Correlating Molecular Phylogeny with Venom Apparatus Occurrence in Panamic Auger Snails (Terebridae)

Central to the discovery of neuroactive compounds produced by predatory marine snails of the superfamily Conoidea (cone snails, terebrids, and turrids) is identifying those species with a venom apparatus. Previous analyses of western Pacific terebrid specimens has shown that some Terebridae groups have secondarily lost their venom apparatus. In order to efficiently characterize terebrid toxins, it is essential to devise a key for identifying which species have a venom apparatus. The findings presented here integrate molecular phylogeny and the evolution of character traits to infer the presence or absence of the venom apparatus in the Terebridae. Using a combined dataset of 156 western and 33 eastern Pacific terebrid samples, a phylogenetic tree was constructed based on analyses of 16S, COI and 12S mitochondrial genes. The 33 eastern Pacific specimens analyzed represent four different species: Acus strigatus, Terebra argyosia, T. ornata, and T. cf. formosa. Anatomical analysis was congruent with molecular characters, confirming that species included in the clade Acus do not have a venom apparatus, while those in the clade Terebra do. Discovery of the association between terebrid molecular phylogeny and the occurrence of a venom apparatus provides a useful tool for effectively identifying the terebrid lineages that may be investigated for novel pharmacological active neurotoxins, enhancing conservation of this important resource, while providing supplementary information towards understanding terebrid evolutionary diversification.     

Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity

Conantokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Conantokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like conantokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) conantokin-T has NMDA antagonist activity. A sequence comparison of conantokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide.     

Function and evolution of allelic variations of Sr13 conferring resistance to stem rust in tetraploid wheat (Triticum turgidum L.)

The resistance gene Sr13 is one of the most important genes in durum wheat for controlling stem rust caused by Puccinia graminis f. sp. tritici (Pgt). The Sr13 functional gene CNL13 has haplotypes R1, R2 and R3. The R1/R3 and R2 haplotypes were originally designated as alleles Sr13a and Sr13b, respectively. To detect additional Sr13 alleles, we developed Kompetitive allele specific PCR (KASP™) marker KASPSr13 and four semi-thermal asymmetric reverse PCR markers, rwgsnp37–rwgsnp40, based on the CNL13 sequence. These markers were shown to detect R1, R2 and R3 haplotypes in a panel of diverse tetraploid wheat accessions. We also observed the presence of Sr13 in durum line CAT-A1, although it lacked any of the known haplotypes. Sequence analysis revealed that CNL13 of CAT-A1 differed from the susceptible haplotype S1 by a single nucleotide (C2200T) in the leucine-rich repeat region and differed from the other three R haplotypes by one or two additional nucleotides, confirming that CAT-A1 carries a new (R4) haplotype. Stem rust tests on the monogenic, transgenic and mutant lines showed that R1 differed from R3 in its susceptibility to races TCMJC and THTSC, whereas R4 differed from all other haplotypes for susceptibility to TTKSK, TPPKC and TCCJC. Based on these differences, we designate the R1, R3 and R4 haplotypes as alleles Sr13a, Sr13c and Sr13d, respectively. This study indicates that Sr13d may be the primitive functional allele originating from the S1 haplotype via a point mutation, with the other three R alleles probably being derived from Sr13d through one or two additional point mutations.     

Alpha-RgIA, a novel conotoxin that blocks the alpha9alpha10 nAChR: structure and identification of key receptor-binding residues

α-Conotoxins are small disulfide-constrained peptides from cone snails that act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). The 13-residue peptide α-conotoxin RgIA (α-RgIA) is a member of the α-4,3 family of α-conotoxins and selectively blocks the α9α10 nAChR subtype, in contrast to another well-characterized member of this family, α-conotoxin ImI (α-ImI), which is a potent inhibitor of the α7 and α3β2 nAChR subtypes. In this study, we have altered side chains in both the four-residue and the three-residue loops of α-RgIA, and have modified its C-terminus. The effects of these changes on activity against α9α10 and α7 nAChRs were measured; the solution structures of α-RgIA and its Y10W, D5E, and P6V analogues were determined from NMR data; and resonance assignments were made for α-RgIA [R9A]. The structures for α-RgIA and its three analogues were well defined, except at the chain termini. Comparison of these structures with reported structures of α-ImI reveals a common two-loop backbone architecture within the α-4,3 family, but with variations in side-chain solvent accessibility and orientation. Asp5, Pro6, and Arg7 in loop 1 are critical for blockade of both the α9α10 and the α7 subtypes. In loop 2, α-RgIA [Y10W] had activity near that of wild-type α-RgIA, with high potency for α9α10 and low potency for α7, and had a structure similar to that of wild type. By contrast, Arg9 in loop 2 is critical for specific binding to the α9α10 subtype, probably because it is larger and more solvent accessible than Ala9 in α-ImI. Our findings contribute to a better understanding of the molecular basis for antagonism of the α9α10 nAChR subtype, which is a target for the development of analgesics for the treatment of chronic neuropathic pain.     

Qualitative HPLC‐DAD/ESI‐TOF‐MS Analysis,Cytotoxic, and Apoptotic Effects of Croatian Endemic Centaurea ragusina L. Aqueous Extracts

Centaurea ragusina L., an endemic Croatian plant species, revealed a good cytotoxic activity of aqueous extracts (AE) on human bladder (T24) and human glioblastoma (A1235) cancer cell lines. The chemical constituents were tentatively identified using high performance liquid chromatography HPLC‐DAD/ESI‐TOF‐MS in negative ionization mode. The main compounds of herba extract were sesquiterpene lactones: solstitialin A 3,13‐diacetate and epoxyrepdiolide; organic acid: quinic acid. The main compounds of flower extract were organic acids: quinic acid, citric acid, and malic acid; sesquiterpene lactone: cynaropicrin; phenolic compounds: chlorogenic acid and phenylpropanoid: syringin. The AE of C. ragusina were investigated for correlation of their effects on human bladder (T24) and human glioblastoma (A1235) cancer cell lines using the MTT assay. Although both extracts showed significant dose‐ and time‐dependent cytotoxic activity against both cancer cell lines, the flower extract exhibited slightly higher activity. In order to determine type of cell death induced by treatment, cell lines were exposed subsequently to a treatment with both flower and herba AE. The majority of the cells died by induced apoptosis treatment. Flower AE (26.25%), compared to a leaf AE (22.15%) showed slightly higher percentage of an apoptosis in T24 cells, when compared to a non‐treated cells (0.04%).     

Phytochemical and Cytogenetic Characterization of Centaurea solstitialis L. (Asteraceae) from Croatia

The cytogenetic characterization of Centaurea solstitialis L. (Asteraceae) showed a chromosome number of 2n = 16. Karyotype is composed by four pairs of metacentric, two pairs of submetacentric and two pairs of subtelocentric chromosomes. Physical mapping of two rDNA probes revealed two loci of 35S and one locus of 5S rRNA genes. Chromomycin fluorochrome banding revealed that all rDNA loci were GC rich. The genome size (2C‐value) of 1.95 pg classes this species in the group of very small genomes. Chemical composition of C. solstitialis volatile oil (VO) from Croatia, studied with gas chromatography–mass spectrometry showed dominant components as it follows: hexadecanoic acid, α‐linolenic acid, germacrene D and heptacosane. Antioxidant capacity, measured by ferric reducing power assay and 2,2‐diphenyl‐1‐picrylhydrazyl methods, as well as inhibition of acetyl‐ and butyrylcholinesterase of VO was lower comparing to a standard solutions. Volatile oil tested with disc diffusion method showed good inhibitory potential against Pseudomonas aeruginosa, Escherichia coli and all tested fungi: Candida albicans, Penicillium funiculosum and Aspergillus fumigatus. The microdilution method showed best activity against Chronobacter sakazakii and A. fumigatus.