Interaction of clinically important human DNA topoisomerase I poison, topotecan, with double-stranded DNA

Topotecan (TPT), a water-soluble derivative of camptothecin, is a potent antitumor poison of human DNA topoisomerase I (top1) that stabilizes the cleavage complex between the enzyme and DNA. The role of the recently discovered TPT affinity to DNA remains to be defined. The aim of this work is to clarify the molecular mechanisms of the TPT-DNA interaction and to propose the models of TPT-DNA complexes in solution in the absence of top1. It is shown that TPT molecules form dimers with a dimerization constant of (4.0 +/- 0.7) x 10(3) M(-1) and the presence of DNA provokes more than a 400-fold increase of the effective dimerization constant. Flow linear dichroism spectroscopy accompanied by circular dichroism, fluorescence, and surface-enhanced Raman scattering experiments provide evidence that TPT dimers are able to bind DNA by bridging different DNA molecules or distant DNA structural domains. This effect may provoke modification of the intrinsic geometry of the cruciform DNA structures, leading to the appearance of new crossover points that serve as the sites of the top1 loading position. The data presume the hypothesis of TPT-mediated modulation of top1-DNA recognition before ternary complex formation.     

DNA binding induces conformational transition within human DNA topoisomerase I in solution

We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex.     

Strategies of adaptation of small arteries in diaphragm and gastrocnemius muscle to aerobic exercise training

Aerobic exercise training is associated with adaptive changes in skeletal muscles and their vascular bed; such changes in individual muscles may vary depending on their characteristics and recruitment. This study was aimed at comparing the effects of eight-week treadmill training on the locomotor and respiratory muscles in rats. The training course increased the aerobic performance in rats, which was evidenced by an increase in maximum O₂ consumption and a decrease in the blood lactate concentration in ramp test. The succinate dehydrogenase activity was increased in the red portion of the gastrocnemius muscle, but not in the diaphragm of trained rats. Arterial segments were isolated from feed arteries and studied by wire myography. The relaxation in response to acetylcholine in gastrocnemius arteries in trained animals was higher as compared with controls (due to higher NO production), while contractile responses to noradrenaline (in the presence of propranolol) were not changed. On the contrary, the endothelial function of diaphragm arteries was not affected by training, but contractile responses to activation of α-adrenoceptors were markedly increased. Thus, aerobic training may increase the blood supply rate to both locomotor and respiratory muscles, but the underlying regulatory mechanisms are different. The results obtained allow us to reveal the physiological mechanisms that determine the physical performance of the body under conditions of compromised functioning of the respiratory system.     

Successions in forest coenoses after windfall: Models of tree competition

Based on the concept of competition for resources, the distribution of trees upon reforestation in windfall areas is studied. As a theoretical model for competition, a Zipf-Pareto model of ranking the distribution of resources is used. Analysis shows that the processes resulting from competitive interactions between the trees of different species proceed slowly in a windfall area where coniferous species get replaced by deciduous ones. In the territory where deciduous species initially dominated, competitive interactions between trees of different species turn out to be formed almost immediately upon natural reforestation after the windfall. By the time the ratio of species stabilizes and becomes a steady state, the ranks of individual species also stabilize. This result is obtained on the basis of a quantitative assessment of the change in leadership between competing species in time using Spearman’s rank correlation coefficient.     

Kinetics of the lactone-carboxylate transition of hybrid camptothecin-netropsin molecules

The kinetics of the hydrolysis of the lactone ring of a hybrid molecule containing the molecules of the antitumor drug camptothecin and a derivative of the antibiotic netropsines, which is highly affine and specific to the DNA A-T sequences was investigated. It was shown that intramolecular interaction significantly slows down the rate of hydrolysis but does not change the equilibrium ratio of concentrations of the lactone and carboxylate forms of the camptothecin fragment of the hybrid molecule, which corresponds to the pH value. The use of intramolecular interaction for controlling the kinetics of the lactone/carboxylate transition makes it possible to create the drugs of the camptothecin family, which preserve the biologically active lactone form under the physiological conditions for a longer time and, therefore, are more effective as anticancer agents.     

The contribution of protein kinase C and rho-kinase to the regulation of receptor-dependent contraction of arteries decreases with age independently of sympathetic innervation

The age-related dynamics of the activity of signaling pathways coupled with α₁-adrenoreceptors and their dependence on sympathetic innervation of arterial smooth muscle has been studied. For this purpose, the effects of protein kinase C inhibitor (GF 109203X, 10^?6 M and Rho-kinase inhibitor (Y27632, 10^?5 M) on isometric contraction of the rat saphenous artery in response to the α₁-adrenoreceptor agonist methoxamine were determined. The rats in the age of two weeks (with partially developed sympathetic innervation) had the lower vascular sensitivity to methoxamine than adult rats, but the effects of both inhibitors were more prominent. The denervation induced by excision of sympathetic ganglia in adult rats increased the arterial sensitivity to methoxamine but the sensitivity to inhibitors was unchanged. Thus, the postnatal development of the arterial smooth muscle is accompanied by diminution of the role of protein kinase C and Rho-kinase in the regulation of contraction, but these changes do not correlate with the changes in arterial sensitivity to α₁-adrenergic stimulation and development of sympathetic innervation.     

Comparative immunological characteristics of Daphnia allergens

Materials on the study of Daphnia allergens are presented. Daphnia allergens have been shown to possess considerable sensitizing properties. The optimum method for the preparation of the allergen has been selected. The method of measuring the electrophoretic mobility of cells with a view to the evaluation of the specific activity of Daphnia allergen is proposed.     

Investigation of the Distribution of Magnetic Nanoparticles in Tumor Tissues by the Method of Scanning Magnetic Force Nanotomography

Doklady Biochemistry and Biophysics - The development of effective biomedical technologies using magnetic nanoparticles (MNPs) for the tasks of oncotherapy and nanodiagnostics requires the...