Novel alpha-conotoxins from Conus spurius and the alpha-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.     

Focusing on optic tectum circuitry through the lens of genetics

The visual pathway is tasked with processing incoming signals from the retina and converting this information into adaptive behavior. Recent studies of the larval zebrafish tectum have begun to clarify how the 'micro-circuitry' of this highly organized midbrain structure filters visual input, which arrives in the superficial layers and directs motor output through efferent projections from its deep layers. The new emphasis has been on the specific function of neuronal cell types, which can now be reproducibly labeled, imaged and manipulated using genetic and optical techniques. Here, we discuss recent advances and emerging experimental approaches for studying tectal circuits as models for visual processing and sensorimotor transformation by the vertebrate brain.     

Resistance to stem rust Ug99 in six bread wheat cultivars maps to chromosome 6DS

Key message

Identified SSR markers ( Xcfd49 and Xbarc183 ) linked with stem rust resistance for efficient use in marker-assisted selection and stacking of resistance genes in wheat breeding programs.

Abstract

More than 80 % of the worldwide wheat (Triticum aestivum L.) area is currently sown with varieties susceptible to the Ug99 race group of stem rust fungus. However, wheat lines Niini, Tinkio, Coni, Pfunye, Blouk, and Ripper have demonstrated Ug99 resistance at the seedling and adult plant stages. We mapped stem rust resistance in populations derived from crosses of a susceptible parent with each of the resistant lines. The segregation of resistance in each population indicated the presence of a single gene. The resistance gene in Niini mapped to short arm of chromosome 6D and was flanked by SSR markers Xcfd49 at distances of 3.9 cM proximal and Xbarc183 8.4 cM distal, respectively. The chromosome location of this resistance was validated in three other populations: PBW343/Coni, PBW343/Tinkio, and Cacuke/Pfunye. Resistance initially postulated to be conferred by the SrTmp gene in Blouk and Ripper was also linked to Xcfd49 and Xbarc183 on 6DS, but it was mapped proximal to Xbarc183 at a similar position to previously mapped genes Sr42 and SrCad. Based on the variation in diagnostic marker alleles, it is possible that Niini and Pfunye may carry different resistance genes/alleles. Further studies are needed to determine the allelic relationships between various genes located on chromosome arm 6DS. Our results provide valuable molecular marker and genetic information for developing Ug99 resistant wheat varieties in diverse germplasm and using these markers to tag the resistance genes in wheat breeding.     

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.     

A novel conotoxin from Conus delessertii with posttranslationally modified lysine residues

A major peptide, de13a from the crude venom of Conus delessertii collected in the Yucatan Channel, Mexico, was purified. The peptide had a high content of posttranslationally modified amino acids, including 6-bromotryptophan and a nonstandard amino acid that proved to be 5-hydroxylysine. This is the first report of 5-hydroxylysine residues in conotoxins. The sequence analysis, together with cDNA cloning and a mass determination (monoisotopic mass of 3486.76 Da), established that the mature toxin has the sequence DCOTSCOTTCANGWECCKGYOCVNKACSGCTH, where O is 4-hydroxyproline, W 6-bromotryptophan, and K 5-hydroxylysine, the asterisk represents the amidated C-terminus, and the calculated monoisotopic mass is 3487.09 Da. The eight Cys residues are arranged in a pattern (C-C-C-CC-C-C-C) not described previously in conotoxins. This arrangement, for which we propose the designation of framework #13 or XIII, differs from the ones (C-C-CC-CC-C-C and C-C-C-C-CC-C-C) present in other conotoxins which also contain eight Cys residues. This peptide thus defines a novel class of conotoxins, with a new posttranslational modification not previously found in other Conus peptide families.     

QTL mapping of slow-rusting, adult plant resistance to race Ug99 of stem rust fungus in PBW343/Muu RIL population

Races of stem rust fungus pose a major threat to wheat production worldwide. We mapped adult plant resistance (APR) to Ug99 in 141 lines of a PBW343/Muu recombinant inbred lines (RILs) population by phenotyping them for three seasons at Njoro, Kenya in field trials and genotyping them with Diversity Arrays Technology (DArT) markers. Moderately susceptible parent PBW343 and APR parent Muu displayed mean stem rust severities of 66.6 and 5 %, respectively. The mean disease severity of RILs ranged from 1 to 100 %, with an average of 23.3 %. Variance components for stem rust severity were highly significant (p < 0.001) for RILs and seasons and the heritability (h ²) for the disease ranged between 0.78 and 0.89. Quantitative trait loci (QTL) analysis identified four consistent genomic regions on chromosomes 2BS, 3BS, 5BL, and 7AS; three contributed by Muu (QSr.cim-2BS, QSr.cim-3BS and QSr.cim-7AS) and one (QSr.cim-5BL) derived from PBW343. RILs with flanking markers for these QTLs had significantly lower severities than those lacking the markers, and combinations of QTLs had an additive effect, significantly enhancing APR. The QTL identified on chromosome 3BS mapped to the matching region as the known APR gene Sr2. Four additional QTLs on chromosomes 1D, 3A, 4B, and 6A reduced disease severity significantly at least once in three seasons. Our results show a complex nature of APR to stem rust where Sr2 and other minor slow rusting resistance genes can confer a higher level of resistance when present together.     

Cytotoxic immunotherapy strategies for cancer: mechanisms and clinical development

Traditional therapies for cancer include surgery, chemotherapy, and radiation. Chemotherapy has widespread systemic cytotoxic effects against tumor cells but also affects normal cells. Radiation has more targeted local cytotoxicity but is limited to killing cells in the radiation field. Immunotherapy has the potential for systemic, specific killing of tumor cells. However, if the immune response is specific to a single antigen, tumor evasion can occur by down-regulation of that antigen. An immunotherapy approach that induces polyvalent immunity to autologous tumor antigens can provide a personalized vaccine with less potential for immunologic escape. A cytotoxic immunotherapy strategy creates such a tumor vaccine in situ. Immunogenic tumor cell death provides tumor antigen targets for the adaptive immune response and stimulates innate immunity. Attraction and activation of antigen presenting cells such as dendritic cells is important to process and present tumor antigens to T cells. These include cytotoxic T cells that kill tumor cells and T cells which positively and negatively regulate immunity. Tipping the balance in favor of anti-tumor immunity is an important aspect of an effective strategy. Clinically, immunotherapies may be most effective when combined with standard therapies in a complimentary way. An example is gene-mediated cytotoxic immunotherapy (GMCI) which uses an adenoviral vector, AdV-tk, to deliver a cytotoxic and immunostimulatory gene to tumor cells in vivo in combination with standard therapies creating an immunostimulatory milieu. This approach, studied extensively in animal models and early stage clinical trials, is now entering a definitive Phase 3 trial for prostate cancer.     

Assembly of lamina-specific neuronal connections by slit bound to type IV collagen

The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.