Biomedical subjects

Yuri E Nesmelov

Publications and source records attributed to Yuri E Nesmelov.

4 recordsLinked to original sources

PubMed2006

Measurement of myocardial free radical production during exercise using EPR spectroscopy.

Exercise is associated with an increase in oxygen flux through the mitochondrial electron transport chain that has recently been demonstrated to increase the production of reactive oxygen species (ROS) in skeletal muscle. This study examined whether exercise also causes free radical production in the heart. We measured ROS production in seven chronically instrumented dogs during rest and treadmill exercise (6.4 km/h at 10 degrees grade; and heart rate, 204 +/- 3 beats/min) using electron paramagnetic resonance spectroscopy in conjunction with the spin trap alpha-phenyl-tert-butylnitrone (PBN) (0.14 mol/l) in blood collected from the aorta and coronary sinus (CS). To improve signal detection, the free radical adducts were deoxygenated over a nitrogen stream for 15 min and extracted with toluene. The hyperfine splitting constants of the radicals were alpha(N) = 13.7 G and alpha(H) = 1.0 G, consistent with an alkoxyl or carbon-centered radical. Resting aortic and CS PBN adduct concentrations were 6.7 and 6.3 x 10(8) arbitrary units (P = not significant). Both aortic and CS adduct concentrations increased during exercise, but there was no significant difference between the aortic and CS concentrations. Thus, in contrast to skeletal muscle, submaximal treadmill exercise did not result in detectable free radical production by the heart.

Animals↗

PubMed2005

Multibore sample cell increases EPR sensitivity for aqueous samples.

We have performed calculations, verified by experiment, to explain why the sensitivity of biological EPR can be dramatically increased by dividing the aqueous sample into separate compartments. In biological EPR, the major factor affecting sensitivity is the number of spins in the sample. For an aqueous sample at ambient temperature, this is limited by the requirement for a small volume, due to strong non-resonant absorption of microwaves by water. However, recent empirical studies have shown that this volume limitation can be greatly relieved by dividing the aqueous sample into separate volumes, which allows much more aqueous sample to be loaded into a resonant cavity without significant degradation of the cavity quality factor. Calculations, based on the Bruggeman mixing rule, show quantitatively that the composite aqueous sample has a permittivity much less than that of bulk water, depending on the aqueous volume fraction f. Analysis for X-band EPR spectroscopy shows that the optimal volume fraction of an aqueous composite sample, producing maximum sensitivity, is f=0.15, increasing the sensitivity by a factor of 8.7, compared with an aqueous sample in a single tube.

Electron Spin Resonance Spectroscopy↗

PubMed2005

Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin.

We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.

Animals↗

PubMed2004

Aqueous sample in an EPR cavity: sensitivity considerations.

The radial mode matching (RMM) method has been used to calculate accurately the microwave field distribution of the TE(011) mode in a spherical EPR cavity containing a linear aqueous sample, in order to understand in detail the factors affecting sensitivity in EPR measurements at X band. Specific details of the experiment were included in the calculations, such as the cavity geometry, the presence of a quartz dewar, the size of the aqueous sample, and the sample's dielectric properties. From the field distribution, several key physical parameters were calculated, including cavity Q, filling factor, mean microwave magnetic field at the sample, and cavity efficiency parameter Lambda. The dependence of EPR signal intensity on sample diameter for a cylindrical aqueous sample was calculated and measured experimentally for non-saturated and half-saturated samples. The optimal aqueous sample diameter was determined for both cases. The impact of sample temperature, conductivity, and cavity Q on sensitivity of EPR is discussed.

Algorithms↗