Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

Nitrogen and water affect direct and indirect plant systemic induced defense in cotton

Plants have direct and indirect constitutively produced and inducible defenses against herbivores and pathogens, which can substantially aid in their ability to defend themselves. However, very little is known about the influence of agronomic factors on such defenses. Here, we tested the effects of nitrogen levels and water availability on the ability of cotton plants to deter feeding by Spodoptera exigua through induction of anti-feedants, and to attract Microplitis croceipes through systemic induction of volatile emission. Cotton plants were grown with various nitrogen levels and were either exposed to water stress or normal water before being exposed to S. exigua for 48 h for induction of defenses. Dual choices of various nitrogen and water treatments were provided to M. croceipes in flight tunnel bioassays. Dual choices of leaf tissue from the various nitrogen and water treatments were provided to S. exigua larvae. Both water stress and nitrogen levels under and over the recommended levels increased leaf tissue consumption and decreased attraction of M. croceipes to the plants. Analyses of induced volatiles released from herbivore damaged plants indicate that their concentrations differ among the nitrogen levels tested with plants receiving no nitrogen or twice the recommended dose having amounts much lower than plants receiving the recommended dose. Because both direct and indirect plant defense mechanisms are negatively affected by improper nitrogen and insufficient water, we argue that these factors should be considered for a better natural control of pests in cotton and most probably in other crops.     

Coevolution of Interacting Fertilization Proteins

Reproductive proteins are among the fastest evolving in the proteome, often due to the consequences of positive selection, and their rapid evolution is frequently attributed to a coevolutionary process between interacting female and male proteins. Such a process could leave characteristic signatures at coevolving genes. One signature of coevolution, predicted by sexual selection theory, is an association of alleles between the two genes. Another predicted signature is a correlation of evolutionary rates during divergence due to compensatory evolution. We studied female–male coevolution in the abalone by resequencing sperm lysin and its interacting egg coat protein, VERL, in populations of two species. As predicted, we found intergenic linkage disequilibrium between lysin and VERL, despite our demonstration that they are not physically linked. This finding supports a central prediction of sexual selection using actual genotypes, that of an association between a male trait and its female preference locus. We also created a novel likelihood method to show that lysin and VERL have experienced correlated rates of evolution. These two signatures of coevolution can provide statistical rigor to hypotheses of coevolution and could be exploited for identifying coevolving proteins a priori. We also present polymorphism-based evidence for positive selection and implicate recent selective events at the specific structural regions of lysin and VERL responsible for their species-specific interaction. Finally, we observed deep subdivision between VERL alleles in one species, which matches a theoretical prediction of sexual conflict. Thus, abalone fertilization proteins illustrate how coevolution can lead to reproductive barriers and potentially drive speciation.     

The conserved C-terminal tail of FtsZ is required for the septal localization and division inhibitory activity of MinCC/MinD

The Escherichia coli Min system contributes to spatial regulation of cytokinesis by preventing assembly of the Z ring away from midcell. MinC is a cell division inhibitor whose activity is spatially regulated by MinD and MinE. MinC has two functional domains of similar size, both of which have division inhibitory activity in the proper context. However, the molecular mechanism of the inhibitory action of either domain is not very clear. Here, we report that the septal localization and division inhibitory activity of MinC^C/MinD requires the conserved C-terminal tail of FtsZ. This tail also mediates interaction with two essential division proteins, ZipA and FtsA, to link FtsZ polymers to the membrane. Overproduction of MinC^C/MinD displaces FtsA from the Z ring and eventually disrupts the Z ring, probably because it also displaces ZipA. These results support a model for the division inhibitory action of MinC/MinD. MinC/MinD binds to ZipA and FtsA decorated FtsZ polymers located at the membrane through the MinC^C/MinD–FtsZ interaction. This binding displaces FtsA and/or ZipA, and more importantly, positions MinC^N near the FtsZ polymers making it a more effective inhibitor.     

Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A

Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Rapid quantitative determination of fat-soluble vitamins and coenzyme Q-10 in human serum by reversed phase ultra-high pressure liquid chromatography with UV detection

We are presenting the first ultra-high pressure LC (UHPLC) method for rapid quantitative measurement of vitamin A, E (α- and γ-tocopherol), β-carotene and CoQ₁₀ from human serum. The chromatography was performed on Shield RP₁₈ UHPLC column with UV detection. The method was validated based on linearity, accuracy, matrix effects study, precision and stability. The calibration was linear over the following range: 0.09–10.0 for retinol and γ-tocopherol, 0.05–5 for β-carotene, 0.9–100 for α-tocopherol and 0.14–15 mg/L for CoQ₁₀. The limit of detection and quantitation for retinol, γ-tocopherol, β-carotene, α-tocopherol and CoQ₁₀ were as follows 0.07/0.024, 0.018/0.06, 0.004/0.12, 0.078/0.261, 0.008/0.028 mg/L. The recoveries were above 85%. The inter- and intra-assay precision was below 10%. Reference intervals were established for children and adults. Because of its low cost, extremely short analysis time (2 min) and excellent chromatographic reproducibility this UHPLC method can easily be adopted for high-throughput clinical and pharmacokinetics studies.