Rosiglitazone stimulates nitric oxide synthesis in human aortic endothelial cells via AMP-activated protein kinase

The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-gamma inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1.     

Traits associated with naturalization in <Emphasis Type=Italic>Anolis</Emphasis> lizards: comparison of morphological,distributional, anthropogenic,and phylogenetic models

The worldwide spread of invasive species affects native biodiversity and causes economic loss, but also allows better understanding of historical biogeographic patterns. Prediction of likely invaders facilitates economic and conservation decisions and gives insight into characteristics that have allowed natural colonization over evolutionary time. However, it is not clear what types of characters best predict naturalization or even whether naturalization is predictable at all. Squamate reptiles have been understudied subjects for invasion biology. Lizards of the genus Anolis have been highly successful colonizers both recently and over evolutionary time. Nineteen of the approximately 350 described species of Anolis have established naturalized populations. We constructed models of naturalization using morphological, distributional, anthropogenic, and phylogenetic characters and compared these single character class models to each other and to a composite model incorporating all four classes. We show that (1) each class of variables significantly predicts invasion, (2) a composite model significantly outperforms each of the submodels, and (3) the final composite model displays extraordinary ability to objectively identify naturalized species of Anolis.     

Amplification ratio control system for copy number variation genotyping

We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP') in each strand form a hairpin. The bases immediately beyond the 3'-end of the UP and 5' of UP' are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3'-ends of amplicon strands. The resultant 'amplification ratio control system' (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and 'chemokine (C-C motif) ligand 3-like 1' gene.     

Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe,Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.     

Effect of Marine Bacterial Isolates on the Growth and Morphology of Axenic Plantlets of the Green Alga Ulva linza

The green marine macroalga, Ulva linza, adopts an “atypical” form when grown in the absence of bacteria. Twenty unique strains of periphytic bacteria, isolated from three species of Ulva, were identified by 16S rDNA sequencing. These isolates were assessed for their effect on the growth and morphological development of axenic plantlets of U. linza. Results showed that the effect of bacterial strains was strain- but not taxon-specific. Thirteen isolates returned the aberrant morphology to normal and of these, five also significantly increased growth rate. One isolate increased growth, but had no effect on morphology. Biofilms of some of these isolates stimulated the settlement of Ulva zoospores but there was no correlation between bacterial isolates that stimulated zoospore settlement and those that initiated changes in morphology and/or growth of the cultured alga.     

Investigating a Mystery Disease: Tales from a Viral Detective

Viral outbreak investigation is challenging logistically as well as scientifically. In the context of addressing a fictional emerging viral disease, I describe the process of discovery, from the initial report of a problem through discussions of intellectual property and sample management, study design, management, experimental execution, and reporting of results.     

JA but not JA‐Ile is the cell‐nonautonomous signal activating JA mediated systemic defenses to herbivory in Nicotiana attenuata

The whole‐plant activation of defense responses to wounding and herbivory requires systemic signaling in which jasmonates (JAs) play a pivotal role. To examine the nature of the slower cell‐nonautonomous as compared to the rapid cell‐autonomous signal in mediating systemic defenses in Nicotiana attenuata, reciprocal stem grafting‐experiments were used with plants silenced for the JA biosynthetic gene ALLENE OXIDE CYCLASE (irAOC) or plants transformed to create JA sinks by ectopically expressing Arabidopsis JA‐O‐methyltransferase (ovJMT). JA‐impaired irAOC plants were defective in the cell‐nonautonomous signaling pathway but not in JA transport. Conversely, ovJMT plants abrogated the production of a graft‐transmissible JA signal. Both genotypes displayed unaltered cell‐autonomous signaling. Defense responses (17‐hydroxygeranyllinalool diterpene glycosides, nicotine, and proteinase inhibitors) and metabolite profiles were differently induced in irAOC and ovJMT scions in response to graft‐transmissible signals from elicited wild type stocks. The performance of Manduca sexta larvae on the scions of different graft combinations was consistent with the patterns of systemic defense metabolite elicitations. Taken together, we conclude that JA and possibly MeJA, but not JA‐Ile, either directly functions as a long‐distance transmissible signal or indirectly interacts with long distance signal(s) to activate systemic defense responses.