Associations between the CYBA 242C/T and the MPO -463G/A Polymorphisms, Oxidative Stress and Cardiovascular Disease in Chronic Kidney Disease Patients

Genetic variations in the NADPH/MPO system in chronic kidney disease (CKD) patients might lead to altered activity of these enzymes, and thus to altered risk for oxidative stress (OS) and cardiovascular disease (CVD). We evaluated the impact of 242C/T CYBA and -463G/A MPO polymorphisms on OS and CVD mortality in stage 5 CKD patients starting dialysis. Two hundred and fifty-seven patients were genotyped using Pyrosequencing. Plasmalogen [dimethylacetal (DMA) 16/C16:0] was used as OS marker. CVD was assessed from patient history and clinical symptoms. Prevalence of CVD was higher (35%) in GG patients (MPO) compared to AG (26%) and AA (0%) patients (p < 0.01). Patients with CC genotype (CYBA) had lower levels of DMA 16/C16:0 (ratio 0.071 +/- 0.003) compared to TT patients (0.089 +/- 0.006; p < 0.05). These patients also had increased CVD mortality compared to CT and TT patients (chi(2) 2.19; p < 0.05). We conclude that genetic variations in the NADPH/MPO system are associated with OS, presence of CVD and CVD-related mortality in CKD patients. Copyright (c) 2007 S. Karger AG, Basel.     

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from approximately 2 ms(-1) at approximately 37 degrees C to approximately 40 ms(-1) at approximately 10 degrees C and correspond to an activation energy of approximately 14 +/- 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF.