Implementing the equality agenda: the scale of the practical problems involved and the power of the opposition should not be underestimated

Mike O’Donnell argues that reducing social inequality is a vitally important objective for the UK. In this article he examines the sizeable obstacles to substantive and meaningful reform

Nucleotide-Induced Conformational Changes in an Isolated Escherichia coli DNA Polymerase III Clamp Loader Subunit

AbstractSliding clamps are loaded onto DNA by ATP-driven clamp loader complexes. The structure of the E. coli clamp loader in a nucleotide-free state has been determined previously. We now report crystal structures of a truncated form of the isolated γ-ATPase subunit, γ1–243, of the E. coli clamp loader, in nucleotide-free and bound forms. The γ subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked. The binding of either ATPγS or ADP, which are shown to bind with equal affinity to γ1–243, induces a change in the relative orientation of the two domains such that nucleotides can be accommodated. This change would break one of the γ:γ interfaces seen in the empty clamp loader complex, and may represent one step in the activation process

Analysis of the role of PCNA-DNA contacts during clamp loading

<p>Abstract</p> <p>Background</p> <p>Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.</p> <p>Results</p> <p>We built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 Å resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.</p> <p>Conclusion</p> <p>Mutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.</p

Crystal structure of a DNA polymerase sliding clamp from a Gram-positive bacterium

BACKGROUND: Sliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously. RESULTS: We have determined the crystal structure of the dimeric β subunit of the DNA polymerase III holoenzyme of Streptococcus pyogenes. The sliding clamp from this Gram-positive organism forms a ring-shaped dimeric assembly that is similar in overall structure to that of the sliding clamps from Gram-negative bacteria, bacteriophage T4, eukaryotes and archaea. The dimer has overall dimensions of ~90 Å × ~70 Å × ~25 Å with a central chamber that is large enough to accommodate duplex DNA. In comparison to the circular shape of other assemblies, the S. pyogenes clamp adopts a more elliptical structure. CONCLUSION: The sequences of sliding clamps from S. pyogenes and E. coli are only 23% identical, making the generation of structural models for the S. pyogenes clamp difficult in the absence of direct experimental information. Our structure of the S. pyogenes β subunit completes the catalog of clamp structures from all the major sequence grouping of sliding clamps. The more elliptical rather than circular structure of the S. pyogenes clamp implies that the topological nature of encircling DNA, rather than a precise geometric shape, is the most conserved aspect for this family of proteins

The Availability of Prior ECGs Improves Paramedic Accuracy in Recognizing ST-Segment Elevation Myocardial Infarction

Introduction Early and accurate identification of ST-elevation myocardial infarction (STEMI) by prehospital providers has been shown to significantly improve door to balloon times and improve patient outcomes. Previous studies have shown that paramedic accuracy in reading 12 lead ECGs can range from 86% to 94%. However, recent studies have demonstrated that accuracy diminishes for the more uncommon STEMI presentations (e.g. lateral). Unlike hospital physicians, paramedics rarely have the ability to review previous ECGs for comparison. Whether or not a prior ECG can improve paramedic accuracy is not known. Study hypothesis The availability of prior ECGs improves paramedic accuracy in ECG interpretation. Methods 130 paramedics were given a single clinical scenario. Then they were randomly assigned 12 computerized prehospital ECGs, 6 with and 6 without an accompanying prior ECG. All ECGs were obtained from a local STEMI registry. For each ECG paramedics were asked to determine whether or not there was a STEMI and to rate their confidence in their interpretation. To determine if the old ECGs improved accuracy we used a mixed effects logistic regression model to calculate p-values between the control and intervention. Results The addition of a previous ECG improved the accuracy of identifying STEMIs from 75.5% to 80.5% (p = 0.015). A previous ECG also increased paramedic confidence in their interpretation (p = 0.011). Conclusions The availability of previous ECGs improves paramedic accuracy and enhances their confidence in interpreting STEMIs. Further studies are needed to evaluate this impact in a clinical setting

Cultured gill epithelia as models for the freshwater fish gill

AbstractWe review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral “influx” of Cl− (and not of Na+). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na+,K+-ATPase activity and promoting active Na+ and Cl− influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na+ uptake demonstrate that both NH3 and NH4+ diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na+-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested