Identification of a novel RAMP-independent CGRP receptor antagonist

Identification of an HIV integrase inhibitor with micromolar affinity for the CGRP receptor led to the discovery of a series of structurally novel CGRP receptor antagonists. Optimization of this series produced compound 16, a low-molecular weight CGRP receptor antagonist with good pharmacokinetic properties in both rat and dog. In contrast to other nonpeptide antagonists, the activity of 16 was affected by the presence of divalent cations and showed evidence of an alternative, RAMP-independent CGRP receptor binding site.     

Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process

Adult conifers are notoriously recalcitrant in vegetative propagation and micropropagation that would result in the regeneration of juvenile propagules through somatic embryogenesis (SE) has not been demonstrated to date. Because SE-derived material is more amenable in subsequent tissue culture experiments compared with seed-derived material, a multi-year study was conducted to investigate induction of SE from primordial shoot (PS) explants that were excised from shoot buds of somatic embryo-derived white spruce. The SE induction experiments were carried out first with greenhouse-grown and later with field-grown trees each year from 2002 (2-year-old) to 2010 (10-year-old). Of the four genotypes tested, 893-2 and 893-12 never responded, 893-1 responded up to year 4 and 893-6 consistently responded every year. In 2010, for the first time, three of the 17 893-6 clonal trees produced male strobili as well as SE from cultured PS explants. SE induction was associated with formation of a nodule on the surface of an elongated needle primordium or in callus. Early somatic embryos were detectable after about 3 weeks of culture. Of 11 genes whose expression profiles were followed during the PS cultures, CHAP3A, VP1, WOX2 and SAP2C were expressed exclusively in the early stages of SE, and could potentially be used as markers of embryogenecity. Mature somatic embryos and plants were produced from the explants of responding genotype. Implication of these results for future research on adult conifer recalcitrance in micropropagation is discussed.     

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.     

Pdr5-mediated multidrug resistance requires the CPY-vacuolar sorting protein Vps3: are xenobiotic compounds routed from the vacuole to plasma membrane transporters for efflux?

In Saccharomyces cerevisiae several members of the ATP-binding cassette transporter superfamily efflux a broad range of xenobiotic substrates from cells. The vacuole also plays a critical role in multidrug resistance. Mutations in genes such as VPS3 that are essential for vacuolar acidification and carboxypeptidase Y vacuolar protein-sorting are multidrug sensitive. A similar phenotype is also observed with deletions of VPS15, VPS34, and VPS38, which encode essential members of the carboxypeptidase Y vacuolar protein-sorting pathway. Prior to the work described herein, detoxification by transporters and the vacuole were presumed to function independently. We demonstrate that this is not the case. Significantly, Vps3 has an epistatic relationship with Pdr5, a major yeast multidrug transporter. Thus, a double pdr5, vps3 deletion mutant is no more multidrug sensitive than its isogenic single-mutant counterparts. Subcellular fractionation experiments and analysis of purified plasma membrane vesicles indicate, however, that a vps3 mutation does not affect the membrane-localization or ATPase activity of Pdr5 even though rhodamine 6G efflux is reduced significantly. This suggests that Vps3 and probably other members of the carboxypeptidase Y vacuolar protein-sorting pathway are required for relaying xenobiotic compounds to transporters in the membrane.     

Interactions of isopenicillin N synthase with cyclopropyl-containing substrate analogues reveal new mechanistic insight

Isopenicillin N synthase (IPNS), a non-heme iron oxidase central to penicillin and cephalosporin biosynthesis, catalyzes an energetically demanding chemical transformation to produce isopenicillin N from the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV). We describe the synthesis of two cyclopropyl-containing tripeptide analogues, delta-(l-alpha-aminoadipoyl)-l-cysteinyl-beta-methyl-d-cyclopropylglycine and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-cyclopropylglycine, designed as probes for the mechanism of IPNS. We have solved the X-ray crystal structures of these substrates in complex with IPNS and propose a revised mechanism for the IPNS-mediated turnover of these compounds. Relative to the previously determined IPNS-Fe(II)-ACV structure, key differences exist in substrate orientation and water occupancy, which allow for an explanation of the differences in reactivity of these substrates.     

Research and conservation of the larger parrots of Africa and Madagascar: a review of knowledge gaps and opportunities

Parrot populations in Africa and Madagascar are declining and the need for conservation actions to address threats is increasingly recognised. Effective conservation requires a robust knowledge base on which decisions over appropriate actions can be made, yet at present there is no current and readily accessible synthesis of the status of populations, the threats they face and knowledge gaps. Here we begin to address this shortfall for the larger species in the region belonging to the genera Coracopsis, Poicephalus, Psittacus and Psittacula. We review developments since the production of the IUCN Parrot Action Plan published in 2000, identify areas where critical knowledge is lacking and highlight opportunities to address them. While advances have been made over the last decade, progress has not been evenly spread, with a strong bias towards populations in southern Africa. To date much research has focused on describing aspects of ecology and behaviour and there remains a need for studies determining the current status of populations and the factors limiting distributions and abundance. This review aims to provide a basis upon which progress towards an improved understanding of the conservation needs of the larger parrots of Africa and Madagascar can be made.