Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA.

The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1. These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system. Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR. A second Epstein-Barr virus RNA, EBER-2, also regulates PKR. EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by U.V. crosslinking experiments and filter binding assays. The affinities of all three RNAs for PKR in vitro are similar (Kd = ca. 0.3 nM). Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus.     

Environmental Drivers of Culicoides Phenology: How Important Is Species-Specific Variation When Determining Disease Policy?

Since 2006, arboviruses transmitted by Culicoides biting midges (Diptera: Ceratopogonidae) have caused significant disruption to ruminant production in northern Europe. The most serious incursions involved strains of bluetongue virus (BTV), which cause bluetongue (BT) disease. To control spread of BTV, movement of susceptible livestock is restricted with economic and animal welfare impacts. The timing of BTV transmission in temperate regions is partly determined by the seasonal presence of adult Culicoides females. Legislative measures therefore allow for the relaxation of ruminant movement restrictions during winter, when nightly light-suction trap catches of Culicoides fall below a threshold (the ‘seasonally vector free period’: SVFP). We analysed five years of time-series surveillance data from light-suction trapping in the UK to investigate whether significant inter-specific and yearly variation in adult phenology exists, and whether the SVFP is predictable from environmental factors. Because female vector Culicoides are not easily morphologically separated, inter-specific comparisons in phenology were drawn from male populations. We demonstrate significant inter-specific differences in Culicoides adult phenology with the season of Culicoides scoticus approximately eight weeks shorter than Culicoides obsoletus. Species-specific differences in the length of the SVFP were related to host density and local variation in landscape habitat. When the Avaritia Culicoides females were modelled as a group (as utilised in the SFVP), we were unable to detect links between environmental drivers and phenological metrics. We conclude that the current treatment of Avaritia Culicoides as a single group inhibits understanding of environmentally-driven spatial variation in species phenology and hinders the development of models for predicting the SVFP from environmental factors. Culicoides surveillance methods should be adapted to focus on concentrated assessments of species-specific abundance during the start and end of seasonal activity in temperate regions to facilitate refinement of ruminant movement restrictions thereby reducing the impact of Culicoides-borne arboviruses.     

Predictome: a database of putative functional links between proteins

The current deluge of genomic sequences has spawned the creation of tools capable of making sense of the data. Computational and high-throughput experimental methods for generating links between proteins have recently been emerging. These methods effectively act as hypothesis machines, allowing researchers to screen large sets of data to detect interesting patterns that can then be studied in greater detail. Although the potential use of these putative links in predicting gene function has been demonstrated, a central repository for all such links for many genomes would maximize their usefulness. Here we present Predictome, a database of predicted links between the proteins of 44 genomes based on the implementation of three computational methods—chromosomal proximity, phylogenetic profiling and domain fusion—and large-scale experimental screenings of protein–protein interaction data. The combination of data from various predictive methods in one database allows for their comparison with each other, as well as visualization of their correlation with known pathway information. As a repository for such data, Predictome is an ongoing resource for the community, providing functional relationships among proteins as new genomic data emerges. Predictome is available at http://predictome.bu.edu.     

Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion

In mice, immunoregulatory APCs express the dendritic cell (DC) marker CD11c, and one or more distinctive markers (CD8alpha, B220, DX5). In this study, we show that expression of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) is selectively induced in specific splenic DC subsets when mice were exposed to the synthetic immunomodulatory reagent CTLA4-Ig. CTLA4-Ig did not induce IDO expression in macrophages or lymphoid cells. Induction of IDO completely blocked clonal expansion of T cells from TCR transgenic mice following adoptive transfer, whereas CTLA4-Ig treatment did not block T cell clonal expansion in IDO-deficient recipients. Thus, IDO expression is an inducible feature of specific subsets of DCs, and provides a potential mechanistic explanation for their T cell regulatory properties.     

Female preference in the context of male–male interactions in Maylandia zebra of Lake Malawi

The mbuna cichlids of Lake Malawi are a diverse, monophyletic, and recently derived clade. Sexual selection is thought to have accelerated their rapid diversification, though the mechanism by which this has occurred remains unknown. In this study, we examine the effect that male–male interactions have on female preference. We first used a short interaction experiment as a proxy for male dominance. We then measured female preference for (1) a single, isolated male; (2) a group of three, highly ranked males, relative to a group of lower ranked males; and (3) a group of three, mixed-ranked males against a similar group of mixed-rank males. We found that male dominance was highly correlated with male standard length. Female preference for male standard length and for dominance rank was significant in both isolated and group interactions. However, females only showed preference for groups of interacting males when males were segregated by dominance rank, not when groups were composed of both dominant and subordinate males. The results suggest that male–male contests influence female mating decisions. By investigating the rules that dictate the outcomes of such interactions we can elucidate the role that behavior plays in the diversification of this species-rich lineage.     

VEGFR1 (Flt1) Regulates Rab4 Recycling to Control Fibronectin Polymerization and Endothelial Vessel Branching

The cell's main receptor for VEGF, VEGFR2 (Kdr) is one of the most important positive regulators of new blood vessel growth and its downstream signalling is well characterized. By contrast, VEGFR1 (Flt1) and the mechanisms by which this VEGF receptor promotes branching morphogenesis in angiogenesis remain relatively unclear. Here we report that engagement of VEGFR1 activates a Rab4A-dependent pathway that transports αvβ3 integrin from early endosomes to the plasma membrane, and that this is required for VEGF-driven fibronectin polymerization in endothelial cells. Furthermore, VEGFR1 acts to promote endothelial tubule branching in an organotypic model of angiogenesis via a mechanism that requires Rab4A and αvβ3 integrin. We conclude that a recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during branching morphogenesis of the vasculature.     

PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca²⁺/Zn²⁺-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.Oxygen and glucose deprivation (OGD)³ associated with transient global ischemia induces delayed cell death, particularly in hippocampal CA1 pyramidal cells (1–3), a phenomenon that involves Ca²⁺/Zn²⁺-permeable, GluR2-lacking AMPARs (4). AMPARs are heteromeric complexes of subunits GluR1–4 (5), and most AMPARs in the hippocampus contain GluR2, which renders them calcium-impermeable and results in a marked inward rectification in their current-voltage relationship (6–8). Ischemia induces a delayed down-regulation of GluR2 mRNA and protein expression (4, 9–11), resulting in enhanced AMPAR-mediated Ca²⁺ and Zn²⁺ influx into CA1 neurons (10, 12). In these neurons, AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification 1–2 days following ischemia and increased sensitivity to 1-naphthyl acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs (13–16). Blockade of these channels at 9–40 h following ischemia is neuroprotective, indicating a crucial role for Ca²⁺-permeable AMPARs in ischemic cell death (16).In addition to delayed changes in AMPAR subunit composition as a result of altered mRNA expression, it was recently reported that Ca²⁺-permable, GluR2-lacking AMPARs are targeted to synaptic sites via membrane trafficking at much earlier times during OGD (17). This subunit rearrangement involves endocytosis of AMPARs containing GluR2 complexed with GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3 (17). However, the molecular mechanisms behind this trafficking event are unknown, and furthermore, it is not known whether these trafficking-mediated changes in AMPAR subunit composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial involvement in long term depression (LTD) and long term potentiation (LTP), activity-dependent forms of synaptic plasticity thought to underlie learning and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching events (brought about by trafficking that involves endo/exocytosis) are central to the necessary changes in synaptic receptor complement (7, 18–20). It is possible that similar mechanisms regulate AMPAR trafficking during OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that binds, via the PDZ domain, to a number of membrane proteins including AMPAR subunits GluR2/3. This interaction is required for AMPAR internalization from the synaptic plasma membrane in response to Ca²⁺ influx via NMDAR activation in hippocampal neurons (21–23). This process is the major mechanism that underlies the reduction in synaptic strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged as a mechanism that regulates the GluR2 content of synaptic receptors, which in turn determines their Ca²⁺ permeability (7, 20). This is likely to be of profound importance in both plasticity and pathological mechanisms. Importantly, PICK1 overexpression has been shown to induce a shift in synaptic AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1 (24). This suggests that PICK1 may mediate the rapid switch in subunit composition occurring during OGD (17). Here, we demonstrate that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1 PDZ interactions, and importantly, that this early trafficking event that occurs during OGD contributes to the signaling that results in delayed neuronal death.     

Molecular differentiation of the Old World Culicoides imicola species complex (Diptera, Ceratopogonidae), inferred using random amplified polymorphic DNA markers

Samples of seven of the 10 morphological species of midges of the Culicoides imicola complex were considered. The importance of this species complex is connected to its vectorial capacity for African horse sickness virus (AHSV) and bluetongue virus (BTV). Consequently, the risk of transmission may vary dramatically, depending upon the particular cryptic species present in a given area. The species complex is confined to the Old World and our samples were collected in Southern Africa, Madagascar and the Ivory Coast. Genomic DNA of 350 randomly sampled individual midges from 19 populations was amplified using four 20-mer primers by the random amplified polymorphic DNA (RAPD) technique. One hundred and ninety-six interpretable polymorphic bands were obtained. Species-specific RAPD profiles were defined and for five species diagnostic RAPD fragments were identified. A high degree of polymorphism was detected in the species complex, most of which was observed within populations (from 64 to 76%). Principal coordinate analysis (PCO) and cluster analysis provided an estimate of the degree of variation between and within populations and species. There was substantial concordance between the taxonomies derived from morphological and molecular data. The amount and the different distributions of genetic (RAPD) variation among the taxa can be associated to their life histories, i.e. the abundance and distribution of the larval breeding sites and their seasonality.     

Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro.

The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5.