Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors

ATP-gated purinergic P2X4 receptors (P2X4Rs) are the most alcohol-sensitive P2XR subtype. We recently reported that ivermectin (IVM), an antiparasitic used in animals and humans, antagonized ethanol inhibition of P2X4Rs. Furthermore, IVM reduced ethanol intake in mice. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed an action pocket for both ethanol and IVM formed by Asp331, Met336 in TM2 and Trp46, and Trp50 in TM1 segments. The role of Asp331 and Met336 was experimentally confirmed. The present study tested the hypothesis that Trp46 plays a role in ethanol and IVM modulation of P2X4Rs. Trp46 was mutated to residues with different physicochemical properties and the resultant mutants tested for ethanol and IVM responses using Xenopus oocyte expression system and two-electrode voltage clamp. Nonaromatic substitutions at position 46 reduced ethanol inhibition at higher concentrations and switched IVM potentiation to inhibition. Simultaneous substitution of alanine at positions Trp46 and Met336 also resulted in similar changes in ethanol and IVM responses. Furthermore, a new molecular model based on the open pore conformation of zebrafish P2X4R suggested a role for Tyr42 that was further supported experimentally. Our previous and current findings, combined with our preliminary evidence of increased ethanol consumption in P2X4R knockout mice, suggest that the ethanol and IVM action pocket in P2X4Rs formed by positions 42, 46, 331, and 336 presents a potential target for medication development for alcohol use disorders.     

Discovery of VU0409106: A negative allosteric modulator of mGlu5 with activity in a mouse model of anxiety

Development of SAR in an aryl ether series of mGlu₅ NAMs leading to the identification of tool compound VU0409106 is described in this Letter. VU0409106 is a potent and selective negative allosteric modulator of mGlu₅ that binds at the known allosteric binding site and demonstrates good CNS exposure following intraperitoneal dosing in mice. VU0409106 also proved efficacious in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu₅ antagonists as well as clinically efficacious anxiolytics.     

Circumscription of species in the genus Sirodotia (Batrachospermales,Rhodophyta) based on molecular and morphological data

Species level taxonomy and phylogeographical distribution patterns in the freshwater rhodophyte Sirodotia are resolved through phylogenetic inferences based on rbcL and cox2–3 sequence data. Previous studies focused on the taxonomy of specific Sirodotia species or the distributions across a limited geographical region. Our molecular phylogenies included samples attributable to five recognized Sirodotia species and include collections from Australia, Brazil, Costa Rica, Canada, Finland, Mexico, New Zealand, South Africa and the United States. Both rbcL and cox2–3 phylogenies inferred S. suecica, S. tenuissima and S. goebelii as a monophyletic group with little sequence divergence. This result supports the synonymy of S. tenuissima and S. goebelii with S. suecica (the species name with priority). Within this clade, samples collected from Australia and New Zealand formed a monophyletic group with no other discernible phylogeographical patterns within S. suecica. This result seems to be somewhat unusual in the Batrachospermales, as other species have shown greater genetic variation among geographically distant locations. As in previous studies, S. huillensis and S. delicatula were inferred as a separate species based on the rbcL phylogeny, supporting the current taxonomy. A specimen of S. aff. huillensis from South Africa, may represent a new species but further research is necessary before it can be designated as such.     

Tethered Domains and Flexible Regions in tRNase ZL,the Long Form of tRNase Z

tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes the pre-tRNA 3′ trailer in a step central to tRNA maturation. The short form (tRNase Z^S) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase Z^L), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase Z^L arose from a tandem duplication of tRNase Z^S followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase Z^S reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase Z^L structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase Z^L performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase Z^L function.     

Inhibition of Fast Axonal Transport by Pathogenic SOD1 Involves Activation of p38 MAP Kinase

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.     

Identification and characterization of ErbB-3-binding protein-1 as a target for immunotherapy

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp1(45-54) and Ebp1(59-67)) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp1(59) epitope gave rise to CD8(+) T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8(+) T cell responsiveness against the Ebp1(59) epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.     

Effects of sustained length-dependent activation on in situ cross-bridge dynamics in rat hearts

The cellular basis of the length-dependent increases in contractile force in the beating heart has remained unclear. Our aim was to investigate whether length-dependent mediated increases in contractile force are correlated with myosin head proximity to actin filaments, and presumably the number of cross-bridges activated during a contraction. We therefore employed x-ray diffraction analyses of beat-to-beat contractions in spontaneously beating rat hearts under open-chest conditions simultaneous with recordings of left ventricle (LV) pressure-volume. Regional x-ray diffraction patterns were recorded from the anterior LV free wall under steady-state contractions and during acute volume loading (intravenous lactate Ringers infusion at 60 ml/h, <5 min duration) to determine the change in intensity ratio (I_1,0/I_1,1) and myosin interfilament spacing (d_1,0). We found no significant change in end-diastolic (ED) intensity ratio, indicating that the proportion of myosin heads in proximity to actin was unchanged by fiber stretching. Intensity ratio decreased significantly more during the isovolumetric contraction phase during volume loading than under baseline contractions. A significant systolic increase in myosin head proximity to actin filaments correlated with the maximum rate of pressure increase. Hence, a reduction in interfilament spacing at end-diastole (∼0.5 nm) during stretch increased the proportion of cross-bridges activated. Furthermore, our recordings suggest that d_1,0 expansion was inversely related to LV volume but was restricted during contraction and sarcomere shortening to values smaller than the maximum during isovolumetric relaxation. Since ventricular volume, and presumably sarcomere length, was found to be directly related to interfilament spacing, these findings support a role for interfilament spacing in modulating cross-bridge formation and force developed before shortening.     

Histone tails and the H3 alphaN helix regulate nucleosome mobility and stability

Nucleosomes fulfill the apparently conflicting roles of compacting DNA within eukaryotic genomes while permitting access to regulatory factors. Central to this is their ability to stably associate with DNA while retaining the ability to undergo rearrangements that increase access to the underlying DNA. Here, we have studied different aspects of nucleosome dynamics including nucleosome sliding, histone dimer exchange, and DNA wrapping within nucleosomes. We find that alterations to histone proteins, especially the histone tails and vicinity of the histone H3 alphaN helix, can affect these processes differently, suggesting that they are mechanistically distinct. This raises the possibility that modifications to histone proteins may provide a means of fine-tuning specific aspects of the dynamic properties of nucleosomes to the context in which they are located.     

A trait-based approach across the native and invaded range to understand plant invasiveness and community impact

Alien plant species invasiveness and impact on diversity (i.e. species richness and composition) can be driven by the altered competitive interactions experienced by the invader in its invaded range compared to its native range. Trait-based competition effects on invasiveness can be mediated through size-asymmetric competition, i.e. a trait suit of the invader that drives competitive dominance, and through ‘niche differences', i.e. trait differentiation and thus minimized competition between invader and the invaded community. In terms of invasion impact, size-asymmetric competition is expected to result in competitive exclusion of co-occurring subordinate species, whereas ‘niche differences' might result in competitive exclusion of the most functionally similar co-occurring species. Although observational work does not allow the full disentanglement of both trait-based effects, it does allow to verify the occurrence of expected theoretical trait patters. In this study, we explored the trait-based competition effects on invasiveness and diversity impact for Rosa rugosa in both its invaded range in Belgium and its native range in Japan, based on seven functional traits across 100 vegetation plots. Following the predictions for enhanced invasiveness, we found much lower functional overlap between R. rugosa and the co-occurring species in the invaded range compared to the native range. This likely also explains the absence of diversity impact in its native range. Despite the absence of changes in species richness in the invaded range, the invader did strongly impact species composition of invaded communities. This impact occurred through strong shade tolerance responses, suggesting size-asymmetric competition effects and cover loss of co-occurring dominant species, next to exclusion of co-occurring species most functionally similar to the invader; suggesting niche difference effects. In conclusion, this case-study illustrates how exploring functional trait patterns across a species native and invaded range can help in understanding how trait-based competition processes can affect invasiveness and community impact.     

Streamflow Impacts of Biofuel Policy-Driven Landscape Change

Likely changes in precipitation (P) and potential evapotranspiration (PET) resulting from policy-driven expansion of bioenergy crops in the United States are shown to create significant changes in streamflow volumes and increase water stress in the High Plains. Regional climate simulations for current and biofuel cropping system scenarios are evaluated using the same atmospheric forcing data over the period 1979–2004 using the Weather Research Forecast (WRF) model coupled to the NOAH land surface model. PET is projected to increase under the biofuel crop production scenario. The magnitude of the mean annual increase in PET is larger than the inter-annual variability of change in PET, indicating that PET increase is a forced response to the biofuel cropping system land use. Across the conterminous U.S., the change in mean streamflow volume under the biofuel scenario is estimated to range from negative 56% to positive 20% relative to a business-as-usual baseline scenario. In Kansas and Oklahoma, annual streamflow volume is reduced by an average of 20%, and this reduction in streamflow volume is due primarily to increased PET. Predicted increase in mean annual P under the biofuel crop production scenario is lower than its inter-annual variability, indicating that additional simulations would be necessary to determine conclusively whether predicted change in P is a response to biofuel crop production. Although estimated changes in streamflow volume include the influence of P change, sensitivity results show that PET change is the significantly dominant factor causing streamflow change. Higher PET and lower streamflow due to biofuel feedstock production are likely to increase water stress in the High Plains. When pursuing sustainable biofuels policy, decision-makers should consider the impacts of feedstock production on water scarcity.