Conus vexillum venom induces oxidative stress in Ehrlich's ascites carcinoma cells: an insight into the mechanism of induction

Background: It is estimated that venoms of marine cone snails (genus Conus) contain more than 100,000 different small peptides with a wide range of pharmacological and biological actions. Some of these peptides were developed into potential therapeutic agents and as molecular tools to understand biological functions of nervous and cardiovascular systems. In this study we examined the cytotoxic and anticancer properties of the marine vermivorous cone snail Conus vexillum (collected from Hurgada and Sharm El-Shaikh, Red Sea, Egypt) and suggest the possible mechanisms involved. The in vitro cytotoxic effects of Conus venom were assessed against Ehrlich's ascites carcinoma (EAC) cells. Results: Conus venom treatment resulted in concentration-dependent cytotoxicity as indicated by a lactate dehydrogenase leakage assay. Apoptotic effects were measured in vivo by measuring levels of reactive oxygen species and oxidative defense agents in albino mice injected with EAC cells. Conus venom (1.25 mg/kg) induced a significant increase (p < 0.05) in several oxidative stress biomarkers (lipid peroxidation, protein carbonyl content and reactive nitrogen intermediates) of EAC cells after 3, 6, 9 and 12 hours of venom injection. Conus venom significantly reduced (p < 0.05) the activities of oxidative defense enzymes (catalase and superoxide dismutase) as well as the total antioxidant capacity of EAC cells, as evidenced by lowered levels of reduced glutathione. Conclusions: These results demonstrate the cytotoxic potential of C. vexillum venom by inducing oxidative stress mediated mechanisms in tumor cells and suggest that the venom contains novel molecules with potential anticancer activity.(AU)

Solution structure of a novel α-conotoxin with a distinctive loop spacing pattern.

α-Pharmacological conotoxins are among the most selective ligands of nicotinic acetylcholine receptors with typical cysteine frameworks. They are characterized by the intercysteine loop and classified into various subfamilies, such as α3/5 and α4/7 conotoxins. A novel α-conotoxin, Pu14a (DCPPHPVPGMHKCVCLKTC), with a distinct loop spacing pattern between cysteines was reported recently. Pu14a belongs to the Cys framework 14 (-C-C-C-C) family containing four proline residues in the loop 1 region. Similar to another framework 14 conotoxin Lt14a (MCPPLCKPSCTNC-NH2), Pu14a has C1-C3/C2-C4 disulfide linkage, and can inhibit some subtypes of nicotinic acetylcholine receptors. In this study, the solution structure of Pu14a was investigated using 1H nuclear magnetic resonance spectroscopy to understand the structure-activity relationship of this conotoxin. 20 converged structures of this conopeptide, with RMSD value of 0.77 Å, were obtained based on distance constraints, dihedral angles and disulfide bond constraints. The three-dimensional structure of Pu14a showed remarkable difference from typical α-conotoxins because of a large intercysteine loop between C2 and C13, as well as a 3(10)-helix near the C-terminal. Furthermore, four proline residues in Pu14a adopted the trans conformation that may correlate with the large loop configuration and the biological activity of this conopeptide. The distinct structural characteristics of Pu14a will be very useful for studying the structure-activity relationship of α-conotoxins.

Metamorphic remodeling of a planktotrophic larva to produce the predatory feeding system of a cone snail (Mollusca, Neogastropoda).

I used histological sections and 3D reconstructions to document development through metamorphosis of the foregut and proboscis in the conoidean neogastropod Conus lividus. A goal was to determine how highly derived features of the post-metamorphic feeding system of this gastropod predator develop without interfering with larval structures for microherbivory. A second goal was to compare foregut development in this conoidean with previous observations on foregut development in the buccinoidean neogastropod Nassarius mendicus. These two neogastropods both have a feeding larval stage, but they show major differences in post-metamorphic foregut morphology. Basic events in development of the proboscis and proboscis sheath in C. lividus and N. mendicus were similar. However, the elongate buccal tube of C. lividus forms during metamorphosis as a composite of apical epidermal tissue that grows inward and ventral foregut tissue that extends outward. The larval mouth is not carried through metamorphosis. Comparative observations on foregut development in caenogastropods, which now include data on C. lividus, suggest that the foregut incorporates dorsal and ventral modules having different ontogenetic and functional fates. This developmental modularity may have facilitated evolutionary diversification of the post-metamorphic foregut. Foregut diversification in predatory gastropods may have been further fast-tracked by developmental uncoupling of larval and post-metamorphic mouths.

Definition and characterization of the short alphaA-conotoxins: a single residue determines dissociation kinetics from the fetal muscle nicotinic acetylcholine receptor.

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