NMR structural characterization of a minimal peptide antagonist bound to the extracellular domain of the corticotropin-releasing factor1 receptor

Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF(1) receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF(1) receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30-41 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.     

Foliar water supply of tall trees: evidence for mucilage-facilitated moisture uptake from the atmosphere and the impact on pressure bomb measurements

The water supply to leaves of 25 to 60 m tall trees (including high-salinity-tolerant ones) was studied. The filling status of the xylem vessels was determined by xylem sap extraction (using jet-discharge, gravity-discharge, and centrifugation) and by (1)H nuclear magnetic resonance imaging of wood pieces. Simultaneously, pressure bomb experiments were performed along the entire trunk of the trees up to a height of 57 m. Clear-cut evidence was found that the balancing pressure (P(b)) values of leafy twigs were dictated by the ambient relative humidity rather than by height. Refilling of xylem vessels of apical leaves (branches) obviously mainly occurred via moisture uptake from the atmosphere. These findings could be traced back to the hydration and rehydration of mucilage layers on the leaf surfaces and/or of epistomatal mucilage plugs. Xylem vessels also contained mucilage. Mucilage formation was apparently enforced by water stress. The observed mucilage-based foliar water uptake and humidity dependency of the P(b) values are at variance with the cohesion-tension theory and with the hypothesis that P(b) measurements yield information about the relationships between xylem pressure gradients and height.     

Using Confidence Set Heuristics During Topology Search Improves the Robustness of Phylogenetic Inference

We examine the impact of likelihood surface characteristics on phylogenetic inference. Amino acid data sets simulated from topologies with branch length features chosen to represent varying degrees of difficulty for likelihood maximization are analyzed. We present situations where the tree found to achieve the global maximum in likelihood is often not equal to the true tree. We use the program covSEARCH to demonstrate how the use of adaptively sized pools of candidate trees that are updated using confidence tests results in solution sets that are highly likely to contain the true tree. This approach requires more computation than traditional maximum likelihood methods, hence covSEARCH is best suited to small to medium-sized alignments or large alignments with some constrained nodes. The majority rule consensus tree computed from the confidence sets also proves to be different from the generating topology. Although low phylogenetic signal in the input alignment can result in large confidence sets of trees, some biological information can still be obtained based on nodes that exhibit high support within the confidence set. Two real data examples are analyzed: mammal mitochondrial proteins and a small tubulin alignment. We conclude that the technique of confidence set optimization can significantly improve the robustness of phylogenetic inference at a reasonable computational cost. Additionally, when either very short internal branches or very long terminal branches are present, confident resolution of specific bipartitions or subtrees, rather than whole-tree phylogenies, may be the most realistic goal for phylogenetic methods. [Reviewing Editor: Dr. Nicolas Galtier]     

Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D

Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines are currently being developed for both human immunodeficiency virus type 1 and other pathogens. The potential limitations associated with rAd5 vectors, however, have led to the construction of novel rAd vectors derived from rare Ad serotypes. Several rare serotype rAd vectors have already been described, but a detailed comparison of multiple rAd vectors from subgroups B and D has not previously been reported. Such a comparison is critical for selecting optimal rAd vectors for advancement into clinical trials. Here we describe the construction of three novel rAd vector systems from Ad26, Ad48, and Ad50. We report comparative seroprevalence and immunogenicity studies involving rAd11, rAd35, and rAd50 vectors from subgroup B; rAd26, rAd48, and rAd49 vectors from subgroup D; and rAd5 vectors from subgroup C. All six rAd vectors from subgroups B and D exhibited low seroprevalence in a cohort of 200 individuals from sub-Saharan Africa, and they elicited Gag-specific cellular immune responses in mice both with and without preexisting anti-Ad5 immunity. The rAd vectors from subgroup D were also evaluated using rhesus monkeys and were shown to be immunogenic after a single injection. The rAd26 vectors proved the most immunogenic among the rare serotype rAd vectors studied, although all rare serotype rAd vectors were still less potent than rAd5 vectors in the absence of anti-Ad5 immunity. These studies substantially expand the portfolio of rare serotype rAd vectors that may prove useful as vaccine vectors for the developing world.     

Randomized trial of a multifaceted commercial weight loss program

Objective: To test whether a commercial weight loss program promotes greater weight loss in overweight or obese women compared with control conditions and to describe the effect on plasma lipids, carotenoids, hormones, and fitness. Research Methods and Procedures: Overweight or obese women were randomized to commercial weight loss program or control conditions (n = 35 each). Results: At randomization, participants were 41.1 (11.4) (mean [standard deviation]) years, BMI 34.0 (3.5) kg/m², and weight 92.0 (11.1) kg. At 6 months, change in weight by intent‐to‐treat (ITT) analysis was ?7.2 (6.7) kg and ?7.8% (7.2%) in the intervention group vs. ?0.3 (3.9) kg and ?0.3% (4.5%) in the control group (n = 35 for each; p < 0.01). One‐year ITT analysis revealed significantly greater change in weight, percent weight, BMI, and waist and hip circumferences in the intervention vs. control group. Completers at 1 year exhibited change in weight of ?7.3 (10.4) kg for the intervention group (n = 32) vs. ?0.7 (5.6) kg for controls (n = 33) (p < 0.01), and ?7.8% (11.1%) weight change for the intervention group vs. ?0.7% (6.2%) for controls (p < 0.01). High‐density lipoprotein (HDL) cholesterol concentration increased significantly in the intervention group. Fasting serum insulin decreased in the intervention but increased in the control group at 6 months (p < 0.01), remaining different at 1 year (p = 0.05). Discussion: The commercial program successfully facilitated weight loss, which was notably maintained at 1 year, and promoted favorable changes in plasma lipid and hormone concentrations.     

Evidences of Moran effect in the population synchrony: a demonstration in experimental microcosm

Three main causes to population synchrony are proposed: exogenous factors, dispersal and inter-specific interactions. This paper had as main goal to test the influence of the exogenous factors in the synchrony in spatially isolated (i.e., no dispersal) populations of Sitophilus zeamais (Mots.) (Coleoptera: Curculionidae), in microcosms with different environmental conditions (humidity, temperature and light intensity). Twelve populations of 20 individuals each, were randomly assigned between two treatment conditions: with or without light. Population size and environmental factors (temperature and relative humidity) were weekly assessed for seven months. Temporal trend in populations increase was eliminated adjusting autoregressive models. Population synchrony, detected by means of Pearson's and Spearman's correlation coefficients, was higher within than between treatments, although the populations kept without lamp were more synchronized than populations with lamp. Besides demonstrating the influence of environment on population fluctuations, these results suggest that metabolism and intra-specific interactions are important factors in population dynamic. Organisms exposed to unsuitable environmental conditions may have abnormal metabolic rates, which negatively influences the population grow. Thus, small populations are more likely to suffer from demographic stochasticity, decreasing the probability of the synchrony among populations. On the other hand, in more suitable environments, individuals are expected to have normal metabolic functions, and so, to achieve higher rates of population grow. In this case, the demographic stochasticity has smaller influence, leading populations without lamp to fluctuate synchronously.     

Regulation of drug transporters during infection and inflammation

Inflammation-induced changes in the pharmacokinetics and dynamics of numerous drugs have been reported. Altered drug disposition during inflammatory disease has traditionally been ascribed primarily to changes in drug metabolism and protein binding. Emerging evidence within the last decade, however, has demonstrated that the inflammatory response affects the expression of several important drug transporters and these changes significantly impact the disposition and activity of drug substrates.     

Engineering linear,branched‐chain triterpene metabolism in monocots

Triterpenes are thirty‐carbon compounds derived from the universal five‐carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large‐scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T₀ mature plants) were obtained using the cytosolic‐targeting strategy. Plastid‐targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid‐targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.