Disulfide transfer between two conserved cysteine pairs imparts selectivity to protein oxidation by Ero1

The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.     

Genome-wide identification of human functional DNA using a neutral indel model

It has become clear that a large proportion of functional DNA in the human genome does not code for protein. Identification of this non-coding functional sequence using comparative approaches is proving difficult and has previously been thought to require deep sequencing of multiple vertebrates. Here we introduce a new model and comparative method that, instead of nucleotide substitutions, uses the evolutionary imprint of insertions and deletions (indels) to infer the past consequences of selection. The model predicts the distribution of indels under neutrality, and shows an excellent fit to human–mouse ancestral repeat data. Across the genome, many unusually long ungapped regions are detected that are unaccounted for by the neutral model, and which we predict to be highly enriched in functional DNA that has been subject to purifying selection with respect to indels. We use the model to determine the proportion under indel-purifying selection to be between 2.56% and 3.25% of human euchromatin. Since annotated protein-coding genes comprise only 1.2% of euchromatin, these results lend further weight to the proposition that more than half the functional complement of the human genome is non-protein-coding. The method is surprisingly powerful at identifying selected sequence using only two or three mammalian genomes. Applying the method to the human, mouse, and dog genomes, we identify 90 Mb of human sequence under indel-purifying selection, at a predicted 10% false-discovery rate and 75% sensitivity. As expected, most of the identified sequence represents unannotated material, while the recovered proportions of known protein-coding and microRNA genes closely match the predicted sensitivity of the method. The method's high sensitivity to functional sequence such as microRNAs suggest that as yet unannotated microRNA genes are enriched among the sequences identified. Futhermore, its independence of substitutions allowed us to identify sequence that has been subject to heterogeneous selection, that is, sequence subject to both positive selection with respect to substitutions and purifying selection with respect to indels. The ability to identify elements under heterogeneous selection enables, for the first time, the genome-wide investigation of positive selection on functional elements other than protein-coding genes.     

Effects of excess selenomethionine on selenium status indicators in pregnant long-tailed macaques (Macaca fascicularis)

Forty pregnant long-tailed macaques were treated daily for 30 d with 0, 25, 150 or 300 μg selenium as L-selenomethionine/kg body weight. Erythrocyte and plasma selenium and glutathione peroxidase specific activities, hair and fecal selenium, and urinary selenium excretion were increased by and were linearly related to L-selenomethionine dose. Hair selenium was most sensitive to L-selenomethionine dose, with an 84-fold increase in the 300 μg selenium/(kg-d) group relative to controls (r=0.917). Daily urinary selenium excretion (80-fold,r=0.958), plasma selenium (22-fold,r=0.885), erythrocyte selenium (24-fold,r=0.920), and fecal selenium (18-fold,r=0.911) also responded strongly to L-selenomethionine. Erythrocyte and plasma glutathione peroxidase specific activities increased 154% and 69% over controls, respectively. Toxicity was associated with erythrocyte selenium >2.3 μg/mL, plasma selenium >2.8 μg/mL, and hair selenium >27 μg/g. Plasma, erythrocyte, and hair selenium concentrations may be useful for monitoring and preventing the toxicity of L-selenomethionine administered to humans in cancer chemoprevention trials.     

TEP on the tide in killifish (<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>): effects of progressively changing salinity and prior acclimation to intermediate or cycling salinity

Transepithelial potentials (TEP) were measured in killifish, acclimated to freshwater (FW), seawater (SW), 33% SW or cycling salinities relevant to tidal cycles in an estuary, and subsequently subjected to salinity changes in progressive or random order. Random compared to progressive salinity changes in an upward or downward direction in FW- and SW-acclimated fish, respectively, did not greatly influence responses to salinity change. Fish acclimated to SW or 33% SW as well as those acclimated to cycling salinities behaved similarly (TEP more positive than +15 mV in 100% SW, decreasing to ~0 mV at 20–40% SW, and more negative than −30 mV in FW). In contrast, FW-acclimated fish displayed a less pronounced TEP response to salinity (0 mV in FW through 20% SW, increasing thereafter to values more positive than +10 mV at 100% SW). We conclude that when evaluated under estuarine tidal conditions, the killifish gill exhibits adaptive electrical characteristics, opposing Na⁺ loss at low salinity and favouring Na⁺ extrusion at high salinity, changes explained at least in part by the Cl⁻ to Na⁺ permeability ratio. Thus animals living in the estuaries can move to lower and higher salinities for short periods with little physiological disturbance, but this ability is lost after acclimation to FW.     

Efficacy of several insecticides alone and with horticultural mineral oils on light brown apple moth (Lepidoptera: Tortricidae) eggs

The aim of the research was to identify efficacious and less environmentally harmful treatments than the standard chlorpyrifos sprays used for the control light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), eggs on nursery stock. A series of dip experiments showed a range of responses when comparing the efficacy of insecticides on egg hatch of E. postvittana. The insecticides that compared most favorably with chlorpyrifos were lamda-cyhalothrin and gamma-cyhalothrin, and thiacloprid. Indoxacarb, novaluron, and spinosad caused significant mortality only when combined with All Seasons mineral oil. All Seasons, showed ovicidal properties when evaluated alone and demonstrated adjuvant properties when combined with the above-mentioned insecticides, except gamma-cyhalothrin and thiacloprid. Several other horticultural mineral oils performed similarly, except the efficacy of spinosad varied with the oil product used, suggesting that the oil type selected is important for some insecticide and oil combinations. Several insecticides evaluated in this study are likely candidates for further work to develop treatments against E. postvittana eggs on nursery plants. Mineral oils are ovicidal and combinations with insecticides are likely to be advantageous.     

The X-linked tumor suppressor TSPX interacts and promotes degradation of the hepatitis B viral protein HBx via the proteasome pathway

Hepatitis B virus (HBV) infection is a major risk for hepatocellular carcinoma (HCC), and it is a serious global health problem with two billion people exposed to it worldwide. HBx, an essential factor for viral replication and a putative oncoprotein encoded by the HBV genome, has been shown to promote oncogenic properties at multiple sites in HBV-infected liver cells. The expression level of HBx closely associates with the development and progression of HCC, therefore the mechanism(s) regulating the stability of HBx is important in oncogenesis of HBV-infected cells. We demonstrate that the X-linked tumor suppressor TSPX enhances the degradation of HBx through the ubiquitin-proteasome pathway. TSPX interacts with both HBx and a proteasome 19S lid subunit RPN3 via its C-terminal acidic tail. Most importantly, over-expression of RPN3 protects HBx from, and hence acts as a negative regulator for, proteasome-dependent degradation. TSPX abrogates the RPN3-depedent stabilization of HBx, suggesting that TSPX and RPN3 act competitively in regulation of HBx stability. Since mutation and/or epigenetic repression of X-located tumor suppressor gene(s) could significantly predispose males to human cancers, our data suggest that TSPX-induced HBx degradation could play key role(s) in hepatocarcinogenesis among HBV-infected HCC patients.     

Low and annually variable migratory connectivity in a long‐distance migrant: Whinchats Saxicola rubetra may show a bet‐hedging strategy

The spatial scale of non‐breeding areas used by long‐distance migrant animals can vary from specific, relatively small non‐breeding areas for each independent breeding population (high connectivity) to a distribution over a large non‐breeding area with mixing of breeding populations (low connectivity). Measuring variation in the degree of connectivity and how it arises is crucial to predict how migratory animals can respond to global habitat and climate change because low connectivity is likely to be an adaptation to environmental uncertainty. Here, we assess whether use of non‐breeding areas in a long‐distance migrant may be stochastic by measuring the degree of connectivity, and whether it is annually variable. Twenty‐nine wintering Whinchats tagged with geolocators over 2 years within 40 km² in central Nigeria were found to be breeding over 2.55 million km² (26% of the land area of Europe), without an asymptote being approached in the relationship between area and sample size. Ranges differed in size between years by 1.51 million km² and only 15% of the total breeding range across both years overlapped (8% overlap between years when only first‐year birds were considered), well above the range size difference and below the proportion of overlap that would be predicted from two equivalent groups breeding at random locations within the observed range. Mean distance between breeding locations (i.e. migratory spread) differed significantly between years (604 ± 18 km in 2013 and 869 ± 33 km in 2014). The results showed very low and variable connectivity that was reasonably robust to the errors and assumptions inherent in the use of geolocators, but with the caveat of having only ranges of 2 years to compare, and the sensitivity of range to the breeding locations of a small number of individuals. However, if representative, the results suggest the scope for between‐year variation (cohort effects) to determine migrant distribution on a large scale. Furthermore, for species with similarly low connectivity, we would predict breeding population trends to reflect average conditions across large non‐breeding areas: thus, as large areas of Africa become subject to habitat loss, migrant populations throughout Europe will decline.     

Using haplotypes to monitor the migration of fall armyworm (Lepidoptera: Noctuidae) corn-strain populations from Texas and Florida

Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), infestations in most of North America north of Mexico arise from annual migrations of populations that overwinter in southern Texas and Florida. A comparison of the cytochrome oxidase I haplotype profiles within the fall armyworm corn-strain, the subgroup that preferentially infests corn (Zea mays L.) and sorghum (Sorghum vulgare Pers.), identified significant differences in the proportions of certain haplotypes between the Texas and Florida populations. These proportional differences were preserved as the populations migrated, providing a molecular metric by which the source of a migrant population could be identified. The migratory pattern derived from this method for several southeastern states was shown to be consistent with predictions based on analysis of historical agricultural and fall armyworm infestation data. These results demonstrate the utility of haplotype proportions to monitor fall armyworm migration, and they also introduce a potential method to predict the severity of cotton crop infestations in the short term.