The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Phi = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.     

Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.     

Spatial and temporal population genetic structure of four northeastern Pacific littorinid gastropods: the effect of mode of larval development on variation at one mitochondrial and two nuclear DNA markers

We investigated the effect of development mode on the spatial and temporal population genetic structure of four littorinid gastropod species. Snails were collected from the same three sites on the west coast of Vancouver Island, Canada in 1997 and again in 2007. DNA sequences were obtained for one mitochondrial gene, cytochrome b ( Cyt b ), and for up to two nuclear genes, heat shock cognate 70 ( HSC70 ) and aminopeptidase N intron ( APN54 ). We found that the mean level of genetic diversity and long-term effective population sizes ( N _e) were significantly greater for two species, Littorina scutulata and L. plena , that had a planktotrophic larval stage than for two species, Littorina sitkana and L. subrotundata , that laid benthic egg masses which hatched directly into crawl-away juveniles. Predictably, two poorly dispersing species, L. sitkana and L. subrotundata , showed significant spatial genetic structure at an 11- to 65-km geographical scale that was not observed in the two planktotrophic species. Conversely, the two planktotrophic species had more temporal genetic structure over a 10-year interval than did the two direct-developing species and showed highly significant temporal structure for spatially pooled samples. The greater temporal genetic variation of the two planktotrophic species may have been caused by their high fecundity, high larval dispersal, and low but spatially correlated early survivorship. The sweepstakes-like reproductive success of the planktotrophic species could allow a few related females to populate hundreds of kilometres of coastline and may explain their substantially larger temporal genetic variance but lower spatial genetic variance relative to the direct-developing species.     

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease ^1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest ^5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5''mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) ⁸. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA ^{2, 4, 9-11}.     

Mechanochemical coupling in spin-labeled, active, isometric muscle

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force?. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.     

Indoline derivatives as 5-HT(2C) receptor agonists

A series of 1-(1-indolinyl)-2-propylamines was synthesised and evaluated as 5-HT(2C) receptor agonists for the treatment of obesity. The general methods of synthesis of the precursor indoles are described. The functional efficacy and radioligand binding data for all of the compounds at 5-HT(2) receptor subtypes are reported. A number of compounds were found to reduce food intake in rats after oral administration.     

A limited survey of aflatoxins and fumonisins in retail maizebased products in the UK using immunoassay detection

A total of 27 maize-based products destined for human consumption were collected from retail outlets within the city of Glasgow in the UK and were analysed for the presence of aflatoxins using immunoaftinity column chromatography with fluorescence detection and for fumonisins by competitive ELISA. Aflatoxins were detected at a trace level below 4 in eight (30%) of the 27 samples tested, no sample contained aflatoxins at a high level although one sample of sweetcorn did contain aflatoxins at a level of 5-10 Fumonisins were detected in eight (30%) of the samples at levels from 1 to 8mgkg^-1 and a further eight samples contained fumonisin at a level below 1 mgkg^-1 but above the detectable level. The highest concentration of fumonisins was found in a sample of fine corn meal at 8-12mgkg^-1.     

Phospholipid composition of human monocytes and alterations occurring due to culture and stimulation by C3b

The phospholipid composition of human peripheral blood monocytes has not been previously reported, due to difficulty in isolating these cells in a purified state. In this study, monocytes were purified by counterflow centrifugation without selective adherence, and were characterized with the use of fluorescent monoclonal antibodies to T and B lymphocytes and monocytes by flow cytometry. These platelet-free cell preparations contained less than 5% T cells and less than 3% B cells. Isolated monocytes, which were rapidly frozen after isolation, contained phospholipids (in order of decreasing concentrations) as follows: phosphatidylcholine greater than phosphatidylethanolamine greater than sphingomyelin greater than phosphatidylserine greater than phosphatidylinositol greater than cardiolipin. A small amount of lyso-PC, but no lyso-PE, phosphatidic acid or lyso-PI, was found. The effect of culturing these cells in the presence or absence of a known stimulant of monocyte prostaglandin E and thromboxane release, the C3b fragment of the third component of human complement (C3), was studied with regard to phospholipid composition. Monocytes cultured without stimulant for 24 h contained 3-4% more sphingomyelin than did uncultured cells, and lyso-PC concentrations were consistently elevated. The addition of the stimulant C3b to cultured cells resulted in enhancement of release of immunoreactive prostaglandin E into culture supernatants, without affecting the release of lysosomal enzymes. Analysis of the phospholipid content of cells cultured in the presence of C3b revealed that there was a significant decrease in total PI compared to cells cultured in the absence of C3b, in addition to an increased concentration of sphingomyelin and lyso-PC when compared to freshly isolated cells. These changes occurred in the absence of elevated concentrations of phosphatidic acid.