Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads

Lipobeads are hydrogel beads surrounded by a lipid bilayer membrane and have been developed to act as a cell analogue. The FLAG-tagged M(2) muscarinic receptor was incorporated onto the surface of the Lipobead by incubating pre-Lipobeads with proteoliposomes containing the receptor. Receptors reconstituted onto the surface of the Lipobeads were functional in that they bound the antagonists quinuclidinylbenzilate and scopolamine with characteristic muscarinic affinities. This demonstrates the feasibility of using Lipobeads to study the binding properties of the M(2) muscarinic receptor and offers a promising approach to the study of transmembrane protein biology in general.     

Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches

The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm.     

Goldfish color vision sensitivity is high under light-adapted conditions

The wavelength discrimination threshold of three goldfish was examined in a series of behavioral experiments. Using an auto-shaping technique, detection thresholds were established for 531 and 648 nm spectral increments presented on a 6.6 cd m^–2 white background. Next, discrimination between the wavelengths was established at equal, suprathreshold, intensities. Finally, the intensities of the two stimuli were reduced to establish the intensity threshold for the wavelength discrimination. The results indicate that goldfish, like several mammalian species, can discriminate wavelength at detection threshold intensity. This finding suggests that high color sensitivity is not confined to mammals or dependent upon a very high percentage of wavelength opponent ganglion cells. Rather, high color vision sensitivity may be based upon an inherent sensitivity advantage of wavelength opponent receptive fields compared to non-wavelength opponent receptive fields and be an important selective advantage of wavelength opponency and color vision.     

Haplotype‐based genotyping‐by‐sequencing in oat genome research

In a de novo genotyping‐by‐sequencing (GBS) analysis of short, 64‐base tag‐level haplotypes in 4657 accessions of cultivated oat, we discovered 164741 tag‐level (TL) genetic variants containing 241224 SNPs. From this, the marker density of an oat consensus map was increased by the addition of more than 70000 loci. The mapped TL genotypes of a 635‐line diversity panel were used to infer chromosome‐level (CL) haplotype maps. These maps revealed differences in the number and size of haplotype blocks, as well as differences in haplotype diversity between chromosomes and subsets of the diversity panel. We then explored potential benefits of SNP vs. TL vs. CL GBS variants for mapping, high‐resolution genome analysis and genomic selection in oats. A combined genome‐wide association study (GWAS) of heading date from multiple locations using both TL haplotypes and individual SNP markers identified 184 significant associations. A comparative GWAS using TL haplotypes, CL haplotype blocks and their combinations demonstrated the superiority of using TL haplotype markers. Using a principal component‐based genome‐wide scan, genomic regions containing signatures of selection were identified. These regions may contain genes that are responsible for the local adaptation of oats to Northern American conditions. Genomic selection for heading date using TL haplotypes or SNP markers gave comparable and promising prediction accuracies of up to r = 0.74. Genomic selection carried out in an independent calibration and test population for heading date gave promising prediction accuracies that ranged between r = 0.42 and 0.67. In conclusion, TL haplotype GBS‐derived markers facilitate genome analysis and genomic selection in oat.     

A MULTI‐GENE MOLECULAR INVESTIGATION OF THE KELP (LAMINARIALES,PHAEOPHYCEAE) SUPPORTS SUBSTANTIAL TAXONOMIC RE‐ORGANIZATION1

Every year numerous ecological, biochemical, and physiological studies are performed using members of the order Laminariales. Despite the fact that kelp are some of the most intensely studied macroalgae in the world, there is significant debate over the classification within and among the three “derived” families, the Alariaceae, Laminariaceae, and Lessoniaceae (ALL). Molecular phylogenies published for the ALL families have generated hypotheses strongly at odds with the current morphological taxonomy; however, conflicting phylogenetic hypotheses and consistently low levels of support realized in all of these studies have resulted in conservative approaches to taxonomic revisions. In order to resolve relationships within this group we have sequenced over 6000 bp from regions in the nuclear, chloroplast, and mitochondrial genomes and included 42 taxa in Bayesian, neighbor‐joining, and parsimony analyses. The result is the first comprehensive and well‐supported molecular phylogeny for the ALL complex of the Laminariales. We maintain the three recognized families (Alariaceae, Laminariaceae, and Lessoniaceae), but with vastly different compositions, as well as propose the Costariaceae fam. nov. for Agarum, Costaria, Dictyoneurum, and Thalassiophyllum, the only genera in the Laminariales with flattened, occasionally terete, stipes and either a perforate or reticulate blade. In addition, our data strongly support a split of the genus Laminaria. We resurrect the genus Saccharina Stackhouse for the Laminaria clade that does not contain L. digitata (Hudson) J.V. Lamouroux, the type of the genus.     

The roles of the hybrid cluster protein,Hcp and its reductase,Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress

The hybrid cluster protein, Hcp, contains a 4Fe‐2S‐2O iron‐sulfur‐oxygen cluster that is currently considered to be unique in biology. It protects various bacteria from nitrosative stress, but the mechanism is unknown. We demonstrate that the Escherichia coli Hcp is a high affinity nitric oxide (NO) reductase that is the major enzyme for reducing NO stoichiometrically to N₂O under physiologically relevant conditions. Deletion of hcp results in extreme sensitivity to NO during anaerobic growth and inactivation of the iron‐sulfur proteins, aconitase and fumarase, by accumulated cytoplasmic NO. Site directed mutagenesis revealed an essential role in NO reduction for the conserved glutamate 492 that coordinates the hybrid cluster. The second gene of the hcp‐hcr operon encodes an NADH‐dependent reductase, Hcr. Tight interaction between Hcp and Hcr was demonstrated. Although Hcp and Hcr purified individually were inactive or when recombined, a co‐purified preparation reduced NO in vitro with a Km for NO of 500 nM. In an hcr mutant, Hcp is reversibly inactivated by NO concentrations above 200 nM, indicating that Hcr protects Hcp from nitrosylation by its substrate, NO.     

Unmasking alpha diversity,cladogenesis and biogeographical patterning in an ancient panarthropod lineage (Onychophora: Peripatopsidae: Opisthopatus cinctipes) with the description of five novel species

Speciation and biogeographical patterning in the velvet worm Opisthopatus cinctipes was examined under a null hypothesis that numerous discrete lineages are nested within the species. A total of 184 O. cinctipes specimens, together with a single specimen of each of the two congeneric point endemic sister species (O. roseus and O. herbertorum), were collected throughout the forest archipelago in the Eastern Cape, KwaZulu‐Natal and Mpumalanga provinces of South Africa. All specimens were sequenced for two partial mitochondrial DNA loci (COI and 12S rRNA), while a single specimen from each locality was sequenced for the nuclear 18S rRNA locus. Evolutionary relationships were assessed using maximum‐likelihood and Bayesian inferences, while divergence time estimations were conducted using BEAST. A Bayesian species delimitation approach was undertaken to explore the number of possible novel lineages nested within Opisthopatus, while population genetic structure was examined for the COI locus using ARLEQUIN. Phylogenetic results revealed that O. cinctipes is a species complex comprising seven geographically discrete and statistically well‐supported clades. An independent statistical approach to species delimitations circumscribed ca. 67 species. Results from divergence time estimation and rate constancy tests revealed near constant net diversification occurring throughout the Eocene and Oligocene with subdivision of ranges during the Miocene. Gross morphological characters such as leg pair number within O. cinctipes were invariant, while dorsal and ventral integument colour was highly polymorphic. However, scanning electron microscopy revealed considerable differences both between and within clades. The caveats associated with both morphological and algorithmic delineation of species boundaries are discussed. The five novel Opisthopatus species are described.     

Sodium Selenite Prevents Paraquat-Induced Neurotoxicity in Zebrafish

Considering the antioxidant properties of sodium selenite (Na₂SeO₃) and the involvement of oxidative stress events in paraquat-induced neurotoxicity, this study investigated the protective effect of dietary Na₂SeO₃ on biochemical and behavioral parameters of zebrafish exposed to paraquat (PQ). Fish were pretreated with a Na₂SeO₃ diet for 21 days and then PQ (20 mg/kg) was administered intraperitoneally with six injections for 16 days. In the novel tank test, the Na₂SeO₃ diet prevented the locomotor impairments, as well as the increase in the time spent in the top area of the tank, and the exacerbation of freezing episodes. In the preference for conspecifics and in the mirror-induced aggression (MIA) tasks, Na₂SeO₃ prevented the increase in the latency to enter the area closer to conspecifics and the agonistic behavior of PQ-treated animals, respectively. Na₂SeO₃ prevented the increase of carbonylated protein (CP), reactive oxygen species (ROS), and nitrite/nitrate (NOx) levels, as well as the decrease in non-protein thiols (NPSH) levels. Regarding the antioxidant enzymatic defenses, Na₂SeO₃ prevented the increase in catalase (CAT) and glutathione peroxidase (GPx) activities caused by PQ. Altogether, dietary Na₂SeO₃ improves behavioral and biochemical function impaired by PQ treatment in zebrafish, by modulating not only redox parameters, but also anxiety- and aggressive-like phenotypes in zebrafish.     

A rapid and efficient method to remove labelled molecular probes from nucleic acids immobilized on solid supports

A rapid and efficient method is described for the removal of radio-active molecular probes from nucleic acids immobilized on nylon membranes. This method involves boiling in distilled water in a microwave oven. This procedure can be completed within ten minutes, does not require the use of any buffers or reagents, and produces results comparable with conventional buffer-wash procedures recommended by the suppliers of the transfer membranes.