Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins

FcγRs are involved in regulating a multitude of innate and adaptive immune responses, which makes them attractive targets for the development of novel immunotherapeutic approaches. In this report, we describe a simple method for the production of a large quantity of recombinant porcine FcγRII. The extracellular domain of the porcine FcγRII (poFcγRII) gene was constructed and cloned into the Escherichia coli expression vector pET-28a. The recombinant protein was expressed at high level in E. coil BL21 (DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine hydrochloride and purified by Ni-chelation, and refolded by rapid dilution. After purification and renaturation, the recombinant soluble protein (rsFcγRII) coated on high-binding ELISA plates, showed concentration dependent binding of porcine IgG and the binding of porcine IgG to the surface bound rsFcγRII was inhibited in a dose-dependent manner by soluble rsFcγRII itself. Then by the inhibition assay we evaluated the effectiveness of the rsFcγRII in inhibiting the IgG binding to the whole molecule of poFcγRII expressed on the Marc-145 cell surface, the rsFcγRII inhibited the binding of porcine IgG to the transfected Marc-145 cell’s surface, with an IC₅₀ value of 0.87 μM, demonstrating that rsFcγRII manifests the similar specificity as native poFcγRII. The method for highly efficient production of biologically active poFcγRII may be employed for both basic research and potential clinical applications.     

Characterization of a thermostable NADPH:FMN oxidoreductase from the mesophilic bacterium Bacillus subtilis

The gene yhdA from Bacillus subtilis encoding a putative flavin mononucleotide (FMN)-dependent oxidoreductase was cloned and heterologously expressed in Escherichia coli. The purified enzyme has a noncovalently bound FMN cofactor, which is preferentially reduced by NADPH, indicating that YhdA is a NADPH:FMN oxidoreductase. The rate of NADPH oxidation is enhanced by the addition of external FMN, and analysis of initial rate measurements reveals the occurrence of a ternary complex in a bi-bi reaction mechanism. YhdA has also been shown to reductively cleave the -N=N- bond in azo dyes at the expense of NADPH, and hence, it possesses azoreductase activity, however, at a rate 100 times slower than that found for FMN. Using Cibacron Marine as a model compound, we could demonstrate that the dye is a competitive inhibitor of NADPH and FMN. The utilization of NADPH and the absence of a flavin semiquinone radical distinguish YhdA from flavodoxins, which adopt the same structural fold, i.e., a five-stranded beta sheet sandwiched by five alpha helices. The native molecular-mass of YhdA was determined to be 76 kDa, suggesting that the protein occurs as a tetramer, whereas the YhdA homologue in Saccharomyces cerevisiae (YLR011wp) forms a dimer in solution. Interestingly, the different oligomerization of these homologous proteins correlates to their thermostability, with YhdA exhibiting a melting point of 86.5 degrees C, which is 26.3 degrees C higher than that for the yeast protein. This unusually high melting point is proposed to be the result of increased hydrophobic packing between dimers and the additional presence of four salt bridges stabilizing the dimer-dimer interface.     

Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.     

Allergen-specific MHC class II tetramer+ cells are detectable in allergic, but not in nonallergic, individuals

Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-gamma. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.     

Life‐history strategy varies with the strength of competition in a food‐limited ungulate population

Fluctuating population density in stochastic environments can contribute to maintain life‐history variation within populations via density‐dependent selection. We used individual‐based data from a population of Soay sheep to examine variation in life‐history strategies at high and low population density. We incorporated life‐history trade‐offs among survival, reproduction and body mass growth into structured population models and found support for the prediction that different life‐history strategies are optimal at low and high population densities. Shorter generation times and lower asymptotic body mass were selected for in high‐density environments even though heavier individuals had higher probabilities to survive and reproduce. In contrast, greater asymptotic body mass and longer generation times were optimal at low population density. If populations fluctuate between high density when resources are scarce, and low densities when they are abundant, the variation in density will generate fluctuating selection for different life‐history strategies, that could act to maintain life‐history variation.     

Intronic variation at the CHD1‐Z gene in Black‐tailed Godwits Limosa limosa limosa: correlations with fitness components revisited

Recently, Schroeder et al. (2010, Ibis 152: 368–377) suggested that intronic variation in the CHD1‐Z gene of Black‐tailed Godwits breeding in southwest Friesland, The Netherlands, correlated with fitness components. Here we re‐examine this surprising result using an expanded dataset (2088 birds sampled from 2004 to 2010 vs. 284 birds from 2004 to 2007). We find that the presence of the Z* allele (9% of the birds) is not associated with breeding habitat type, egg size, adult survival, adult body mass or adult body condition. The results presented here, when used in synergy with the previously reported results by Schroeder et al., suggest that there might be a tendency towards female adults with the Z* allele laying earlier clutches than adult females without the Z* allele. The occurrence of the Z* allele was also associated with a higher chick body mass and return rate. Chicks with the Z* allele that had hatched early in the breeding season were heavier at birth than chicks without the Z* allele and chicks with the Z* allele that had hatched late. Collectively, the results suggest that variation in the CHD1‐Z gene may indeed have arisen as a byproduct of selection acting on females during the egg fase and on chicks during the rearing stages of the reproductive cycle.     

Cytogeography of European perennial species of Cyanus (Asteraceae)

To reveal the general cytogeographical pattern of Cyanus section Protocyanus in Europe, DNA ploidy and/or chromosome numbers were newly examined for 160 populations by flow cytometry (450 plants) and/or chromosome counting (30 plants). Furthermore, previously published karyological data were revised (236 records). Our analyses confirmed chromosome counts of 2n = 22 for all newly investigated samples of the C. triumfetti group (the records for C. semidecurrens and C. ternopoliensis are new), C. diospolitanus and C. achtarovii; 2n = 44 for C. montanus and C. mollis; and 2n = 20 for C. lingulatus, C. napulifer, C. nissanus, C. orbelicus, C. thirkei, C. tuberosus and C. velenovskyi. The chromosome count of 2n = 20 is the first report for C. epirotus. The cytotype 2n = 40 was newly recorded for the Crimean endemic C. fuscomarginatus and Calabrian and Greek populations of C. graminifolius. The cytotypes 2n = 20 and 2n = 40 were confirmed for C. pindicola. For the first time triploidy (2n～3x～30) was found in C. nissanus, C. thirkei and in a newly discovered hybrid, C. epirotus × C. graminifolius. Two contrasting ecogeographical patterns emerged: cytotypes derived from the base chromosome number x = 11 (2n = 22, 44) are widespread in northern latitudes and ecologically diverse, whereas cytotypes with x = 10 (2n = 20, 30, 40) are confined to mountains in southern Europe. In general, tetraploids have smaller ranges than diploids. The new combinations Cyanus section Protocyanus (Dobrocz.) Ol?avská comb. nov. and Cyanus ternopoliensis (Dobrocz.) Ol?avská comb. nov. are provided. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 230–257.     

Abundance of amino acid transporters involved in mTORC1 activation in skeletal muscle of neonatal pigs is developmentally regulated

Previously we demonstrated that the insulin- and amino acid-induced activation of the mammalian target of rapamycin complex 1 (mTORC1) is developmentally regulated in neonatal pigs. Recent studies have indicated that members of the System A transporter (SNAT2), the System N transporter (SNAT3), the System L transporters (LAT1 and LAT2), and the proton-assisted amino acid transporters (PAT1 and PAT2) have crucial roles in the activation of mTORC1 and that the abundance of amino acid transporters is positively correlated with their activation. This study aimed to determine the effect of the post-prandial rise in insulin and amino acids on the abundance or activation of SNAT2, SNAT3, LAT1, LAT2, PAT1, and PAT2 and whether the response is modified by development. Overnight fasted 6- and 26-day-old pigs were infused for 2 h with saline (Control) or with insulin or amino acids to achieve fed levels while amino acids or insulin, respectively, as well as glucose were maintained at fasting levels. The abundance of SNAT2, SNAT3, LAT1, LAT2, PAT1, and PAT2 was higher in muscle of 6- compared with 26-day-old pigs. The abundance of the PAT2–mTOR complex was greater in 6- than in 26-day-old pigs, consistent with the higher activation of mTORC1. Neither insulin nor amino acids altered amino acid transporter or PAT2–mTOR complex abundance. In conclusion, the amino acid transporters, SNAT 2/3, LAT 1/2, and PAT1/2, likely have important roles in the enhanced amino acid-induced activation of mTORC1 in skeletal muscle of the neonate.     

Pharmacologic uncoupling of angiogenesis and inflammation during initiation of pathological corneal neovascularization

Pathological neovascularization occurs when a balance of pro- and anti-angiogenic factors is disrupted, accompanied by an amplifying inflammatory cascade. However, the interdependence of these responses and the mechanism triggering the initial angiogenic switch have remained unclear. We present data from an epithelial debridement model of corneal neovascularization describing an initial 3-day period when a substantial component of neovascular growth occurs. Administration of selective inhibitors shows that this initial growth requires signaling through VEGFR-2 (vascular endothelial growth factor receptor-2), independent of the accompanying inflammatory response. Instead, increased VEGF production is found prominently in repair epithelial cells and is increased prior to recruitment of neutrophil/granulocytes and macrophage/monocytes. Consequently, early granulocyte and monocyte depletion has little effect on corneal neovascularization outgrowth. These data indicate that it is possible to pharmacologically uncouple these mechanisms during early injury-driven neovascularization in the cornea and suggest that initial tissue responses are coordinated by repair epithelial cells.     

Evolutionary diversification of reef corals: a comparison of the molecular and fossil records

Understanding historical patterns of diversity dynamics is of paramount importance for many evolutionary questions. The fossil record has long been the only source of information on patterns of diversification, but the molecular record, derived from time-calibrated phylogenies, is becoming an important additional resource. Both fossil and molecular approaches have shortcomings and biases. These have been well studied for fossil data but much less so for molecular data and empirical comparisons between approaches are lacking. Here, we compare the patterns of diversification derived from fossil and molecular data in scleractinian reef coral species. We also assess the robustness of molecular diversification rates to poor taxon sampling. We find that the temporal pattern of molecular diversification rates is robust to incomplete sampling when rates are calculated per interval. The major obstacle of molecular methods is that rate estimates are distorted because diversification rates can never be negative, whereas the fossil record suffers from incomplete preservation and inconsistent taxonomy. Nevertheless, the molecular pattern of diversification is comparable to the pattern we observe in the fossil record, with the timing of major diversification pulses coinciding in each dataset. For example, both agree that the end-Triassic coral extinction was a catastrophic bottleneck in scleractinian evolution.