Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).
The concentration of circulating DNA (cirDNA) and deoxyribonuclease activity in blood plasma of healthy donors and patients with colon or stomach cancer were analyzed. The concentration of DNA was measured using Hoechst 33258 fluorescent assay after the isolation by the glass-milk protocol. A 1-kbp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers was used as a substrate for DNase. DNase activity was estimated from the data of immunochemical detection of the nonhydrolyzed amplicon. The average concentration of cirDNA in the plasma of healthy donors was low (34 +/- 34 ng/mL), and was accompanied with high DNase activity (0.356 +/- 0.410 U/mL). The increased concentrations of cirDNA in blood plasma of patients with colon and stomach cancer were accompanied by a decrease in DNase activity below the detection level of the assay. The data obtained demonstrate that low DNase activity in blood plasma of cancer patients can cause an increase in the concentration of cirDNA.
Calcium phosphate bions (CPB) are biomimetic mineralo-organic nanoparticles which represent a physiological mechanism regulating the function, transport and disposal of calcium and phosphorus in the human body. We hypothesised that CPB may be pathogenic entities and even a cause of cardiovascular calcification. Here we revealed that CPB isolated from calcified atherosclerotic plaques and artificially synthesised CPB are morphologically and chemically indistinguishable entities. Their formation is accelerated along with the increase in calcium salts-phosphates/serum concentration ratio. Experiments in vitro and in vivo showed that pathogenic effects of CPB are defined by apoptosis-mediated endothelial toxicity but not by direct tissue calcification or functional changes in anti-calcification proteins. Since the factors underlying the formation of CPB and their pathogenic mechanism closely resemble those responsible for atherosclerosis development, further research in this direction may help us to uncover triggers of this disease.
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