Thermodynamic contributions of single internal rA·dA, rC·dC, rG·dG and rU·dT mismatches in RNA/DNA duplexes

The thermodynamic contributions of rA·dA, rC·dC, rG·dG and rU·dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the ΔG°37, ΔH° and melting temperature (Tm) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4°C, respectively. The general trend in decreasing stability for the single internal mismatches is rG·dG > rU·dT > rA·dA > rC·dC. The stability trend for the base pairs 5′ of the single internal mismatch is rG·dC > rC·dG > rA·dT > rU·dA. The stability trend for the base pairs 3′ of the single internal mismatch is rC·dG > rG·dC >> rA·dT > rU·dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST

Expression, purification and characterization of the LEC-rat N-terminal metal binding domain from Atp7b, an orthologue to ATP7B, a copper transporting P-type ATPase implicated in Wilson disease

grantor: University of TorontoWilson disease is an autosomal disorder caused by mutations in the 'ATP7B' gene encoding a copper-transporting P-type ATPase, resulting in compromised copper transport across the trans-Golgi network and plasma membrane of hepatocytes. The Long Evans Cinnamon (LEC) rat is an established animal model for Wilson disease. As in all rats, the N-terminal copper-binding domain of the LEC rat atp7b protein (LEC-rCBD) contains 5 putative copper binding motifs (GMTCXXC), as compared to 6 in the human orthologue. Competition 65Zn-blot experiments with Cu(I) and Ag(I) have strongly implicated LEC-rCBD to display cooperativity, a phenomenon exclusive to these two metals and not observed among other transition metals examined. Near-UV Circular Dichroism (CD) spectra suggest secondary and tertiary structural changes in the protein with the gradual saturation of copper, the most significant structural transformation occurring between 2 and 3 moles copper per mole of LEC-rCBD. Far-UV CD demonstrates that Ag(I) induces similar structural deviations and that the addition of copper to a 1:5, protein:metal ratio is directly proportional to a decrease in LEC-rCBD disulfide bonds. These results together with those obtained from competition studies establish a model where LEC-rCBD undergoes specific metal dependent structural changes.M.Sc

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes