Against expectation: a short sequence with high signal elucidates cone snail phylogeny

A short (259 nucleotide) conserved intronic sequence (CIS) is surprisingly informative for delineating deep phylogenetic relationships in cone snails. Conus species previously have been assigned to clades based on the evidence from mitochondrial 12S and 16S rRNA gene sequences (1129 bp). Despite their length, these genes lack the phylogenetic information necessary to resolve the relationships among the clades. Here we show that the relationships can be inferred from just 46 sites in the very short CIS sequence (a portion of "intron 9" of the γ-glutamyl carboxylase gene). This is counterintuitive because in short sequences sampling error (noise) often drowns out phylogenetic signal. The intron 9 CIS is rich in synapomorphies that define the divergence patterns among eight clades of worm- and fish-hunting Conus, and it contains almost no homoplasy. Parsimony, maximum likelihood and Bayesian analyses of the combined sequences (mt rRNA+CIS) confirm most of the relationships among 23 Conus sequences. This phylogeny implies that fish-hunting behavior evolved at least twice during the history of Conus-once among New World species and independently in the Indo-Pacific clades.     

Phylogeny of the genus Turris: correlating molecular data with radular anatomy and shell morphology

There are over 10,000 species of venomous marine molluscs, the vast majority of these, which are generally referred to as "turrids", are traditionally assigned to a single family, Turridae (Powell 1966). Here, we provide an initial molecular analysis of the type genus of the family, Turris R?ding, 1798, thought to be among the most well characterized groups in the family. We show that the type genus is not monophyletic. We analyzed specimens conventionally assigned to 9 different Turris species using molecular markers, combined with the shell morphology and radular anatomy whenever feasible. The results suggest that species assigned to the genus Turris, provisionally assigned to two different subgenera are not monophyletic. Five previously described species belong to the subgenus Turris (s.s.) R?ding 1798: Turris babylonia, (Linne, 1758), Turris grandis, (J. E. Gray, 1834), Turris dollyae, (Olivera, 1999), Turris normandavidsoni (Olivera, 1999) and Turris spectabilis (Reeve, 1843). With a change in species designation, Turris assyria (formerly T. babylonia1010) is added to a well-defined clade, which is in turn more closely related to Lophiotoma and Gemmula species than to the other five Turris species. We show that these five species conventionally assigned to Turris do not belong in the same subgenus, and form a clade provisionally designated as AnnulaturrisPowell, 1966: Turris annulata, (Reeve, 1843), Turris undosa, (Lamarck, 1816), Turris cristata, (Vera-Peláez, Vega-Luz, and Lozano-Francisco 2000) Turris cryptorrhaphe (G. B. Sowerby, 1825) and Turris nadaensis (Azuma, 1973). Implications of the molecular phylogenetic results and its correlation with radular morphology are discussed.     

Synthesis and anti-aromatase activity of some new steroidal D-lactones

Starting from D-seco derivatives of 5-androstene 1-3, the D-homo lactones, 4 and 5, were synthesized. By the Oppenauer oxidation and/or by dehydration of 4 and 5 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), the corresponding D-lactones 6-12 were obtained. The structures of 6 and 10 were unambiguously proved by the appropriate X-ray structural analysis. Anti-aromatase assay showed that tested compounds possess inhibition potency, however, two to four times smaller (IC50 from 0.2 to 0.7 microM, respectively) in comparison to aminoglutethimide (AG).     

Matrix metalloproteinase secretion by gastric epithelial cells is regulated by E prostaglandins and MAPKs

Because matrix metalloproteinases (MMPs) play roles in inflammatory tissue injury, we asked whether MMP secretion by gastric epithelial cells may contribute to gastric injury in response to signals involved in Helicobacter pylori-induced inflammation and/or cyclooxygenase inhibition. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and epidermal growth factor (EGF) stimulated gastric cell MMP-1 secretion, indicating that MMP-1 secretion occurs in inflammatory as well as non-inflammatory situations. MMP-1 secretion required activation of the MAPK Erk and subsequent protein synthesis but was down-regulated by the alternate MAPK, p38. In contrast, secretion of MMP-13 was stimulated by TNF-alpha/IL-1beta but not EGF and was Erk-independent and mediated by p38. MMP-13 secretion was more rapid (peak, 6 h) than MMP-1 (peak > or =30 h) and only partly depended on protein synthesis, suggesting initial release of a pre-existing MMP-13 pool. Therefore, MMP-1 and MMP-13 secretion are differentially regulated by MAPKs. MMP-1 secretion was regulated by E prostaglandins (PGEs) in an Erk-dependent manner. PGEs enhanced Erk activation and MMP-1 secretion in response to EGF but inhibited Erk and MMP-1 when TNF-alpha and IL-1beta were the stimuli, indicating that the effects of PGEs on gastric cell responses are context-dependent. These data show that secretion of MMPs is differentially regulated by MAPKs and suggest mechanisms through which H. pylori infection and/or cyclooxygenase inhibition may induce epithelial cell signaling to contribute to gastric ulcerogenesis.     

Molecular interaction of delta-conotoxins with voltage-gated sodium channels

Various neurotoxic peptides modulate voltage-gated sodium (Na(V)) channels and thereby affect cellular excitability. Delta-conotoxins from predatory cone snails slow down inactivation of Na(V) channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of Na(V) channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. Delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its "on" position.     

Alpha S-conotoxin RVIIIA: a structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.     

Multiple 6-bromotryptophan residues in a sleep-inducing peptide

We have characterized a novel sleep-inducing peptide comprising 33 amino acids with three residues of the unusual posttranslationally modified amino acid, 6-bromotryptophan. The peptide, termed "light sleeper" or the r7a conotoxin, was purified from the venom of the fish-hunting Conus radiatus. The light sleeper peptide has additional notable biochemical properties; it equilibrates slowly between two distinct conformers, and has four gamma-carboxyglutamate residues. The pattern of posttranslational bromination in the light sleeper peptide suggests that tryptophan residues at N- and C-termini may be preferential sites for posttranslational bromination.     

Zebrafish collapsin response mediator protein (CRMP)-2 is expressed in developing neurons

The collapsin response mediator proteins (CRMPs) are highly expressed in the vertebrate nervous system. CRMP2 has been shown to function in Semaphorin and lysophosphatidic acid induced growth cone collapse. Correspondingly, the highest levels of CRMP2 protein are found in the distal portion of growing axons. To understand the role of CRMP2 during embryonic development we have documented its expression pattern in zebrafish embryos at multiple stages. We find that CRMP2 is expressed in the major neural clusters of the embryonic brain during the primary stages of neurogenesis. From 20 somites through 30 hpf CRMP2 is expressed in the dorsal rostral cluster of the telencephalon, the ventral rostral cluster of the diencephalon, the ventral caudal cluster of the mesencephalon, and the hindbrain clusters. CRMP2 is also expressed in the trigeminal sensory ganglia and the Rohon Beard cells of the neural tube from 15 somites. By 48 hpf, we find expression of CRMP2 throughout the developing brain, trigeminal sensory ganglia, and Rohon Beard cells. CRMP2 is also detected in the retinal ganglion cell layer of the eye, and in the otic vesicle. Finally, we have compared the expression of CRMP2 to PlexinA4, a Semaphorin receptor expressed in sensory neurons, and find that their expression partially overlaps.     

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.