Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.     

Metabolomics Reveals the Origins of Antimicrobial Plant Resins Collected by Honey Bees

The deposition of antimicrobial plant resins in honey bee, Apis mellifera, nests has important physiological benefits. Resin foraging is difficult to approach experimentally because resin composition is highly variable among and between plant families, the environmental and plant-genotypic effects on resins are unknown, and resin foragers are relatively rare and often forage in unobservable tree canopies. Subsequently, little is known about the botanical origins of resins in many regions or the benefits of specific resins to bees. We used metabolomic methods as a type of environmental forensics to track individual resin forager behavior through comparisons of global resin metabolite patterns. The resin from the corbiculae of a single bee was sufficient to identify that resin''s botanical source without prior knowledge of resin composition. Bees from our apiary discriminately foraged for resin from eastern cottonwood (Populus deltoides), and balsam poplar (P. balsamifera) among many available, even closely related, resinous plants. Cottonwood and balsam poplar resin composition did not show significant seasonal or regional changes in composition. Metabolomic analysis of resin from 6 North American Populus spp. and 5 hybrids revealed peaks characteristic to taxonomic nodes within Populus, while antimicrobial analysis revealed that resin from different species varied in inhibition of the bee bacterial pathogen, Paenibacillus larvae. We conclude that honey bees make discrete choices among many resinous plant species, even among closely related species. Bees also maintained fidelity to a single source during a foraging trip. Furthermore, the differential inhibition of P. larvae by Populus spp., thought to be preferential for resin collection in temperate regions, suggests that resins from closely related plant species many have different benefits to bees.     

Deriving how far structural information is transmitted through parallel homodimeric coiled‐coils: A correlation analysis of helical staggers

How local conformation is affected by local sequence is fairly well understood for alpha‐helical coiled‐coils, but less is known about how local conformation is influenced by distant features. Here, I describe an approach to detect such an effect, based on computing correlation coefficients of local out‐of‐register alignments, or so‐called “staggers” between the helices, as a function of the axial distance between the staggers. This approach requires parallel homodimers, in which each stagger can occur with two “signs,” where either one helix or the other is shifted towards the N terminus. The signs of such staggers separated by up to 12 residues are strongly correlated, indicating that the conformations of the ends of coiled‐coils are commonly influenced by attached structures. Thus, the structures of coiled‐coil residues aberrantly attached to alternative proteins, such as those resulting from leukemogenic chromosomal rearrangements, may be distinguishable from those in normal tissues, and in turn serve as targets of selective drug design. The signs of helical staggers separated by between 13 and 30 residues are moderately yet significantly correlated, indicating that some of the coiled‐coils transmit this conformational feature axially for at least 45 Å. A positive, albeit noisy, correlation also exists among tropomyosin coiled‐coils for signed staggers separated by the 40‐residue actin repeat distance, consistent with the semi‐flexible tropomyosin filament binding F‐actin and regulating skeletal muscle contraction in a partially cooperative manner. Communication of the signs of axial staggers is explained in part by minimization of main‐chain hydrogen bond deformations. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Theoretical Analysis of the Footprinting Experiment

Abstract

In the footprinting experiment, an end-radiolabeled DNA restriction fragment is subjected to digest by an endonuclease in the presence and absence of a ligand which alters the endonuclease cleavage rate at sites of ligand-DNA contact. The location of these sites, and the strength of the ligand binding, are then deduced from the measured concentrations of the different oligonucleotides produced by the digest. We analyze the experiment in terms of coupled kinetic equations which take into account the cutting rates of endonuclease for sites with ligand present and absent, and the rates of binding and dissociation of the ligand to a site. As long as the ligand concentration remains essentially constant (which occurs, for example, if digest is terminated early enough to assure that all fragments result from single cuts by the endonuclease), the oligonucleotide concentrations reflect only the ligand binding equilibrium constant (ratio of rate constants) and the cutting rates in the presence and absence of ligand. We also show how the measured oligonucleotide concentrations (from, e.g. an autoradiogram) can be used to deduce the ligand equilibrium binding constants for the various sites on the polymer.     

Studies on Bleomycin-DNA and Bleomycin-Iron Interactions

Abstract

The bleomycins, a group of antitumor antibiotics (Figure 1), cause the degradation of DNA by a process requiring iron(II) and dioxygen (1,2). DNA degradation appears to involve two steps: association of the drug with the nucleic acid and degradation of the DNA. As part of studies directed toward achieving an understanding of how the bleomycins degrade DNA, we have examined various properties of the drug using a variety of chemical and physico- chemical techniques, including NMR and Mössbauer spectroscopy. We have studied both the interaction of the antibiotic with its target (DNA) as well as its association with its metal ion cofactor. This work has been performed on the intact drug and its derivatives as well as on synthetic models of the parent drug. This paper reviews and updates the recent work from this laboratory on the bleomycins.     

Hydrated mucilage reduces post-dispersal seed removal of a sand desert shrub by ants in a semiarid ecosystem

Post-dispersal seed removal by animals can lead to extensive seed loss and thus is an important factor in structuring plant communities. However, we know much less about post-dispersal seed predation than about other forms of herbivory. Mucilage plays many ecological roles in adaptation of plants to diverse environments; nevertheless, until now the role of mucilage in ant-mediated seed movement remains largely hypothetical. We studied the role of mucilage in seed removal of Artemisia sphaerocephala by ants in Mu Us Sandland in Inner Mongolia, China. Messor aciculatus was the most active seed predator of Artemisia sphaerocephala. Time to first ant collecting (T _1st) of wet intact seeds was longest and significantly different from that for dry intact seeds, wet demucilaged seeds, and dry demucilaged seeds; number of seeds removed to ant nests was lowest for wet intact seeds. After they were collected by ants, 5 % of wet intact seeds were dropped during transport. Our results indicate that seed mucilage of Artemisia sphaerocephala may play a significant role in post-dispersal seed removal by (1) making seeds less attractive to ants, thus resulting in a delay of collection time; (2) forming a strong bond to soil particles, making it difficult for ants to remove seeds; and (3) making seeds more likely to be dropped during transport, thereby allowing them to escape from predation even after collection by ants. This study demonstrates the importance of mucilage in reducing seed removal by ants and thus in anchoring seeds of desert plants in the vicinity of mother plants.     

Origin and evolution of the Pseudorhyncocyonidae,a European Paleogene family of insectivorous placental mammals

Two new species of pseudorhyncocyonid, Fordonia lawsoni sp. nov. and Leptictidium prouti sp. nov. from the UK earliest Eocene, described here, are older than any previously recorded member of the family. They are represented by teeth from numerous loci, which allow a better understanding of the sparsely known dentitions of currently known pseudorhyncocyonids. This facilitates the recognition of two further species of Leptictidium, L. listeri sp. nov. from the Middle Eocene of Germany and L. storchi sp. nov. from the Late Eocene of France. Study of occlusal relationships also helps to fill gaps in our knowledge of missing tooth loci. Cladistic analysis of pseudorhyncocyonids with their previously judged closest relatives, the Leptictidae, Pantolesta and Palaeanodonta, shows that two European species, Diaphyodectes prolatus and Palaeictops? levei, formerly thought to be leptictids, are instead primitive pseudorhyncocyonids, extending the range of the family further back in time to the Middle Paleocene. P? levei is placed in the new genus Phakodon gen. nov. The analysis also shows that the Pseudorhyncocyonidae are sister group to the other three groups combined and that family‐level differentiation in this probable clade took place as early as the earliest Paleocene.     

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the K_i in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.     

Formation of Covellite (CuS) Under Biological Sulfate-Reducing Conditions

Sulfate-reducing bacteria (SRB) play a major role in the precipitation of metal sulfides in the environment. In this work, biogenic copper sulfide formation was examined in cultures of SRB and compared to chemically initiated Cu sulfide precipitation as a reference system. Mixed cultures of SRB were incubated at 22, 45, and 60°C in nutrient solutions that contained copper sulfate. Abiotic reference samples were produced by reacting uninoculated liquid media with Na₂S solutions under otherwise identical conditions. Precipitates were collected anaerobically by centrifugation, frozen in liquid N₂, and freeze-dried, followed by analysis using X-ray diffraction (XRD), X-ray fluorescence, and scanning electron microscopy. Covellite (CuS) was the only mineral found in the precipitates. Covellite was less crystalline in the biogenic precipitates than in the abiotic samples based on XRD peak widths and peak to background ratios. Poor crystallinity may be the result of slower precipitation rates in bacterial cultures as compared to the abiotic reference systems. Furthermore, bacterial cells may inhibit the nucleation steps that lead to crystal formation. Incubation at elevated temperatures improved the crystallinity of the biotic specimens.