CJ, Winkfein RJ, Beedle AM, Spafford JD, Zamponi GW. (2003) "Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA." J Biol Chem 2003 Mar 24; [epub ahead of print]
    Abstract: The Conus magus peptide toxin w-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF hand motif in domain IIIS5-H5 region alters block by both w-conotoxin GVIA and MVIIA (Feng et al., 2001, JBC 276:15728), the introduction of five point mutations known to affect GVIA block (and located downstream of the EF hand) affected MVIIA block to a smaller degree compared to GVIA. These data suggest that despite some overlap, MVIIA and GVIA block do not share identical channel structural determinants. At higher concentrations (~3 mM), MVIIA reversibly blocked, L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of wash out was highly variable, ranging from little or no recovery to 55% recovery over a three-minute wash period, and appeared to weakly depend on the holding potential applied during toxin application. Based on these observations, we propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.

    Mediterranean cone yields first functional target for Lys-Trp dyad-containing Contryphan peptide, Contryphan-Vn:
    Dr. Gabriella Raybaudi Massilia and colleagues from the Department of Biology, University 'Roma Tre,' Viale Guglielmo Marconi 446, I-00146, Rome, Italy, have isolated a conopeptide from the Mediterranean cone snail, Conus ventricosus (also known popularly as Conus mediterraneus), that modulates the activity of Ca(2+)-dependent K(+) channels. Contryphan-Vn, as it has been termed, affects both voltage-gated and Ca(2+)-dependent K(+) channel activities in vertebrates and invertebrates.

    Raybaudi Massilia G., Eliseo T., Grolleau F., Lapied B., Barbier J., Bournaud R., Molgo J., Cicero D.O., Paci M., Eugenia Schinina M., Ascenzi P. and Polticelli F. (2003) "Contryphan-Vn: a modulator of Ca(2+)-dependent K(+) channels." Biochem Biophys Res Commun. 303: 238-246
    Abstract:Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.

2 April, 2003

    New Cone Images from Senegal:
    Giancarlo Paganelli has updated his Cone Shells collection (currently currently 687 images), with the following 6 images of cones from Senegal:
    Conus genuanus - mercator f. belairensis LINNAEUS, 1758, Senegal, M'Bour - 60.3 mm; Conus mercator belairensis PIN & TACK, 1989, Senegal, Dakar - 19.7 mm; Conus pulcher LIGHTFOOT, 1786, Senegal, La Somone - 51.8 mm; Conus pulcher f. byssinus RÖDING, 1798, Senegal, Popenguine - 40.0 mm Conus pulcher f. canariensis VAN ROSSUM, 1997, Senegal, Popenguine - 46.9 mm and Conus pulcher f. prometheus HWASS in BRUGUIÈRE, 1792, Senegal, La Somone - 67.5 mm

    Lipophilic analogues of alpha-conotoxin MII:
    Blanchfield JT, Dutton JL, Hogg RC, Gallagher OP, Craik DJ, Jones A, Adams DJ, Lewis RJ, Alewood PF, Toth I. (2003) Synthesis, structure elucidation, in vitro biological activity, toxicity, and Caco-2 cell permeability of lipophilic analogues of alpha-conotoxin MII. J Med Chem. 2003 Mar 27;46(7):1266-72.
    School of Pharmacy, Institute for Molecular Bioscience, and School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Australia.
    Abstract:The alpha-conotoxin MII is a two disulfide bridge containing, 16 amino acid long peptide toxin isolated from the marine snail Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors (nAChRs) of the subtype alpha3beta2. To improve the bioavailability of this peptide, two lipidic analogues of MII have been synthesized, the first by coupling 2-amino-d,l-dodecanoic acid (Laa) to the N terminus (LaaMII) and the second by replacing Asn5 in the MII sequence with this lipoamino acid (5LaaMII). Both lipidic linear peptides were then oxidized under standard conditions. (1)H NMR shift analysis of these peptides and comparison with the native MII peptide showed that the tertiary structure of the N-conjugated analogue, LaaMII, was consistent with that of the native conotoxin, whereas the 5LaaMII analogue formed the correct disulfide bridges but failed to adopt the native helical tertiary structure. The N terminus conjugate was also found to inhibit nAChRs of the subtype alpha3beta2 with equal potency to the parent peptide, whereas the 5LaaMII analogue showed no inhibitory activity. The active LaaMII analogue was found to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII, and both peptides showed negligible toxicity.

12 March, 2003

    Folding of delta Conotoxin PVIA from Conus purpurascens:
    Dr. DeLa Cruz and colleagues from the Department of Biology, University of Utah, Salt Lake City , Utah, USA have shown that detergent can aid in the oxidative folding of delta-conotoxins.

    DeLa Cruz R, Buczek O, Bulaj G, Whitby FG. (2003). Detergent-assisted oxidative folding of delta-conotoxins. J Pept Res 61: 202-212
    Abstract: Conotoxins comprise a diverse group of disulfide-rich peptides found in venoms of predatory Conus species. The native conformation of these peptides is marginally stable in comparison with alternative conformations, often resulting in low folding yields. The oxidative folding of hydrophobic delta-conotoxins was found to produce less than 1% of the native peptide [Bulaj, G. et al. (2001) Biochemistry 40, 13201]. In order to identify factors that might improve folding yields, we screened a number of additives including water-soluble polymers, detergents and osmolytes for their ability to increase steady-state accumulation of the native delta-conotoxin PVIA. The presence of a non-ionic detergent Tween and low temperature appeared to be the most effective factors in improving the oxidative folding. The detergent was also effective in promoting folding of other hydrophobic delta-conotoxins. Based on our findings, we discuss a possible mechanism for detergent-assisted folding and the general applicability of this mechanism to facilitating the proper folding of hydrophobic, cysteine-rich peptides.

    Action of a sulphated O-superfamily contoxin in the brain:
    Dr. Li and colleagues from the Department of Biological Science and Technology, Tsinghua University, Beijing, China have investigated the effect of a sulphated conotoxin from a new O-superfamily on Na and K currents in the brain.
    [see also entry for 12 October, 2003]

    Li Z, He XP, Xie ZP, Dai QY, Huang PT. (2003) Effect of new O-superfamily conotoxin SO3 on sodium and potassium currents of cultured hippocampal neurons. Brain Res 965: 155-158.
    Abstract: The effects of a new O-superfamily conotoxin SO3 on sodium and potassium currents were examined in cultured rat hippocampal neurons using the whole-cell patch clamp technique. SO3 caused a concentration-dependent, rapidly developing and reversible inhibition of sodium currents (I(Na)). The IC(50) value for the blockage of I(Na) was calculated to be 0.49 and the Hill coefficient was 1.7. Using electrophysiological and pharmacological protocols, transient A-type potassium currents (I(A)) and delayed rectifiers potassium currents (I(K)) were isolated. SO3 caused a concentration-dependent, and reversible inhibition of I(K). The IC(50) value for the blockage of I(K) was calculated to be 1.6 and the Hill coefficient was 0.6, with no significant effect on I(A). These results indicate that SO3 can selectively inhibit neuronal sodium and potassium currents.

9 March, 2003

    Conus victoriae yields its secrets:
    This photo montage depicts the fate of a Conus victoriae from Broome, Australia, which was kept in our marine aquarium in Melbourne before processing to remove its venom duct and extract its venom for conotoxins. The photos were taken during a recent visit of Meng Shyan Choy, from Biochemistry Department at NUS. He used a Canon PowerShot G2 with a 4 mega pixels CCD sensors. Only low resolution images are shown here.
    The sequence of the photos shows

    • (1) Dr. Livett examining two Conus victoriae and a Conus novohollandae placed on the grid above the marine aquarium tank;
    • (2) a Conus victoriae (facing) and a Conus novohollandae (on the RHS);
    • (3) a Conus victoriae (side on. Note the markings on the foot) and a Conus novohollandae (from the rear);
    • (4) the same Conus victoriae as in (3) but removed ready for extraction. This cone had previously envenomated and eaten the body of another Conus victoriae some 2-4 hours earlier and during transport to the laboratory it regurgitated the body whole (seen to the left);
    • (5) all good things must come to an end. The shell is gently cracked in a controlled manner using a plumber's tube vice;
    • (6) the body of the cone (above) is carefully removed from the shell and venom duct and bulb are dissected out. In Conus victoriae this can be up to 36 cm in length;
    • (7) the venom is removed and spread on a glass plate to dry (for less than 10 min). Pieces of the broken shell are seen to the right;
    • (8) the dried venom is scraped from the glass surface using a scalpel blade and transferred as a flaky powder to an Ependorf tube for storage;
    • (9) David Sandall (PhD student) and Meng Shyan Choy (visiting researcher from NUS, Singapore) look pleased with their efforts; and
    • (10) Dr. Bruce Livett (bending down) and Dr. John Down (seated at the computer), prepare to write the next paper (with Prof. Bill Sawyer looking on).

8 March, 2003

    Update on clinical trials of Ziconotide:
    Dorene Taqi and colleagues from the Pain Evaluation & Treatment Center, Tulsa, OK have reported at the 18th Annual AAPM meeting on the effects of ziconotide administered to patients with chronic severe pain.

    Dorene Taqi, Ian Gunyea, PA-C, Bhadresh Bhakta, MD, Venkatesh Movva, MD, Sameh Ward,MD, Mike Jenson, PA-C, CPP, Mike Royal, MD (2002) Intrathecal Ziconotide Effect on neuropathic symptoms and potential for adjunctive use with opiates: A retrospective review in 25 patients. Pain Medicine 3 (2), 180-181.
    Abstract: An ongoing, open-label, multicenter clinical trial to assess the long-term safety and tolerability of intrathecally (IT) administered ziconotide to patients with chronic, severe pain was initiated in November, 1998. As part of this study, we evaluated 52 patients with chronic intractable pain on IT ziconotide. Ziconotide is an N-type, voltage-sensitive calcium channel blocker with proven efficacy as an IT analgesic. In this trial, IT ziconotide was initiated at a dose of 0.1mcg/hr, followed by long-term ziconotide infusion with titrations as needed within study parameters. Efficacy was assessed using a visual analog scale. Of our 52 patients, 25 had primarily neuropathic pain symptoms and were on ziconotide for at least 3 months. Following the change in IT therapy, 25 patients were queried during the course of their treatment with ziconotide as to its effect on their neuropathic symptoms (dysesthesia, allodynia, paresthesia, radiculopathic symptoms etc.) and queried after discontinuing ziconotide, which was primarily due to adverse events from uptitration to cover any nociceptive pain component, on the relative effectiveness of their current IT opiate to IT ziconotide on their neuropathic symptoms and on their willingness to go back on lower dose ziconotide with their present IT opiate in an attempt to more effectively manage neuropathic symptoms. Most of the patients (18/25) observed that the neuropathic symptoms improved at lower doses of ziconotide, but when uptitration of dose was done to cover more of their pain, side effects occurred causing eventual discontinuation. These patients also observed that their IT opiate was less effective on these same neuropathic symptoms. Twenty-one of the 25 patients (84%) said they would like to try ziconotide with their opiate and 18 (72%) of them were quite sure that their neuropathic symptoms improved dramatically with the ziconotide at a dose much lower than their termination dose. Studies of low dose IT ziconotide with morphine (and other opiates) are needed to determine if the risk-benefit of ziconotide-based treatment can be enhanced with IT drug combinations.

Elan reports on unintentional overdose with Ziconotide:
Dr. Steven Charapata from Research Medical Center, Kansas City, MO, and Dr. David Ellis from Elan Pharmaceuticals, South San Francisco, CA, USA, present case reports of unintentional overdose in six chronic pain patients treated with IT ziconotide. These unintentional overdoses were attributable to pump programming or dilution errors; none were lethal. One patient received 300-fold the current recommended initial dose rate of 0.1 mcg/hr. Unlike an unintentional overdose with IT morphine, which slows respiration and could potentially lead to hypoxia, coma or death; ziconotide does not produce respiratory depression. No tolerance to the analgesic effect of ziconotide, or withdrawal symptoms after discontinuation of the drug have been reported. It is concluded that Ziconotide has a wide margin of safety as an IT analgesic.

Steven G. Charapata and David Ellis (2002) Unintentional overdose with intrathecal ziconotide.Pain Medicine 3:(2) 189-190.
Abstract:Ziconotide is a novel, N-type, voltage-sensitive calcium channel (VSCC) blocker, with well-documented efficacy as an intrathecal (IT) analgesic. Ziconotide has been administered to over 1000 chronic pain patients in nine clinical trials. Over 350 patients have been on ziconotide IT therapy for more than three months in a long-term safety and tolerability study. Common adverse events for ziconotide include dizziness, nausea, nystagmus, abnormal gait, constipation, urinary retention, somnolence, postural hypotension, vomiting, confusion and abnormal vision. Ziconotide adverse events are recognizable, reversible and manageable, by dose adjustment and slow dose titration. Case reports of unintentional overdose in six chronic pain patients treated with IT ziconotide are presented. These unintentional overdoses were attributable to pump programming or dilution errors; none were lethal. The patient who received the highest overdose was administered 31 mcg/hr over 24 hours, or nearly 750 mcg ziconotide, total. This hourly dose rate is 300-fold the current recommended initial dose rate of 0.1 mcg/hr. This patient was sedated, but arousable; vital signs were stable and patient had no change in blood pressure. His symptoms resolved within 24 hours. His Visual Analog Score of Pain Intensity (VASPI) was reduced from 82 at baseline to 2.5 at the end of the titration period. The patient elected to continue in the long-term IT ziconotide study. The other 5 cases of inadvertent overdose were less severe, with dose rate at 5 mcg/hr or less. Associated adverse events also resolved within 24-hours of discontinuing ziconotide infusion. Unlike an unintentional overdose with IT morphine, which slows respiration and could potentially lead to hypoxia, coma or death; ziconotide does not produce respiratory depression. No tolerance to the analgesic effect of ziconotide, or withdrawal symptoms after discontinuation of the drug have been reported. Ziconotide has a wide margin of safety as an IT analgesic.

28 February, 2003

    Cone shells from the French Caribbean Islands:
    Guy Boltz maintains a web site on French Caribbean Islands Seashells in which he has a very nice display of Conus burryae Clench 1942, from Martinique. At this site you can also download a shareware version of a program that he is developing as an interactive Cd-Rom of the "Seashells of the French West-Indies" with more than 330 species of shells. Guy would welcome your feedback.

23 February, 2003

    Cones from the Americas:
    Giancarlo Paganelli has updated his Cone Shells collection (currently currently 681 images), with the following 6 images of cones from the Americas:
    Conus archon BRODERIP, 1833 Panama, Canal de Afuera - 38.1 mm; Conus arcuatus BRODERIP & SOWERBY, 1829 Panama, Canal de Afuera - 38.1 mm; Conus atractus TOMLIN, 1937 Brazil, Vitoria, Espiritu Santo - 60.1 mm; Conus baccatus SOWERBY III, 1876 Panama, Canal de Afuera - 20.8 mm; Conus kalafuti DA MOTTA, 1986 Honduras, Roatan Is. - 13.0 mm; and Conus mindanus agassizii DALL, 1889 Brazil, Santos - 36.8 mm

    New addition to Cones on Stamps:
    Tom Walker has notified me of a new addition for the Cones on Stamps. Guinea-Bissau isued a beautiful set showing shells in 2002, one of which was Conus genuanus. I have added this to the web site.

    There are one or two amendments to the Cones on Stamps site as a result of Tom discussing some of the stamps with Guido and his colleagues. Guido tells us that that all the stamps from ANGOLA, BURKINO FASO and SOUTH WEST AFRICA stamps show portions of Conus pulcher as this shell is the "routine" cone used for jewellery in this area of Africa. Accordingly, I have moved these images from Conus sp. to Conus pulcher. Tom now has his complete collection of Shells on Stamps in a searchable database on Guido's site. There are over 5080 stamps figured, 1600 details highlighted. You can search by country, by theme, family, species etc... Each stamps is documented (when you click on it). A magnificent maintained resource.

21 February, 2003

    New O-superfamily of Conotoxins:
    Dr. Li and colleagues from the Department of Biological Science and Technology, Tsinghua University, 100084, Beijing, China have reported on the effects of a new O-superfamily conotoxin SO3 which blocks neuronal sodium and potassium channels.
    Li, Z., He, X.P., Xie, Z.P., Dai, Q.Y. and Huang, P.T. (2003) "Effect of new O-superfamily conotoxin SO3 on sodium and potassium currents of cultured hippocampal neurons." Brain Res. 7:965(1-2):155-8.
    The effects of a new O-superfamily conotoxin SO3 on sodium and potassium currents were examined in cultured rat hippocampal neurons using the whole-cell patch clamp technique. SO3 caused a concentration-dependent, rapidly developing and reversible inhibition of sodium currents (I(Na)). The IC(50) value for the blockage of I(Na) was calculated to be 0.49 and the Hill coefficient was 1.7. Using electrophysiological and pharmacological protocols, transient A-type potassium currents (I(A)) and delayed rectifiers potassium currents (I(K)) were isolated. SO3 caused a concentration-dependent, and reversible inhibition of I(K). The IC(50) value for the blockage of I(K) was calculated to be 1.6 and the Hill coefficient was 0.6, with no significant effect on I(A). These results indicate that SO3 can selectively inhibit neuronal sodium and potassium currents.

17 February, 2003

    "Target Pain" published:
    The Association of British Pharmaceutical Industries have produced an informative booklet called "Target Pain", the 14th in a series of guides charting the pharmaceutical industry's prgress in major disease areas. Written by the well-respected medical writer, Dr. Mike Hall, Target Pain is available free from the Publications Department, ABPI, 12 Whitehall, London, SW1A 2DY, or email mfleming@abpi.org.uk. The article includes information about How medicine researchers turn prey-killers into pain-killers. Predatory cone snails have proven particularly interesting to researchers. This report summarizes advances made in the use of cone snail toxins and venom components from tarantula spiders and poison-dart frogs. Included is information about products being developed by:

  • Cognetix Inc. USA (CGX-1160 (contulakin G from Conus geographus) for short-term management of post-operative pain and CGX-1007 (conantokin G from Conus geographus) for controling seizures in patients with intractable epilepsy
  • Elan Corporation (Ziconotide (Prialt(TM)) (omega-conotoxin MVIIA from Conus magus) for intractable neuropathic pain, cancer pain
  • AMRAD Operations, Australia, under licence from the Universtity of Queensland (AM336, (w-conotoxin CVID from Conus catus)for severe morphine-resistant pain)
  • Xenome Ltd, Australia (chi-conotoxin MRIA/B from Conus marmoreus) being developed for neuropathic pain, and
  • University of Melbourne, Australia (ACV1 (alpha-conotoxin Vc1.1 from Conus victoriae) being developed for neuropathic pain, and for speeding the rate of functional recovery after a nerve injury.

16 February, 2003

    Conus victoriae on the prowl:
    Bruce Livett has placed a number of images of a live Conus victoriae in the presence of a small prey mollusk looking hungry as it patrols the covering of his aquarium tank at the University of Melbourne in search of food. If that kind of thing interests you, take a look at the following 15 images: Image No. 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 25; 26; 27.

15 February, 2003

10 February, 2003

2 February, 2003

    Cone shells of Louisiana:
    Harry G. Lee of Jacksonville Shell Club has combined with Emilio Garcia to produce an electronic version of the very recent American Conchologist paper "Report on molluscan species found in the offshore waters of Louisiana, including many extensions of known range and un-named species". This report deals with molluscs collected in waters of the Louisiana coast from 1970 to the present. Of the 385 species-level taxa listed, 108 are reported as new records for the northwestern Gulf of Mexico and comprise approximately 28% of the total. Each of these is marked with an asterisk (*). Many of the species have never been reported from the continental United States. Among the 385 species collected to date are the following eight Conus species: Conus armiger Rehder & Abbott, 1951; Conus atractus Tomlin, 1937; Conus ermineus Born, 1778; *Conus mindanus Hwass in Bruguière, 1792; *Conus mus Hwass in Bruguière, 1792; *Conus patae Abbott, 1971; *Conus riosi Petuch, 1986 and *Conus stimpsoni Dall, 1902, five of which had not been reported in the offshore waters of Louisiana before. This paper on Louisiana offshore mollusks is available at http://www.jaxshells.org/efg1030.htm. As time permits, illustrations will be added to accompany the list, in which case the species will be preceded by a "#" sign. (see also, García, E. F. (1974). A variation of Conus ranunculus Hwass. Of Sea and Shore. 5(3):121).

    Cone shells from Madagascar:
    Giancarlo Paganelli has updated his Cone Shells collection (currently currently 667 images), with the following 4 images of Conus textile from Tuléar, Madagascar: Conus textile LINNAEUS, 1758 Madagascar, Tuléar. 50.2 mm, 44.4 mm, 50.3 mm and 67.5 mm.

    In addition, five new images of fossil Conus have been added to Giancarlo's Cone Shells collection: Conus aldrovandi, Conus derelictus, Conus fuscocingulatus jaspideus, and Conus marylandicus. An extensive list of references on cones is available here.

    Novel conotoxin k-BtX from Conus aureus targets BK channels:
    Dr. Fan and colleagues from the Institute of Neuroscience, Shanghai, China, have discovered a novel conotoxin, k-BtX, in the venom of the vermivorous cone snail Conus betulinus. This 31-residue peptide toxin (CRAeGTYCeNDSQCCLNeCCWGGCGHoCRHP*, where e=gamma-hydroxy glutamate, o=hydroxy-proline and P*=amidated proline) has four disulfide bonds and is post-translationally modified, having three gamma-carboxylated glutamates, a hydroxy-proline, and an amidated proline at the C-terminus. It shares no sequence homology with any other conotoxins. Conotoxin k-BtX specifically targets the Ca2+- and voltage-sensitive "BK" K+ channels. Although more than 80 conotoxins have been characterized to date, only three of them, k-conotoxin PVIIA and kA-conotoxin SIVA (Zhou and Misler, 1995; Lawson, 1996) and conotoxin kM-conotoxin RIIK (Ferber et al 2003, see below), target potassium channels. These three conotoxins inhibit voltage-gated potassium channels whereas k-BtX is an up-modulator of BK channels in rat chromaffin cells.

    Fan, C.X., Chen, X.K., Zhang, C., Wang, L.X., Duan, K.L., He, L.L., Cao, Y., Liu, S.Y., Zhong, M.N., Ulens, C., Tytgat, J., Chen, J.S., Chi, C.W., and Zhou, Z. (2003) "A novel conotoxin from Conus betulinus, k-BtX, unique in cysteine pattern and in function as a specific BK channel modulator." J Biol Chem. J Biol Chem. 278: 12624-12633.
    Abstract: A novel conotoxin, k-conotoxin (k-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin [see above]and a 13-residue extra peptide at the C-terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27 and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that (1) among voltage-gated channels, k-BtX is a specific modulator of K+ channels, (2) among the K channels, k-BtX specifically up-modulates the Ca2+- and voltage-sensitive BK channels (252 47%), (3) its EC50 is 0.7 nM with a single binding site (Hill = 0.88), (4) the time constant of wash-out is 8.3 s, and (5) k-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that k-BtX is a novel specific biotoxin against BK channels.

    New Potassium Channel-targeted Conotoxin:
    'Toto' Olivera, Richard DeLaCruz, and Maren Watkins in Utah together with colleagues in Germany (Michael Ferber, Annett Sporning, Gunnar Jeserich, and Heinrich Terlau) have identified and characterized a novel peptide (kM-conotoxin RIIK) from Conus radiatus venom ducts which blocks Shaker potassium channels. This peptide has the same class III (or M-1) disulfide framework as the mu-conotoxins which target voltage-gated sodium channels, and psi-conotoxin that is a non-competitive antagonist of nicotinc acetylcholine receptors. The class III conotoxins can be recognized by the following pattern of Cys residues: -CC---C---C----CC- . However, despite its structural similarity to the mu- and psi-conotoxins, the Conus radiatus peptide has an entirely different pharmacology: it blocks Shaker K+ channels by binding to the outer vestibule, and defines a new family of conotoxins, the kM-conotoxins.

    kM-Conotoxin RIIIK is not the only conopeptide known to inbibit the Shaker K+ channel by binding to the outer vestibule; k-conotoxin PVIIA from Conus purpurascens, another fish hunting cone, was first shown to do this. However, although Conus purpurascens and Conus radiatus are both piscivorous, they are not closely related species as judged by available molecular phylogeny data for the genus Conus. The results presented thereby establish that two different species of cone snails have evolved structurally and genetically unrelated peptides, with different structural scaffolds, both of which block the Shaker K+ channel. These peptides are proposed to serve as components of the "lightning-strike cabal" of toxins that cause excitotoxic shock (equivalent to a tonic/clonic seizure), resulting in a rapid tetanic paralysis and instant immobilization of the prey

    Ferber, M., Sporning, A., Jeserich, G., DeLaCruz, R., Watkins, M., Olivera, B.M. and Terlau, H. (2003) "A novel Conus peptide ligand for K+ channels" J. Biol. Chem. 278: 2177-2183.
    Abstract: Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.

    Conotoxins help define nicotinic receptor subtypes:
    Nai, Q., McIntosh, J.M. and Margiotta, J.F.(2003) Relating neuronal nicotinic acetylcholine receptor subtypes defined by subunit composition and channel function. Mol Pharmacol. 63:311-324.
    Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are widespread, diverse ion channels involved in synaptic signaling, addiction, and disease. Despite their importance, the relationship between native nAChR subunit composition and function remains poorly defined. Chick ciliary ganglion neurons express two major nAChR types: those recognized by alpha-bungarotoxin (alphaBgt), nearly all of which contain only alpha7 subunits (alpha7-nAChRs) and those insensitive to alphaBgt, which contain alpha3, alpha5, beta4, and, in some cases, beta2 subunits (alpha3*-nAChRs). We explored the relationship between nAChR composition and channel function using toxins recognizing alpha7 subunits (alphaBgt), and alpha3/beta4 (alpha-conotoxin-AuIB), or alpha3/beta2 (alpha-conotoxin-MII) subunit interfaces to perturb responses induced by nicotine, alpha7-, or alpha3-selective agonists (GTS-21 or epibatidine, respectively). Using these reagents, fast-decaying whole-cell current components were attributed solely to alpha7-nAChRs, and slow-decaying components mostly to alpha3*-nAChRs. In outside-out patches, nicotine activated brief 60- and 80-pS single nAChR channel events, and mixed-duration 25- and 40-pS nAChR events. Subsequently, 60- and 80-pS nAChR events and most brief 25- and 40-pS events were attributed to alpha7-nAChRs, and long 25- and 40-pS events to alpha3*-nAChRs. alpha3*-nAChRs lacking beta2 subunits seemed responsible for long 25 pS nAChR events, whereas those containing beta2 subunits mediated the long 40 pS nAChR events that dominate single-channel records. These results reveal greater functional heterogeneity for alpha7-nAChRs than previously expected and indicate that beta2 subunits contribute importantly to alpha3*-nAChR function. By linking structural to functional nAChR subtypes, the findings also illustrate a useful pharmacological strategy for selectively targeting nAChRs.

    Functional link between presynaptic N-type Ca(2+) channels and D(2)-like DA receptors in the brain:
    Momiyama T. (2003) "Parallel decrease in omega-conotoxin-sensitive transmission and dopamine-induced inhibition at the striatal synapse of developing rats. J Physiol 546(Pt 2):483-490
    Abstract: Whole-cell patch-clamp recordings of GABAergic IPSCs were made from cholinergic interneurones in slices of striatum from developing rats aged 21-60 days postnatal. In addition, the Ca(2+) channel subtypes involved in synaptic transmission, as well as dopamine (DA)-induced presynaptic inhibition, were investigated pharmacologically with development by bath application of Ca(2+) channel blockers and DA receptor agonists. The IPSC amplitude was reduced by omega-conotoxin GVIA (omega-CgTX) or omega-agatoxin TK (omega-Aga-TK) across the whole age range, suggesting that multiple types of Ca(2+) channels mediate transmission of the synapse. The IPSC fraction reduced by omega-CgTX significantly decreased, whereas that reduced by omega-Aga-TK remained unchanged with development. DA or quinpirole, a D(2)-like receptor agonist, presynaptically reduced the IPSC amplitude throughout development. The DA-induced inhibition decreased with age in parallel with the decrease in N-type Ca(2+) channels. DA showed no further inhibition of IPSCs after the inhibitory effect of omega-CgTX had reached steady state throughout development. These results demonstrate that there is a functional link between presynaptic N-type Ca(2+) channels and D(2)-like DA receptors at inhibitory synapses in the striatum. They also demonstrate that the suppression of GABAergic transmission by D(2)-like receptors is mediated by modulation of N-type Ca(2+) channels and decreases in parallel with the developmental decline in the contribution of N-type Ca(2+) channels to exocytosis.

31 January, 2003

    Nocturnal antics of a Conus aureus:
    Bob Abela from Tamuning, Guam has set up a salt-water aquarium tank to observe the behavior of mollusks. Bob writes in CONCH-L that in one, he has a "pet" Conus aureus that he is interested in photographing, particularly to record its predation habits. "The main problem has been that it is nocturnal". Now, most cones are crepuscular, but not Conus aureus, it is nocturnal says Bob (see, "Natural history and evolution patterns in Conus Californicus Hinds in Reeve, 1844" by Fabio H. A. Costa, in La Conchiglia, Jan-March. 1996, p 44-52).

    "I've offered a few small "feeder" mollusks and they are often consumed before I have a chance to get it on film. Recently, I placed a Murex laciniatus in the tank, which the C. aureus showed interest in. On one occasion, I watched the cone try to dislodge the murex by attempting to wedge itself between the Murex's aperture and the rock it was resting upon. The Murex clamped down hard and, in this instance, the tactic failed. On a seperate occasion, I observed the cone would intake a large volume of water through its extended siphon, slowly raising its body as it did so. Then expel it, presumeably through its rostrum (difficult to tell). This was forceful and quick, pulling its shell back down as it did so. It resulted in stirring sand in the immediate vicinity of the Murex which was resting at the edge of the sand and the base of a rock. This continued for perhaps one-half hour and then the cone resumed roving about. I assume this was intended to irritate and perhaps dislodge the murex. The Murex clamped down the entire time. If anyone else has observed similar behaviors, I'd sure like to know about it". [David Kirsh from Durham,NC replied that he has noticed the slow rise and quick descent behavior (repeatedly) in a Cymatium parthenopeum he found on the sandflats in North Carolina, and Bruce Livett has observed the same behaviour in a Conus textile he was videoing in a marine aquarium]

    "A day later, I did find a nice clean murex resting bottoms up on the sand, the operculum resting by its side. The animal was completely consumed. The C. aureus rested for quite a while, exposed on the surface of the sand, not even bothering to bury itself during daylight hours, as is typical. By the way, the Murex laciniatus is now a well-preserved specimen for my collection. As for my pet Conus (Bob named him "Aureus Linus" because it reminded him of a Monty Python character, from 'The Life of Bryan', "Maximus Buttus,"), it's doing just fine, had 'him' for about three months now. Not a gem, a couple of large breaks on the body whorl. Large though, ~57mm.

    You can view "Aureus Linus" close-up at: http://briefcase.yahoo.com/bc/bobsonguam/vwp?.dir=/Public&.dnm=close-up+of+Conus+aureus.jpg&.view=t .

  • Watch this site for further photographs of this beautiful and active live cone.
  • Bob also has nice close-up photos of other shells (Strombus taurus, Terebra arga, Vexillum costatum, Nassarius papilosus, Strombus fragilis and Cypraea depressa) at this site.

5 January, 2003

    Novel conotoxin from Conus textile:
    High-resolution, high-accuracy precursor ion scanning mass spectrospcopy has enabled H. Steen and M. Mann from Denmark to identify proline-hydroxylated and tryptophan-brominated species in a highly complex Conus textile conotoxin mixture. This lead to the characterization of one novel C. textile conotoxin containing a bromotryptophan residue and one novel C. textile conotoxin carrying two hydroxyproline residues.

    Steen H. and Mann M. (2002) "Analysis of bromotryptophan and hydroxyproline modifications by high-resolution, high-accuracy precursor ion scanning utilizing fragment ions with mass-deficient mass tags". Anal Chem 74:6230-6236
    Abstract: Protein modifications are often detected by precursor ion scanning. When quadrupole TOF mass spectrometers are used for precursor ion scanning with high-resolution, high-accuracy fragment ion selection, "reporter" ions are required to have a unique mass within +/-0.04 Da or less instead of +/-0.5 Da on triple quadrupole mass spectrometers, the traditional instrument used for precursor ion scanning. Thus, characteristic fragment ions can be utilized even if other fragment ions have the same nominal mass as long as the characteristic fragment ions are slightly mass deficient as compared to the other fragments, i.e., when they have an inherent mass-deficient mass tag. Here, the immonium ions of bromotryptophan and hydroxyproline are described as two fragment ions characteristic for tryptophan-brominated and proline-hydroxylated peptides, respectively. The "reporter" ion of trytophan-brominated peptides is highly mass deficient due to the presence of bromine, thereby allowing the selective detection of these species and the distinction from other dipeptidic a-, b-, and y-fragment ions by high-resolution, high-accuracy precursor ion scanning. This strategy also enables the differentiation between precursors giving rise to the oxygen-containing immonium ion of hydroxyproline and precursors of the immonium ions of near-ubiquitous leucine/isoleucine. Both immonium ions have the same nominal mass of 86 Da, but the exact masses differ by less than 0.04 Da. High-resolution, high-accuracy precursor ion scanning enabled the identification of proline-hydroxylated and tryptophan-brominated species and the directed analysis of species carrying these modifications in a highly complex Conus textile conotoxin mixture. This lead to the characterization of one novel C. textile conotoxin containing a bromotryptophan residue and one novel C. textile conotoxin carrying two hydroxyproline residues.

    Similarities between the Conus carboxylase and vertebrate Vitamin K gamma-carboxylases:
    Czerwiec E, Begley GS, Bronstein M, Stenflo J, Taylor K, Furie BC, Furie B. (2002) "Expression and characterization of recombinant vitamin K-dependent gamma-glutamyl carboxylase from an invertebrate, Conus textile". Eur J Biochem 269:6162-6172
    Abstract: The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, gamma-carboxyglutamic acid, have been identified. To examine its biosynthesis of gamma-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and approximately 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin K-dependent epoxidase activity. The recombinant carboxylase has a molecular mass of approximately 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues -18 to +10 of human proprothrombin and proFactor IX with Km values of 420 micro m, 1.7 micro m and 6 micro m, respectively; the Km for vitamin K is 52 micro m. The Km values for peptides based on the sequence of the conotoxin epsilon-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 micro m, 75 micro m and 74 micro m, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent gamma-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of gamma-carboxyglutamic acid synthesis in diverse biological systems.

    Drugs from the seas - Review:
    Proksch P, Edrada RA and Ebel R. (2002) "Drugs from the seas - current status and microbiological implications.". Appl Microbiol Biotechnol 59:125-34
    Abstract: The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.

    Cone shell gallery:
    Alfonso Pina from Málaga, Spain has assembled a fine personal collection of cone shells with images available on the web. The collection includes the following species : Conus archon (Broderip,1833) 33 mm I. Contadora, Panamá; Conus axelrodi (Walls,1978) 16 mm Palawan, Filipinas; Conus borgesi (Trovao, 1979) 26 mm Boavista, Cabo Verde; Conus bullatusf (Linnaeus,1758) 59 mm Samar, Filipinas; Conus canonicus (Hwass,1792) 48 mm Bahía de Nacala, Mozambique; Conus decoratus (Röckel, Rolán & Monteiro 1980) 28,7 mm Sta. Lucía, Cabo Verde; Conus genuanus (Linnaeus,1758) 36 mm S. Vicente, Cabo Verde; Conus guanche (Lauer, 1993) 28 mm Tenerife, I. Canarias; Conus josephinae (Rolán, 1980) 31,6 mm Cabo Verde; Conus longurionis (Kiener, 1849) 33 mm Filipinas; Conus mercator (Linnaeus,1758) 26 mm I. Goree, Senegal; Conus milneedwardsi (Jousseaume,1894) 94 mm Canal de Mozambique; Conus natalis (Sowerby, 1857) 61 mm Sudáfrica; Conus pulcher siamensis (Hwass in Bruguière,1792) 66 mm Tenerife, I. Canarias; Conus sugimotonis (Kuroda, 1928) 74 mm Aliguay, Filipinas; and Conus zapatosensis (Röckel, 1987) 40 mm Filipinas.

4 January, 2003

    Conus collectibles:
    Peter Ackermans has an interest in cone shells and also in collectibles such as cigar bands and phone cards. Here he presents one of each with cone shells clearly featured. The TELECOM phonecard is particularly nice.

    In 1998, Fiji (Post & Telecom) issued a number of phone cards with shells, including two cards, one depicting a collection of different cones and another featuring Conus geographus.

3 January, 2003

1 January, 2003

Continued in What's new in 2002
See also : What's new in 2007, What's new in 2006, What's new in 2005, What's new in 2004, What's new in 2001, What's new in 2000, What's new in 1999, What's new in 1998, What's New in 1997 and What's New in 1996

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Continued in What's new in 2002


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