Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.     

Stimulation of central β2-adrenoceptors suppresses NFκB activity in rat brain: A role for IκB

In this study we examined the impact of systemic treatment with the long-acting brain penetrant β₂-adrenoceptor agonist clenbuterol on NFκB activity and IκB expression in rat brain. Clenbuterol decreased NFκB activity (p65 DNA binding) in nuclear extracts prepared from rat cortex and hippocampus for up to 8 h following a single treatment. This was accompanied by increased expression of IκBα mRNA and protein. The temporal increase in IκB protein expression paralleled the suppression of NFκB activity, suggesting that IκBα mediates the suppression NFκB activity observed. These actions of clenbuterol were prevented by pre-treatment with the non-selective β-adrenoceptor antagonist propranolol, the β₂-adrenoceptor antagonist ICI-118,551, but not the β₁-adrenoceptor antagonist metoprolol, suggesting that the effects of clenbuterol on IκBα expression and NFκB activity are mediated specifically by the β₂-adrenoceptor. In addition, the actions of clenbuterol were mimicked by systemic administration of another highly selective long-acting β₂-adrenoceptor agonist formoterol. As neurodegenerative diseases are associated with inflammation we determined if clenbuterol could suppress NFκB activation that occurs in response to an inflammatory stimulus. In this regard we demonstrate that clenbuterol inhibited IκB phosphorylation and IκB degradation and inhibited NFκB activity in hippocampus and cortex of rats following a central injection of the inflammagen bacterial lipopolysaccharide (LPS). In tandem, clenbuterol blocked expression of the NFκB-inducible genes TNF-α and ICAM-1 following LPS administration. Our finding that clenbuterol and formoterol inhibit NFκB activity in the CNS further supports the idea that β₂-adrenoceptors may be an attractive target for treating neuroinflammation and combating inflammation-related neurodegeneration.     

Anion exchange membrane adsorbers for flow‐through polishing steps: Part I. clearance of minute virus of mice

Membrane adsorbers may be a viable alternative to the packed‐bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18–22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands. Biotechnol. Bioeng. 2013; 110: 491–499. © 2012 Wiley Periodicals, Inc.     

Characterization of three loci for homologous gene targeting and transgene expression

Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare‐cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site‐specific transgene integration, suitable for a variety of biotechnological applications. Biotechnol. Bioeng. 2013; 110: 2225–2235. © 2013 Wiley Periodicals, Inc.     

A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators

The objective of the current study is to develop a new cryomacroscope prototype for the study of vitrification in large-size specimens. The unique contribution in the current study is in developing a cryomacroscope setup as an add-on device to a commercial controlled-rate cooler and in demonstration of physical events in cryoprotective cocktails containing synthetic ice modulators (SIM)—compounds which hinder ice crystal growth. Cryopreservation by vitrification is a highly complex application, where the likelihood of crystallization, fracture formation, degradation of the biomaterial quality, and other physical events are dependent not only upon the instantaneous cryogenic conditions, but more significantly upon the evolution of conditions along the cryogenic protocol. Nevertheless, cryopreservation success is most frequently assessed by evaluating the cryopreserved product at its end states—either at the cryogenic storage temperature or room temperature. The cryomacroscope is the only available device for visualization of large-size specimens along the thermal protocol, in an effort to correlate the quality of the cryopreserved product with physical events. Compared with earlier cryomacroscope prototypes, the new Cryomacroscope-III evaluated here benefits from a higher resolution color camera, improved illumination, digital recording capabilities, and high repeatability in tested thermal conditions via a commercial controlled-rate cooler. A specialized software package was developed in the current study, having two modes of operation: (a) experimentation mode to control the operation of the camera, record camera frames sequentially, log thermal data from sensors, and save case-specific information; and (b) post-processing mode to generate a compact file integrating images, elapsed time, and thermal data for each experiment. The benefits of the Cryomacroscope-III are demonstrated using various tested mixtures of SIMs with the cryoprotective cocktail DP6, which were found effective in preventing ice growth, even at significantly subcritical cooling rates with reference to the pure DP6.     

A calcaneus attributable to the primitive late eocene anthropoid Proteopithecus sylviae: Phenetic affinities and phylogenetic implications

A well‐preserved calcaneus referrable to Proteopithecus sylviae from the late Eocene Quarry L‐41 in the Fayum Depression, Egypt, provides new evidence relevant to this taxon's uncertain phylogenetic position. We assess morphological affinities of the new specimen using three‐dimensional geometric morphometric analyses with a comparative sample of primate calcanei representing major extinct and extant radiations (n = 58 genera, 106 specimens). Our analyses reveal that the calcaneal morphology of Proteopithecus is most similar to that of the younger Fayum parapithecid Apidium. Principal components analysis places Apidium and Proteopithecus in an intermediate position between primitive euprimates and crown anthropoids, based primarily on landmark configurations corresponding to moderate distal elongation, a more distal position of the peroneal tubercle, and a relatively “unflexed” calcaneal body. Proteopithecus and Apidium are similar to cercopithecoids and some omomyiforms in having an ectal facet that is more tightly curved, along with a larger degree of proximal calcaneal elongation, whereas other Fayum anthropoids, platyrrhines and adapiforms have a more open facet with less proximal elongation. The similarity to cercopithecoids is most plausibly interpreted as convergence given the less tightly curved ectal facets of stem catarrhines. The primary similarities between Proteopithecus and platyrrhines are mainly in the moderate distal elongation and the more distal position of the peroneal tubercle, both of which are not unique to these groups. Proteopithecus and Apidium exhibit derived anthropoid features, but also a suite of primitive retentions. The calcaneal morphology of Proteopithecus is consistent with our cladistic analysis, which places proteopithecids as a sister group of Parapithecoidea. Am J Phys Anthropol 151:372–397, 2013.© 2013 Wiley Periodicals, Inc.     

Pollen and Mould Allergy in Southern Sardinia (Italy): Comparison of Skin-Test Frequencies and Air Sampling Data

A study was carried out to investigate the influence of atmospheric pollen and fungi in determining allergic diseases by comparing the frequency of skin reactions to air sampling data over a 6-year period.

48% of our population reacted to at least one of the pollen and fungal extracts used. Among pollen, Gramineae gave the most frequent positive reactions, followed by Parietaria, Olea and Compositae. The most common positive skin tests in fungus sensitive patients were by extracts of Alternaria, Cladosporium, Aspergillus and Candida

As for the aerobiological survey, the general trend of pollen and molds was similar during the sampling period. The annual pollen catch did not show remarkable differences during the years sampled, whereas the total fungal spore count was highest in 1988 and 1990.

A comparison between aerobiological and clinical data revealed a good degree of concordance between total pollen counts and positive skin test frequencies for Urticaceae, Gramineae and Oleaceae but not for Compositae (high positive skin reactions and very low counts) and Cupressaceae (high counts and few skin reactions).

A less marked correlation has been found between fungal spore counts and positive skin-test frequencies as compared to pollen. Spores such as Cladosporium, which are present in large number in the air, appear to be less sensitizing, while certain spore types (e.g. Alternaria), seem to be able to sensitize patients in spite of their low atmospheric concentrations.     

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK)

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.     

‘Manipulation’ without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites

Previous studies have suggested that Plasmodium parasites can manipulate mosquito feeding behaviours such as probing, persistence and engorgement rate in order to enhance transmission success. Here, we broaden analysis of this ‘manipulation phenotype’ to consider proximate foraging behaviours, including responsiveness to host odours and host location. Using Anopheles stephensi and Plasmodium yoelii as a model system, we demonstrate that mosquitoes with early stage infections (i.e. non-infectious oocysts) exhibit reduced attraction to a human host, whereas those with late-stage infections (i.e. infectious sporozoites) exhibit increased attraction. These stage-specific changes in behaviour were paralleled by changes in the responsiveness of mosquito odourant receptors, providing a possible neurophysiological mechanism for the responses. However, we also found that both the behavioural and neurophysiological changes could be generated by immune challenge with heat-killed Escherichia coli and were thus not tied explicitly to the presence of malaria parasites. Our results support the hypothesis that the feeding behaviour of female mosquitoes is altered by Plasmodium, but question the extent to which this is owing to active manipulation by malaria parasites of host behaviour.     

Predictive systems ecology

Human societies, and their well-being, depend to a significant extent on the state of the ecosystems that surround them. These ecosystems are changing rapidly usually in response to anthropogenic changes in the environment. To determine the likely impact of environmental change on ecosystems and the best ways to manage them, it would be desirable to be able to predict their future states. We present a proposal to develop the paradigm of predictive systems ecology, explicitly to understand and predict the properties and behaviour of ecological systems. We discuss the necessary and desirable features of predictive systems ecology models. There are places where predictive systems ecology is already being practised and we summarize a range of terrestrial and marine examples. Significant challenges remain but we suggest that ecology would benefit both as a scientific discipline and increase its impact in society if it were to embrace the need to become more predictive.