Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.     

Steady-state current flow through gap junctions. Effects on intracellular ion concentrations and fluid movement.

Double voltage clamp studies were performed on gap junctions contained in septal membranes of the earthworm median giant axon. The gap junctions exhibited no conductance changes in response to voltages imposed across either the septal membrane or the plasma membrane. However, the trans-septal current displayed a slow (10 s) relaxation in response to transjunctional voltage steps. The experimental evidence suggests that this relaxation is a polarization of the septum due to local accumulation/depletion of permeant ions. A theoretical analysis of this observation suggests that the applied electric field causes accumulation of impermeant anions on one side of the junction and depletion on the other, which leads to a change in concentration of permeant ions to maintain macroscopic electroneutrality. The change in concentration of permeant ions generates a transjunctional equilibrium potential that opposes junctional current flow. These results indicate that currents flowing through gap junctions can have an influence on the distribution of intracellular ions. Moreover, the theoretical analysis suggests that such currents will be accompanied by significant intracellular and intercellular water flow.     

Biocontrol Activity and Plant Growth Promotion Exerted by Aureobasidium pullulans Strains

The most common leguminous plants’ diseases are caused by soil-borne pathogens leading to important economic losses worldwide. Strains L1 and L8, belonging to Aureobasidium pullulans species, were tested in vitro and in vivo as biocontrol agents (BCAs) against Rhizoctonia solani (Rs1) (AG-4) and as plant growth promoters (PGPs). The non-volatile metabolites produced by L1 and L8 strains inhibited the pathogen mycelial growth by 87.9% on average, with no significant differences between the two strains. The lower pathogen diametric growth inhibition was displayed by both yeasts’ volatile metabolites (VOCs) that significantly reduced the colony growth of R. solani, and similarly to the control, with an average of 10.5%. By in vivo assay, L1 and L8 strains showed the ability to control the pathogen virulence probably through the biofilm formation around the bean and soybean plant roots, as confirmed by scanning electron microscope (SEM) analysis. The spectroscopic analysis highlighted the composition of non-volatile compounds: complex carbohydrates (pullulan), degrading enzymes, siderophores and antifungals (aureobasidins). Moreover, the ability of L1 and L8 strains to stimulate the bean and soybean plant roots, stems, and leaves growth was investigated, showing that these yeasts could have an application not only as BCAs but also as plant growth biostimulator.

     

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrP^C. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp^0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn^0/0/Prnp^0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrP^C, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable.     

Potentials and limitations of histone repeat sequences for phylogenetic reconstruction of Sophophora.

Simplified DNA sequence acquisition has provided many new data sets that are useful for phylogenetic reconstruction, including single- and multiple-copy nuclear and organellar genes. Although transcribed regions receive much attention, nontranscribed regions have recently been added to the repertoire of sequences suitable for phylogenetic studies, especially for closely related taxa. We evaluated the efficacy of a small portion of the histone repeat for phylogenetic reconstruction among Drosophila species. Histone repeats in invertebrates offer distinct advantages similar to those of widely used ribosomal repeats. First, the units are tandemly repeated and undergo concerted evolution. Second, histone repeats include both highly conserved coding and variable intergenic regions. This composition facilitates application of "universal" primers spanning potentially informative sites. We examined a small region of the histone repeat, including the intergenic spacer segments of coding regions from the divergently transcribed H2A and H2B histone genes. The spacer (about 230 bp) exists as a mosaic with highly conserved functional motifs interspersed with rapidly diverging regions; the former aid in alignment of the spacer. There are no ambiguities in alignment of coding regions. Coding and noncoding regions were analyzed together and separately for phylogenetic information. Parsimony, distance, and maximum-likelihood methods successfully retrieve the corroborated phylogeny for the taxa examined. This study demonstrates the resolving power of a small histone region which may now be added to the growing collection of phylogenetically useful DNA sequences.     

VORFFIP-Driven Dock: V-D2OCK,a Fast and Accurate Protein Docking Strategy

The experimental determination of the structure of protein complexes cannot keep pace with the generation of interactomic data, hence resulting in an ever-expanding gap. As the structural details of protein complexes are central to a full understanding of the function and dynamics of the cell machinery, alternative strategies are needed to circumvent the bottleneck in structure determination. Computational protein docking is a valid and valuable approach to model the structure of protein complexes. In this work, we describe a novel computational strategy to predict the structure of protein complexes based on data-driven docking: VORFFIP-driven dock (V-D²OCK). This new approach makes use of our newly described method to predict functional sites in protein structures, VORFFIP, to define the region to be sampled during docking and structural clustering to reduce the number of models to be examined by users. V-D²OCK has been benchmarked using a validated and diverse set of protein complexes and compared to a state-of-art docking method. The speed and accuracy compared to contemporary tools justifies the potential use of VD²OCK for high-throughput, genome-wide, protein docking. Finally, we have developed a web interface that allows users to browser and visualize V-D²OCK predictions from the convenience of their web-browsers.     

Three New Species of Phytotelm-Breeding Melanophryniscus from the Atlantic Rainforest of Southern Brazil (Anura: Bufonidae)

Three new species of Melanophryniscus are described from the Serra do Mar mountain range of the state of Santa Catarina, southern Brazil. All species are found at intermediate to high altitudes and share phytotelm-breeding as their reproductive strategy. The new species are distinguished from other phytotelm-breeding Melanophryniscus based on different combinations of the following traits: snout-vent length, presence of white and/or yellow spots on forearms, mouth, belly and cloaca, pattern and arrangement of warts, and presence and number of corneous spines. The discovery of these species in a rather restricted geographical area suggests that the diversity of phytotelm-breeding species of Melanophryniscus might be severely underestimated. The conservation status of these species is of particular concern, given that one of them is at risk of extinction not only due to its restricted habitat, but also because of anthropogenic disturbances.     

How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4–15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by “state of the art” method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.     

The Oxidative Cost of Acoustic Signals: Examining Steroid Versus Aerobic Activity Hypotheses in a Wild Bird

Vertebrate vocalizations are widespread secondary sexual signals used for mate attraction and territory defence, and variation in signal quality is often condition dependent and impacts reproductive outcomes. Although vocal signal performance is known to reflect various aspects of male quality, few studies have examined the underlying mechanisms mediating its costs and hence its honesty. Using a population of Arctic‐breeding snow buntings (Plectrophenax nivalis), we compared the ‘Oxidation Handicap Hypothesis’, which predicts that testosterone‐induced increases in oxidative stress provide a direct mechanistic basis for ensuring the honesty of many secondary sexual signals, to the ‘Aerobic Activity Hypothesis, which predicts that it is the aerobic activity involved with signal production (i.e. vocal performance or defending a large territory) and not testosterone directly that links signal quality and oxidative stress. Males singing at faster rates had higher levels of both reactive oxygen metabolites and non‐enzymatic antioxidant capacity in the plasma (i.e. without an increase in overall oxidative stress), enabling certain males to produce high‐quality signals while also mitigating the costs of an associated increase in oxidative stress. However, these results were completely independent of plasma testosterone levels, supporting the role of aerobic performance in directly affecting oxidative stress. Although song performance was not linked to reproductive parameters in our data set, our research is the first to test these competing hypotheses in a behavioural trait and results suggest that oxidative stress may be an underlying physiological cost preventing low‐quality individuals from producing high‐quality signals.