Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jkappa recognition site. RBP-Jkappa recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jkappa, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jkappa sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch-RBP-Jkappa interaction. In addition to RBP-Jkappa, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.     

Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.     

The influence of facultative endosymbionts on honeydew carbohydrate and amino acid composition of the black bean aphid Aphis fabae

The facultative endosymbionts Hamiltonella defensa and Regiella insecticola are commonly found in aphids. They are linked with various ecological benefits but generally occur at low prevalence, which indicates a possible harbouring cost. Little is known about how the presence of facultative endosymbionts is reflected in honeydew composition. Honeydew is the key mediator of the mutualism between aphids and their tending ants. The present study examines whether endosymbionts have an influence on aphid honeydew quality by comparing the amino acid and carbohydrate concentrations between infected and uninfected aphids. To this end, two genetic lines of the aphid Aphis fabae Scopoli are experimentally infected with different strains of Hamiltonella and Regiella. Infected aphids are shown to have reduced concentrations of amino acids in the honeydew compared with uninfected aphids. However, the presence of endosymbionts has no effect on the absolute amount of carbohydrates produced. Nevertheless, interclonal variation in honeydew composition between aphid genotypes is observed for both carbohydrate and amino acid production. These results imply that the nutritional value of honeydew depends on aphid genotype, as well as on the presence of secondary bacterial endosymbionts, which suggests that there is a physiological cost of harbouring endosymbionts and which could also impact aphid attractiveness to tending ants.     

Somatic embryogenesis from <Emphasis Type="Italic">Arabidopsis</Emphasis> shoot apical meristem mutants

Zygotic embryos of three Arabidopsis thaliana (L.) Heynh. mutants lacking an embryonic shoot apical meristem (SAM), shoot meristemless (stm), wuschel (wus) and zwille/pinhead (zll/pnh) were used as explants to establish embryogenic cell cultures. Somatic embryos of all three mutants showed the same mutant phenotypes as their zygotic equivalents. These results provide genetic evidence that the developmental program of somatic and zygotic embryos is indistinguishable. They also suggest that a functional SAM is not required for somatic embryogenic cell formation in Arabidopsis.     

Multiple testing in candidate gene situations: a comparison of classical, discrete, and resampling-based procedures

In candidate gene association studies, usually several elementary hypotheses are tested simultaneously using one particular set of data. The data normally consist of partly correlated SNP information. Every SNP can be tested for association with the disease, e.g., using the Cochran-Armitage test for trend. To account for the multiplicity of the test situation, different types of multiple testing procedures have been proposed. The question arises whether procedures taking into account the discreteness of the situation show a benefit especially in case of correlated data. We empirically evaluate several different multiple testing procedures via simulation studies using simulated correlated SNP data. We analyze FDR and FWER controlling procedures, special procedures for discrete situations, and the minP-resampling-based procedure. Within the simulation study, we examine a broad range of different gene data scenarios. We show that the main difference in the varying performance of the procedures is due to sample size. In small sample size scenarios,the minP-resampling procedure though controlling the stricter FWER even had more power than the classical FDR controlling procedures. In contrast, FDR controlling procedures led to more rejections in higher sample size scenarios.     

A systematic study of the energetics involved in structural changes upon association and connectivity in protein interaction networks

The study of protein binding mechanisms is a major topic of research in structural biology. Here, we implement a combination of metrics to systematically assess the cost of backbone conformational changes that protein domains undergo upon association. Through the analyses of 2090 unique unbound → bound transitions, from over 12,000 structures, we show that two-thirds of these proteins do not suffer significant structural changes upon binding, and could thus fit the lock-and-key model well. Among the remaining proteins, one-third explores the bound conformation in the unbound state (conformational selection model) and, while most transitions are possible from an energetic perspective, a few do require external help to break the thermodynamic barrier (induced fit model). We also analyze the relationship between conformational transitions and protein connectivity, finding that, in general, domains interacting with many partners undergo smaller changes upon association, and are less likely to freely explore larger conformational changes.