Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.     

Complex traits analysis of chicken growth using targeted genetical genomics

Dissecting the genetic control of complex trait variation remains very challenging, despite many advances in technology. The aim of this study was to use a major growth quantitative trait locus (QTL) in chickens mapped to chromosome 4 as a model for a targeted approach to dissect the QTL. We applied a variant of the genetical genomics approach to investigate genome-wide gene expression differences between two contrasting genotypes of a marked QTL. This targeted approach allows the direct quantification of the link between the genotypes and the genetic responses, thus narrowing the QTL-phenotype gap using fewer samples (i.e. microarrays) compared with the genome-wide genetical genomics studies. Four differentially expressed genes were localized under the region of the QTL. One of these genes is a potential positional candidate gene (AADAT) that affects lysine and tryptophan metabolism and has alternative splicing variants between the two genotypes. In addition, the lysine and glycolysis metabolism pathways were significantly enriched for differentially expressed genes across the genome. The targeted approach provided a complementary route to fine mapping of QTL by characterizing the local and the global downstream effects of the QTL and thus generating further hypotheses about the action of that QTL.     

Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb

Establishment of limb innervation by motor neurons involves a series of hierarchical axon guidance decisions by which motor-neuron subtypes evaluate peripheral guidance cues and choose their axonal trajectory. Earlier work indicated that the pathway into the dorsal limb by lateral motor column (LMC[l]) axons requires the EphA4 receptor, which mediates repulsion elicited by ephrinAs expressed in ventral limb mesoderm. Here, we implicate glial-cell-line-derived neurotrophic factor (GDNF) and its receptor, Ret, in the same guidance decision. In Gdnf or Ret mutant mice, LMC(l) axons follow an aberrant ventral trajectory away from dorsal territory enriched in GDNF, showing that the GDNF/Ret system functions as an instructive guidance signal for motor axons. This phenotype is enhanced in mutant mice lacking Ret and EphA4. Thus, Ret and EphA4 signals cooperate to enforce the precision of the same binary choice in motor-axon guidance.     

Soil factors affecting selenium concentration in wheat grain and the fate and speciation of Se fertilisers applied to soil

UK crops have a low selenium (Se) status, therefore Se fertilisation of wheat (Triticum aestivum L.) at 10 field sites was investigated and the effect on the content and speciation of Se in soils determined. Soil characterisation was carried out at each field site to determine the soil factors that may influence wheat grain Se concentrations in unfertilised plots. Soil samples were taken after harvest from each treatment to determine the fate and speciation of selenate fertiliser applied to soil. Wheat grain Se concentrations could be predicted from soil Se concentration and soil extractable sulphur (S) using the following regression model: Grain Se?=?a?+?b(total soil Se)?+?c(extractable soil Se) - d(extractable soil S), with 86 % of the variance being accounted for, suggesting that these properties control Se concentrations in grain from unfertilised plots. Extractable soil Se concentrations were low (2.4 – 12.4 µg kg^?1) and predominantly consisted of selenite (up to 70 % of extractable Se) and soluble organic forms, whereas selenate was below the detection limit. Little of the added Se, in either liquid or granular form was left in the soil after crop harvest. Se fertilisation up to 20 g ha^?1 did not lead to a significant Se accumulation in the soil, suggesting losses of Se unutilised by the crop.     

Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse cortex

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton–spine interface and an increase in glutamate transporter expression in astrocytic processes.     

Passive flow through an unstalked intertidal ascidian: orientation and morphology enhance suspension feeding in Pyura stolonifera

Passive flow is believed to increase the gains and reduce the costs of active suspension feeding. We used a mixture of field and laboratory experiments to evaluate whether the unstalked intertidal ascidian Pyura stolonifera exploits passive flow. We predicted that its orientation to prevailing currents and the arrangement of its siphons would induce passive flow due to dynamic pressure at the inhalant siphon, as well as by the Bernoulli effect or viscous entrainment associated with different fluid velocities at each siphon, or by both mechanisms. The orientation of P. stolonifera at several locations along the Sydney-Illawarra coast (Australia) covering a wide range of wave exposures was nonrandom and revealed that the ascidians were consistently oriented with their inhalant siphons directed into the waves or backwash. Flume experiments using wax models demonstrated that the arrangement of the siphons could induce passive flow and that passive flow was greatest when the inhalant siphon was oriented into the flow. Field experiments using transplanted animals confirmed that such an orientation resulted in ascidians gaining food at greater rates, as measured by fecal production, than when oriented perpendicular to the wave direction. We conclude that P. stolonifera enhances suspension feeding by inducing passive flow and is, therefore, a facultatively active suspension feeder. Furthermore, we argue that it is likely that many other active suspension feeders utilize passive flow and, therefore, measurements of their clearance rates should be made under appropriate conditions of flow to gain ecologically relevant results.     

Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta

Almost all TGF-beta is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-beta binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-beta cytokine. LAP is the TGF-beta pro-peptide that is cleaved intracellularly prior to secretion, and TGF-beta binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-beta. Previous studies have revealed that the LTBP-LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3(LTBP1). Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs.     

Identification of asteroid genera with species capable of larval cloning

Asexual reproduction in larvae, larval cloning, is a recently recognized component of the complex life histories of asteroids. We compare DNA sequences of mitochondrial tRNA genes (Ala, Leu, Asn, Pro, and Gln) from larvae in the process of cloning collected in the field with sequences from adults of known species in order to identify asteroid taxa capable of cloning. Neighbor-joining analysis identified four distinct groups of larvae, each having no, or very little, sequence divergence (p distances ranging from 0.00000 to 0.02589); thus, we conclude that each larval group most likely represents a single species. These field-collected larvae cannot be identified to species with certainty, but the close assemblage of known taxa with the four larval groups indicates generic or familial identity. We can assign two of the larval groups discerned here to the genera Luidia and Oreaster and another two to the family Ophidiasteridae. This study is the first to identify field-collected cloning asteroid larvae, and provides evidence that larval cloning is phylogenetically widespread within the Asteroidea. Additionally, we note that cloning occurs regularly and in multiple ways within species that are capable of cloning, emphasizing the need for further investigation of the role of larval cloning in the ecology and evolution of asteroids.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.