High affinity inhibitors of the dopamine transporter (DAT): novel biotinylated ligands for conjugation to quantum dots

Compounds capable of inhibiting the dopamine transporter protein (DAT) that can be conjugated to cadmium selenide/zinc sulfide/core shell nanocrystals may be used to image the location and distribution of the DAT in neuronal cell membranes. This letter describes the synthesis of biotinylated analogs of the DAT antagonists GBR 12909 and GBR 12935 that can be attached to streptavidin coated cadmium selenide/zinc sulfide/core shell nanocrystals.     

Assessing strategies for improved superfamily recognition

There are more than 200 completed genomes and over 1 million nonredundant sequences in public repositories. Although the structural data are more sparse (approximately 13,000 nonredundant structures solved to date), several powerful sequence-based methodologies now allow these structures to be mapped onto related regions in a significant proportion of genome sequences. We review a number of publicly available strategies for providing structural annotations for genome sequences, and we describe the protocol adopted to provide CATH structural annotations for completed genomes. In particular, we assess the performance of several sequence-based protocols employing Hidden Markov model (HMM) technologies for superfamily recognition, including a new approach (SAMOSA [sequence augmented models of structure alignments]) that exploits multiple structural alignments from the CATH domain structure database when building the models. Using a data set of remote homologs detected by structure comparison and manually validated in CATH, a single-seed HMM library was able to recognize 76% of the data set. Including the SAMOSA models in the HMM library showed little gain in homolog recognition, although a slight improvement in alignment quality was observed for very remote homologs. However, using an expanded 1D-HMM library, CATH-ISL increased the coverage to 86%. The single-seed HMM library has been used to annotate the protein sequences of 120 genomes from all three major kingdoms, allowing up to 70% of the genes or partial genes to be assigned to CATH superfamilies. It has also been used to recruit sequences from Swiss-Prot and TrEMBL into CATH domain superfamilies, expanding the CATH database eightfold.     

Host diversity and behavior determine patterns of interspecies transmission and geographic diffusion of avian influenza A subtypes among North American wild reservoir species

Wild birds can carry avian influenza viruses (AIV), including those with pandemic or panzootic potential, long distances. Even though AIV has a broad host range, few studies account for host diversity when estimating AIV spread. We analyzed AIV genomic sequences from North American wild birds, including 303 newly sequenced isolates, to estimate interspecies and geographic viral transition patterns among multiple co-circulating subtypes. Our results show high transition rates within Anseriformes and Charadriiformes, but limited transitions between these orders. Patterns of transition between species were positively associated with breeding habitat range overlap, and negatively associated with host genetic distance. Distance between regions (negative correlation) and summer temperature at origin (positive correlation) were strong predictors of transition between locations. Taken together, this study demonstrates that host diversity and ecology can determine evolutionary processes that underlie AIV natural history and spread. Understanding these processes can provide important insights for effective control of AIV.     

Patellaria: A new genus in the Chenopodiaceae

Comparative taxonomic studies within the genus Beta have shown that the section Patellares is distinct and should be regarded as a new genus, Patellaria, with the type Patellaria webbiana. The transference of Beta patellaris to this genus requires a new specific name, P. cordata.     

Helicobacter felis�CAssociated Gastric Disease in Microbiota-Restricted Mice

Human Helicobacter pylori infection leads to multiple pathological consequences, including gastritis and adenocarcinoma. Although this association has led to the classification of H. pylori as a type 1 carcinogen, it is not clear if additional nonhelicobacter gastric microbiota play a role in these diseases. In this study, we utilized either specific pathogen-free C57BL/6 mice (B6.SPF) or mice colonized with altered Schaedler flora (B6.ASF) to evaluate the role of nonhelicobacter gastric microbiota in disease development after Helicobacter felis infection. Despite similar histological changes, H. felis persisted in B6.ASF stomachs, while H. felis could no longer be detected in the majority of B6.SPF mice. The B6.SPF mice also acquired multiple Lactobacillus spp. in their stomachs after H. felis infection. Our data indicate that potential mechanisms responsible for the ineffective H. felis clearance in the B6.ASF model include the absence of new gastric microbiota to compete for the gastric niche, the lack of expression of new gastric mucins, and a reduced ratio of H. felis–specific IgG2c:IgG1 serum antibodies. These data suggest that although H. felis is sufficient to initiate gastric inflammation and atrophy, bacterial eradication and the systemic immune response to infection are significantly influenced by pre-existing and acquired gastric microbiota.     

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.     

Conversion of AFLP markers to high-throughput markers in a complex polyploid,sugarcane

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications.     

A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video-microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step-up and step-down stimuli. After a step-up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step-down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.