Purification,structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen,Phl p 4, with cross-reactive potential

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas

Background  

The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate.     

An ELISA method to measure inhibition of the COX enzymes

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).     

The Conformational Ensembles of α-Synuclein and Tau: Combining Single-Molecule FRET and Simulations

Intrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson’s and Alzheimer’s diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense.Abbreviations used: AAMD, all-atom molecular dynamics; ECMC, experimentally constrained Monte Carlo; ET_eff, energy transfer efficiency; (sm)FRET, (single molecule) Förster resonance energy transfer; IDP, intrinsically disordered protein; LJ, Lennard-Jones; MC, Monte Carlo; MD, molecular dynamics; PRE, paramagnetic relaxation enhancement; SAX(N)S, small-angle x-ray (neutron) scattering; UMC, unconstrained Monte Carlo     

The species–area relationship in centipedes (Myriapoda: Chilopoda): a comparison between Mediterranean island groups

The present study article examines the shapes of centipede species–area relationships (SARs) in the Mediterranean islands, compares the results of the linear form of the power model between archipelagos, discusses biological significance of the power model parameters with other taxa on the Aegean archipelago, and tests for a significant small‐island effect (SIE). We used 11 models to test the SARs and we compared the quality‐of‐fit of all candidate models. The power function ranked first and Z‐values was in the range 0.106–0.334. We assessed the presence of SIEs by fitting both a continuous and discontinuous breakpoint regression model. The continuous breakpoint regression functions never performed much better than the closest discontinuous model as a predictor of centipede species richness. We suggest that the relatively low Z‐values in our data partly reflect better dispersal abilities in centipedes than in other soil invertebrate taxa. Longer periods of isolation and more recent island formation may explain the somewhat lower constant c in the western Mediterranean islands compared to the Aegean islands. Higher breakpoint values in the western Mediterranean may also be a result of larger distance to the mainland and longer separation times. Despite the differences in the geological history and the idiosyncratic features of the main island groups considered, the overall results are quite similar and this could be assigned to the ability of centipedes to disperse across isolation barriers. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 146–159.     

The role of pollinator shifts in the floral diversification of Iochroma (Solanaceae)

Differences in floral traits among plant species have often been attributed to adaptation to pollinators. We explored the importance of pollinator shifts in explaining floral divergence among 15 species of Iochroma. We examined four continuously varying floral traits: corolla length, nectar reward, display size, and flower color. Pollinator associations were characterized with a continuously varying measure of pollinator importance (the product of visitation and pollen deposition) for four groups of pollinators: hummingbirds, Hymenoptera, Lepidoptera, and Diptera. A phylogenetic generalized least squares approach was used to estimate correlations between pollinator groups and floral traits across a sample of Bayesian trees using different models of trait evolution. Multivariate analyses were also employed to identify suites of traits associated with each pollinator group. We found that nonphylogenetic models typically fit the data better than phylogenetic models (Brownian motion, Ornstein-Uhlenbeck), and thus results varied little across trees. Our results indicated that species with high nectar reward and large displays are significantly more likely to be pollinated by hummingbirds and less likely to be pollinated by all groups of insects. Corolla length and flower color did not show any consistently significant associations with pollinator groups. For these two traits, we discuss alternative evolutionary forces, including phylogenetic inertia and community-level factors.