Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT

PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT''s association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis.     

Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [³H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [³H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.     

Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS,DIRECTED MUTAGENESIS,EPR, AND NMR STUDY*

Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn²⁺, and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H₂O₂-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.     

Morphological phylogenetic analysis of Ophrys (Orchidaceae): insights from morpho‐anatomical floral traits into the interspecific relationships in an unresolved clade

Reconstructing the phylogeny of the sexually deceptive orchid genus Ophrys is crucial to our understanding of the evolution of its complex floral morphology. Molecular phylogenetic analyses showed that section Pseudophrys forms a well supported clade with Ophrys bombyliflora, O. tenthredinifera and O. speculum, but were unable to elucidate the relationships between these four groups of taxa. Here we conduct a morphological phylogenetic analysis of this unresolved clade of Ophrys based on a data matrix of 45 macro‐ and micromorphological and anatomical floral characters, using maximum parsimony and Bayesian inference. Our cladistic analysis yielded a single most parsimonious tree and a Bayesian 50% majority‐rule consensus tree which differed in their overall topology but agreed that O. tenthredinifera and O. bombyliflora are not sister groups. The phylogenetic placement of O. tenthredinifera was ambiguous since it shares six valid synapomorphies each with the cluster of O. speculum–O. bombyliflora and with section Pseudophrys. In contrast, O. bombyliflora is most likely the sister group to O. speculum, a finding that rejects an earlier morphological phylogenetic hypothesis and favours the existing molecular trees based on nuclear ITS rather than plastid data. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 454–476.     

The effects of deer exclusion on the development of a Mediterranean plant community affected by a wildfire

Despite the resilience of Mediterranean ecosystems to fire, the ecological restoration of burned plant communities can be hindered by ungulate herbivores, particularly in areas with high population densities. This study compares the postfire development of a shrub community with and without deer, after a wildfire occurred in 2003 in a protected area in Central Portugal. We monitored 12 fenced and 12 unfenced plots 2, 3, 4, and 8 years after fire. Within each plot, we established a linear transect and measured the monospecific canopy projections (plant patches). Five plant community indicators (patch number, average patch length, average patch height, patch cover, and patch phytovolume per square meter) were obtained. The diameter and height of individuals of the most abundant shrub species (Cistus salvifolius, Erica scoparia, Myrtus communis, Pistacia lentiscus, Rubus ulmifolius, and Ulex jussiaei) were also measured. These measurements were used as response variables in generalized linear mixed models in order to assess the effects of time‐after‐fire and fencing, on the development of the plant community. Patch height and phytovolume had a significantly higher growth in fenced plots. At the species level, C. salvifolius, M. communis, R. ulmifolius, and U. jussiaei showed a higher growth across time both in height and in diameter, in the absence of herbivory. This work shows that deer exclusion needs to be considered when aiming at the postfire restoration of Mediterranean shrub communities.     

Risk Perception and Chronic Exposure to Organochlorine Pesticides in Maya Communities of Mexico

In order to analyze risk perception related to the use and handling of organochlorine pesticides (OCP) in agricultural and livestock activities among Maya communities of Yucatan, Mexico, and to study their impact on public health and the environment, we conducted an analytical study applying 274 semi-structured interviews in 11 municipalities in the zone called “Ring of Cenotes.” The harmful effects of agrochemicals on water supplies, soils, air, and human health were considered, including the indoor use of pesticides to preserve harvest products. Recent studies showed high levels of OCP in groundwater. A generalized low risk perception related to human health and the environment due to pesticide use was found. Likewise, social parameters were analyzed, including the educational level, as well as risk factors related to groundwater karst vulnerability. Pesticides that have been banned by international conventions are still in use. The occurrence of some diseases such as cervical cancer, with a very high prevalence at the national level, and the practice of obtaining drinking water from polluted wells and sinkholes, may be associated with this low risk perception and with poor social conditions. Establishing programs on health education, agro-ecological production alternatives, and water chemical quality monitoring is recommended.