Determination of protection from serum nuclease activity by DNA-polyelectrolyte complexes using an electrophoretic method

Polyelectrolyte complexes between cationic polymers and DNA have emerged as potential nonviral vectors for DNA delivery. For successful in vivo delivery, methods for analyzing their ability to prevent digestion of the DNA payload by serum nucleases are essential. We report here a simple assay to determine degradation of DNA in these complexes using standard electrophoretic techniques. The assay is based on a high pH buffer which can dissociate the complexes under standard electrophoretic conditions. This assay can be used qualitatively to determine the time taken for degradation to occur. Alternatively, with a standard gel analysis program it can be used quantitatively to investigate rates of DNA degradation from complexes in the presence of serum nucleases. We have shown that it can distinguish between different formulations with the same polymer, and also to distinguish between the time taken to degradation and the rates of degradation of DNA in complexes formed with two structurally related, linear polyamidoamine polymers. The assay could also distinguish between the time to degradation using poly-l-lysine complexes, although these were less well dissociated by the electrophoresis buffer, and could not be analyzed quantitatively. This assay will be of value in investigating and developing polyelectrolyte formulations for parenteral administration.     

Different anesthetic sensitivities of skeletal and cardiac isoforms of the Ca-ATPase.

We have previously shown that low levels of the volatile anesthetic halothane activate the Ca-ATPase in skeletal sarcoplasmic reticulum (SR), but inhibit the Ca-ATPase in cardiac SR. In this study, we ask whether the differential inhibition is due to (a) the presence of the regulatory protein phospholamban in cardiac SR, (b) different lipid environments in skeletal and cardiac SR, or (c) the different Ca-ATPase isoforms present in the two tissues. By expressing skeletal (SERCA 1) and cardiac (SERCA 2a) isoforms of the Ca-ATPase in Sf21 insect cell organelles, we found that differential anesthetic effects in skeletal and cardiac SR are due to differential sensitivities of the SERCA 1 and SERCA 2a isoforms to anesthetics. Low levels of halothane inhibit the SERCA 2a isoform of the Ca-ATPase, and have little effect on the SERCA 1 isoform. The biochemical mechanism of halothane inhibition involves stabilization of E2 conformations of the Ca-ATPase, suggesting direct anesthetic interaction with the ATPase. This study establishes a biochemical model for the mechanism of action of an anesthetic on a membrane protein, and should lead to the identification of anesthetic binding sites on the SERCA 1 and SERCA 2a isoforms of the Ca-ATPase.     

Structural Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.     

Complete 1H, 13C and 15N NMR assignments for donor-strand complemented AafA, the major pilin of aggregative adherence fimbriae (AAF/II) from enteroaggregative E. coli

Aggregative adherence fimbriae (AAF) are the primary adhesive factors of enteroaggregative Escherichia coli (EAEC) and are required for intestinal colonization. They mediate binding to extracellular matrix proteins of the enteric mucosa and display proinflammatory effects on epithelial cells in vitro. Among the simplest of bacterial fimbriae, these passive hairlike appendages are composed primarily of a single 16-kDa structural and adhesive subunit, AafA. Oligomerization occurs by incorporating the N-terminal strand of each AafA subunit into an otherwise incomplete β-sheet of an adjacent AafA subunit. We have engineered a highly soluble AafA monomer by positioning the N-terminal “donor strand” at the C-terminus, following a turn and short linker that were introduced to allow access of the donor strand to the recipient cleft of the same subunit. The resulting “donor-strand complemented” AafA subunit, or AafA-dsc folds autonomously, is monodisperse in solution, and yields high quality NMR spectral data. Here, we report the ¹H, ¹³C, and ¹⁵N chemical shift assignments for AafA-dsc.     

Investing in Threatened Species Conservation: Does Corruption Outweigh Purchasing Power?

In many sectors, freedom in capital flow has allowed optimization of investment returns through choosing sites that provide the best value for money. These returns, however, can be compromised in countries where corruption is prevalent. We assessed where the best value for money might be obtained for investment in threatened species that occur at a single site, when taking into account corruption. We found that the influence of corruption on potential investment decisions was outweighed by the likely value for money in terms of pricing parity. Nevertheless global conservation is likely to get best returns in terms of threatened species security by investing in "honest" countries than in corrupt ones, particularly those with a high cost of living.     

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination

Background

During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.

Methods and Findings

To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject.

Conclusion

The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.     

SHORT NOTES ON BIRDS FROM KLIP RIVER VALLEY

Watkins, B. P. &; Furness, R. W. 1986. Population status, breeding and conservation of the Gough Moorhen. Ostrich 57: 32–36.

The Gough Moorhen Gallinula comeri holds territories of about 5000m² on Gough Island, where there may be 2 000–3 000 pairs. Scavenging from petrel carcasses partly eaten by Subantarctic Skuas Catharacta antarctica seems to be an important feeding method. Nests are constructed in tussocks with a tunnel entrance. Nest, egg and adult biometrics are given together with observations of incubation behaviour, and we discuss potential threats to this flightless endemic species.