Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of ␣-cobratoxin (a long-chain ␣-neurotoxin) and heterodimers formed by ␣-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26 -30 in polypeptide loop II of ␣-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the ␣-cobratoxin capacity to compete with ␣-bungarotoxin for binding to Torpedo and ␣7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that ␣-cobratoxin dimer not only interacts with ␣7 nAChR but, in contrast to ␣-cobratoxin monomer, also blocks ␣3␤2 nAChR. In the latter activity it resembles -bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric ␣3␤2 nAChRs. Three-fingered toxins (TFTs)2 are the main components of the Elapidae snake venoms. TFTs consist of one polypeptide chain, their spatial structure being characterized by a hydrophobic core stabilized by four disulfide bridges, which confine three polypeptide loops (fingers). In cobra venom TFTs are represented mainly by ␣-neurotoxins and cytotoxins. So-called short-chain ␣-neurotoxins (60 -62 amino acid residues, 4 intramolecular disulfides) effectively block nicotinic acetylcholine receptors (nAChRs) of muscle-type, and long-chain ␣-neurotoxins (65-75 residues with an additional disulfide in central loop II) block neuronal homopentameric ␣7 nAChR as well (1, 2). These toxins are widely used as tools in the nAChR studies. Cytotoxins, structurally related to short-chain ␣-neurotoxins, manifest another activity; they non-selectively disrupt cell membranes and, thus, kill the cells (3).Another example of TFT interacting with nAChR are -bungarotoxins (-Bgts), minor components of the krait (Elapidae) venom (4, 5). All -Bgts consist of 66 amino acid residues and, similar to long-chain ␣-neurotoxins, contain five disulfide bonds. However, in contrast to ␣-neurotoxins, -Bgts practically do not block muscle-type nAChRs and only weakly act on ␣7 nAChR but with high efficiency interact with ␣3␤2 neuronal receptors (6). Despite the vast array of data on structure-activity relationship for ␣-neurotoxins and -Bgts, it is not yet clear what are the main structural features determining the specificity of a toxin to the particular receptor type. Recently, based on the x-ray structure of ␣-cobratoxin (␣-CT) with acetylcholinebinding protein, Bourne et al. (7) suggested that Lys-29 is the main residue determining the difference in specificity between ␣-neurotoxins and -bungarotoxins. However, A29K mut...

Background: Venoms from rare snake species may contain toxins of new structural or/and pharmacological types.Results: Amino acid sequence of the new polypeptide azemiopsin isolated from Azemiops feae viper venom was established, and its biological activity was determined.Conclusion: Azemiopsin is the first natural toxin that blocks nicotinic acetylcholine receptors and does not contain disulfide bridges.Significance: Azemiopsin is the first member of a new toxin group.

Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

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We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.

Human alpha satellite (AS) sequence domains that currently function as centromeres are typically flanked by layers of evolutionarily older AS that presumably represent the remnants of earlier primate centromeres. Studies on several human chromosomes reveal that these older AS arrays are arranged in an age gradient, with the oldest arrays furthest from the functional centromere and arrays progressively closer to the centromere being progressively younger. The organization of AS on human chromosome 21 (HC21) has not been well-characterized. We have used newly-available HC21 sequence data and an HC21p YAC map to determine the size, organization, and location of the AS arrays, and compared them to AS arrays found on other chromosomes. We find that the majority of the HC21 AS sequences are present on the p-arm of the chromosome and are organized into at least five distinct isolated clusters which are distributed over a larger distance from the functional centromere than that typically seen for AS on other chromosomes. Using both phylogenetic and L1 element age estimations, we found that all of the HC21 AS clusters outside the functional centromere are of a similar relatively recent evolutionary origin. HC21 contains none of the ancient AS layers associated with early primate evolution which is present on other chromosomes, possibly due to the fact that the p-arm of HC21 and the other acrocentric chromosomes underwent substantial reorganization about 20 million years ago. AbstractClick here to download Abstract ABSTRACT.docx List of AbbreviationsAS -alpha satellite HCX -human chromosome X HC21 -human chromosome 21HC21p -human chromosome 21 short arm HOR -higher order repeat SD -segmental duplications