Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

A series of non-nucleoside-based 2,4-dinitrophenyl (DNP) phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid-phase synthesis. The length of spacer arm between the DNP label and the oligonucleotide phosphate backbone, and the number of attached DNP groups have both been varied in order to determine the optimum conditions for anti-DNP antibody binding. Detection using enzyme-linked colorimetric techniques showed sensitivity equivalent to that obtainable using biotinylated oligonucleotides.     

Edge effects influence the composition and density of reef residents on subtidal restored oyster reefs

Within estuarine and coastal ecosystems globally, extensive habitat degradation and loss threaten critical ecosystem functions and necessitate widescale restoration efforts. There is abundant evidence that ecological processes and species interactions can vary with habitat characteristics, which has important implications for the design and implementation of restoration efforts aimed at enhancing specific ecosystem functions and services. We conducted an experiment examining how habitat characteristics (presence; edge vs. interior) influence the communities of resident fish and mobile invertebrates on restored oyster (Crassostrea virginica) reefs. Similar to previous studies, we found that restored reefs altered community composition and augmented total abundance and biomass relative to unstructured sand habitat. Community composition and biomass also differed between the edge and interior of individual reefs as a result of species-specific patterns over small spatial scales. These patterns were only weakly linked to oyster density, suggesting that other factors that vary between edge and interior (e.g. predator access or species interactions) are likely more important for community structure on oyster reefs. Fine-scale information on resident species' use of oyster reefs will help facilitate restoration by allowing decision makers to optimize the amount of edge versus interior habitat. To improve the prediction of faunal use and benefits from habitat restoration, we recommend investigations into the mechanisms shaping edge and interior preferences on oyster reefs.     

Trophic ecology of juvenile bull sharks (Carcharhinus leucas) in the Coyote estuary,Costa Rica

Bull shark (Carcharhinus leucas) is a near-threatened elasmobranch species capable of moving between the fresh and salty waters of tropical and subtropical coastal areas, for which we still lack important ecological information. During their first years of life, bull sharks use estuarine systems as nursery areas, making them highly susceptible to environmental and anthropogenic pressures. We studied the trophic ecology of juveniles found in the Coyote estuary, a potential nursery area in Costa Rica, to understand the potential impact of further bull shark declines and gain knowledge that could aid in their conservation. We analysed the trophic ecology of juvenile bull sharks [81–103 cm total length (TL)] in the Coyote estuary, Costa Rica, using stable isotopes of δ¹⁵N and δ¹³C. Since one problem using this technique in juveniles is the confounding effect of the maternal signature, we sampled different tissues (muscle and plasma), verified the status of the shark's umbilical scar and identified the size at which the isotope signature is a result of the animal's current diet. The isotopic values of the muscle tissue reflected the maternal isotopic signature. In contrast, plasma values reflected the diet of juvenile bull sharks >95 cm TL and with a closed umbilical scar. Juvenile bull sharks fed primarily on teleost fishes of the order Anguilliformes and Siluriformes, and have a high trophic position (≥4.0) in the Coyote estuary. Our findings suggest that this estuary is an important feeding site for juvenile bull sharks of the Pacific of Costa Rica. Thus, the protection of essential habitats such as the Coyote estuary will benefit not only bull shark conservation, but also the conservation of an array of fish species that also use this habitat as a rookery, many of which are of commercial interest.     

Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.     

Comparisons of biomass,water use efficiency and water use strategies across five genomic groups of Populus and its hybrids

Genetic improvement and hybridization in the Populus genus have led to the development of genotypes exhibiting fast growth, high rooting ability and disease resistance. However, while large biomass production is important for bioenergy crops, efficient use of resources including water is also important in sites lacking irrigation and for maintaining ecosystem water availability. In addition, comparison of water use strategies across a range of growth rates and genetic variability can elucidate whether certain strategies are shared among the fastest growing and/or most water use efficient genotypes. We estimated tree water use throughout the second growing season via sapflow sensors of 48 genotypes from five Populus taxa; P. deltoides W. Bartram ex Marshall × P. deltoides (D × D), P. deltoides × P. maximowiczii A. Henry (D × M), P. deltoides × P. nigra L. (D × N), P. deltoides × P. trichocarpa Torr. & Gray (D × T) and P. trichocarpa × P. deltoides (T × D) and calculated average canopy stomatal conductance (G_S). We regressed G_S and atmospheric vapor pressure deficit (VPD) wherein the slope of the relationship represents stomatal sensitivity to VPD. At the end of the second growing season, trees were harvested, and their dry woody biomass was used to calculate whole tree water use efficiency (WUE_T). We found that D × D and D × M genotypes exhibited differing water use strategies with D × D genotypes exhibiting high stomatal sensitivity while retaining leaves while D × M genotypes lost leaf area throughout the growing season but exhibited low stomatal sensitivity. Across measured taxa, biomass growth was positively correlated with WUE_T, and genotypes representing each measured taxa except D × N and T × D had high 2-year dry biomass of above 6 kg/tree. Overall, these data can be used to select Populus genotypes that combine high biomass growth with stomatal sensitivity and WUE_T to limit the negative impacts of bioenergy plantations on ecosystem water resources.     

COMPONENTS OF PHENOTYPIC SELECTION: POLLEN EXPORT AND FLOWER COROLLA WIDTH IN IPOMOPSIS AGGREGATA

In the hummingbird-pollinated herb Ipomopsis aggregata, selection through male function during pollination favors wide corolla tubes. We explored the mechanisms behind this selection, using phenotypic selection analysis to compare effects of corolla width on two components of male pollination success, pollinator visit rate and pollen exported per visit. During single visits by captive hummingbirds, flowers with wider corollas exported more pollen, and more dye used as a pollen analogue, to stigmas of recipient flowers. Corolla width was less strongly related to visit rate in the field, and had no direct effect on visit rate after nectar production and corolla length were controlled for. Moreover, the phenotypic selection differential was 80% higher for the effect on pollen exported per visit, suggesting that this is the more important mechanism of selection.     

Identification of cDNAs encoding isoprenylated proteins

Isoprenylated proteins are involved in signal transduction, control of cell growth and differentiation, organization of the nuclear lamina and cytoskeleton, and vesicle sorting. The isoprenoid moiety facilitates the interaction of these proteins with membranes and/or other proteins. However, many isoprenylated proteins remain unidentified. A method is described for identifying novel and known cDNAs encoding isoprenylated proteins. Sufficient details of the screening procedure are given so that this method may be easily used to identify cDNAs encoding other covalently modified proteins or proteins possessing high affinity ligand binding sites.     

The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System

Abstract: The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P₀ mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P₀ gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.     

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall.

Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients.

The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments.

Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.     

Chaperone SecB: Conformational changes demonstrated by circular dichroism

The chaperone SecB, which is involved in protein export inEscherichia coli, is shown by circular dichroism measurements to contain a high content of-pleated sheets. Prediction of the secondary structure of SecB is in good agreement with the observed content of-sheet. In accordance with the previous studies in which changes in conformation were assessed indirectly [Randall (1992),Science 257, 241–245], here we show that the conformation of SecB changes with the concentration of salt in the milieu and also when SecB interacts with a peptide ligand.Abbreviations ANS 1-anilino-naphthalene-8-sulfonate - CD circular dichroism - NMR nuclear magnetic resonance - CCA convex constraint analysis