alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

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 alpha7/5HT(3) 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. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Solution structure of the sodium channel antagonist conotoxin GS: A new molecular caliper for probing sodium channel geometry

Background: The venoms of Conus snails contain small, disulfide-rich inhibitors of voltage-dependent sodium channels. Conotoxin GS is a 34-residue polypeptide isolated from Conus geographus that interacts with the extracellular entrance of skeletal muscle sodium channels to prevent sodium ion conduction. Although conotoxin GS binds competitively with mu conotoxin GIIIA to the sodium channel surface, the two toxin types have little sequence identity with one another, and conotoxin GS has a four-loop structural framework rather than the characteristic three-loop mu-conotoxin framework. The structural study of conotoxin GS will form the basis for establishing a structure-activity relationship and understanding its interaction with the pore region of sodium channels. Results: The three-dimensional structure of conotoxin GS was determined using two-dimensional NMR spectroscopy. The protein exhibits a compact fold incorporating a beta hairpin and several turns. An unusual feature of conotoxin GS is the exceptionally high proportion (100%) of cis-imide bond geometry for the three proline or hydroxyproline residues. The structure of conotoxin GS bears little resemblance to the three-loop mu conotoxins, consistent with the low sequence identity between the two toxin types and their different structural framework. However, the tertiary structure and cystine-knot motif formed by the three disulfide bonds is similar to that present in several other polypeptide ion channel inhibitors. Conclusions: This is the first three-dimensional structure of a 'four-loop' sodium channel inhibitor, and it represents a valuable new structural probe for the pore region of voltage-dependent sodium channels. The distribution of amino acid sidechains in the structure creates several polar and charged patches, and comparison with the mu conotoxins provides a basis for determining the binding surface of the conotoxin GS polypeptide.

Crystal structure at 1.1 angstrom resolution of alpha-conotoxin PnIB: Comparison with alpha-conotoxins PnIA and GI

Conotoxins are small, cysteine-rich peptides isolated from the venom of Conus spp. of predatory marine snails, which selectively target specific receptors and ion channels critical to the functioning of the neuromuscular system. alpha-Conotoxins PnIA and PnIB are both 16-residue peptides (differing in sequence at only two positions) isolated from the molluscivorous snail Conus pennaceus. In contrast to the muscle-selective alpha-conotoxin GI from Conus geographus, PnIA and PnIB block the neuronal nicotinic acetylcholine receptor (nAChR). Here, we describe the crystal structure of PnIB, solved at a resolution of 1.1 Angstrom and phased using the Shake-and-Bake direct methods program. PnIB crystals are orthorhombic and belong to the space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 14.6 Angstrom, b = 26.1 Angstrom, and c = 29.2 Angstrom. The final refined structure of alpha-conotoxin PnIB includes all 16 residues plus 23 solvent molecules and has an overall R-factor of 14.7% (R-free of 15.9%). The crystal structures of the alpha-conotoxins PnIB and PnIA are solved from different crystal forms, with different solvent contents. Comparison of the structures reveals them to be very similar, showing that the unique backbone and disulfide architecture is not strongly influenced by crystal lattice constraints or solvent interactions. This finding supports the notion that this structural scaffold is a rigid support for the presentation of important functional groups. The structures of PnIB and PnIA differ in their shape and surface charge distribution from that of GI.

The synthesis and structure of an n-terminal dodecanoic acid conjugate of a-conotoxin MII

The alpha-conotoxin MII is a 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 of the subtype alpha3beta2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

IMI: ELES COMEM TUDO E NÃO DEIXAM NADA

O IMI é aliás, em si mesmo, um imposto profundamente injusto. A sua aplicação constitui, em muitos casos, uma situação de Abuso do Direito, art. 334º do Código Civil. A sua concretização implica uma violação do fim económico e social da própria Justiça no Direito. O sistema fiscal não objectiva apenas as necessidades financeiras do Estado e doutras entidades públicas, mas também, e sobretudo uma repartição justa dos rendimentos e da riqueza: art. 103º da Constituição. É claro que, para isso, tem que existir também um controlo rigoroso e honesto da gestão e da distribuição constitucional dos dinheiros públicos. § IMI is indeed in itself a profoundly unfair tax. Its use is in many cases a right Abuse situation art. 334 of the Civil Code. Its implementation involves a violation of economic and social justice at the end of its law. The tax system does not seek only the financial needs of the state and of other public authorities, but also and especially a fair distribution of income and wealth: art. 103 of the Constitution. Of course, for this, it must also be a rigorous and honest control of the constitutional management and distribution of public funds.

Práticas sobre o ordenamento e fiscalidade do território : caso de estudo IMI

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia do Ambiente, perfil de Gestão e Sistemas Ambientais

Differential antagonism by conotoxin p-TIA of contractions mediated by distinct alpha(1)-adrenoceptor subtypes in rat vas deferens, spleen and aorta

The ability of the conotoxin p-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha(1A)-adrenoceptors in rat vas deferens, alpha(1B)-adrenoceptors in rat spleen and alpha(ID)-adrenoceptors in rat aorta, and to inhibit the binding of [I-125]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha(1)-adrenoceptors was investigated. p-TIA (100 nM to 1 muM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA(2)similar to7.2, n=4). This suggests that p-TIA is a competitive antagonist of alpha(1A)- and alpha(1D)-adrenoceptors with no selectivity between these subtypes. Incubation of p-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that p-TIA is a non-competitive antagonist at alpha(1B)-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of p-TIA in inhibiting contractions was examined with similar occupancies (similar to25%) at each subtype. Its potency (pIC(50)) was 12 times higher in spleen (8.3 +/- 0.1, n=4) than in vas deferens (7.2 +/- 0.1, n=4) or aorta (7.2 0.1, n=4). In radioligand binding assays, p-TIA decreased the number of binding sites (B,,,,,,) in membranes from HEK293 cells expressing the rat alpha(1B)-adrenoceptors without affecting affinity (K-D), In contrast, in HEK293 cells expressing rat alpha(1A)- or alpha(1D)-adrenoceptors, p-TTA decreased the KD without affecting the B-max. It is concluded that p-TIA will be useful for distinguishing the role of particular alpha(1)-adrenoceptor subtypes in native tissues. (C) 2004 Elsevier B.V. All rights reserved.

Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only.

Joan Eckii ... De materia iuramenti accutiss[imi] decisio ad Georgium Kungspergium augustanum.

Mode of access: Internet.

Doklad ob otvi︠e︡tstvennosti obshchestv zheli︠e︡znykh dorog za srochnui︠u︡ dostavku i t︠s︡i︠e︡lostʹ pervozimykh imi gruzov /

Mode of access: Internet.

Opisanīe kubanskago imi͡enīi͡a "Khutorok" barona V.R. Shteĭngelʹ : 1900 g. /

Mode of access: Internet.