Electrostatic potentials of DNA. Comparative analysis of promoter and nonpromoter nucleotide sequences.

Distribution of electrostatic potential of DNA fragments was evaluated. A method for calculation of electrostatic potential distribution based on Coulomb's law is proposed for long DNA fragments (approximately 1000 nucleotide pairs). For short DNA sequences, this technique provides a good correlation with the results obtained using Poisson-Boltzmann equation thus justifying its application in comparative studies for long DNA fragments. Calculation was performed for several DNA fragments from E. coli and bacteriophage T7 genomes containing promoter and nonpromoter regions. The results obtained indicate that coding regions are characterized by more homogeneous distribution of electrostatic potential whereas local inhomogeneity of DNA electrostatic profile is typical for promoter regions. The possible role of electrostatic interactions in RNA polymerase-promoter recognition is discussed.     

Synthesis and biological evaluation of copper (II) complexes of sterically hindered o-aminophenol derivatives as antimicrobial agents

Cu(II) complexes with 4,6-di(tert-butyl)-2-aminophenol (I) and 2-anilino-4,6-di(tert-butyl)phenol (II) have been synthesized and characterized by means of elemental analysis, TG/DTA, FT-IR, UV-vis, ESR, and conductance measurements. The compounds I and II can coordinate in their singly deprotonated forms and behave as bidentate O,N-coordinated ligands; their CuL(2) complexes are characterized by CuN(2)O(2) coordination modes and square planar geometry. In vitro antimicrobial screening against Gram-positive and Gram-negative bacteria, yeasts, and moulds indicated that the compound I and its Cu(II) complex were more active than Questiomycin B, the compound II, and its Cu(II) complex.     

Effect of alpha-lipoic acid on 3T3 and 3T3-SV40 fibroblasts: Comparison with N-acetylcysteine

In this study, we have investigated the intracellular level of reduced glutathione, cell-cycle phase distribution, and microfilament and microtubule structures in normal (3T3) and transformed (3T3-SV40) fibroblasts exposed to alpha-lipoic acid (ALA) in concentrations of 0.7–5 mM. It was found that ALA treatment increased the glutathione content in transformed cells, but did not affect its level in normal cells; moreover, it also induced the cell-cycle arrest of 3T3 cells (but not 3T3-SV40 cells) and disrupted actin microfilaments in cells of both lines. The ALA effect was compared to that of N-acetylcysteine (NAC), another antioxidant we examined previously. The findings allow us to assert that each of these antioxidants impacts on distinct target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. However, intermediate steps of ALA and NAC action may be common (altered intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).     

Novel NMR tools to study structure and dynamics of biomembranes

Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13C-(1)H dipolar couplings, benefit from the excellent resolution of 13C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.     

Effects of phenolic compounds on reactions involving various organic radicals

Investigation of effects produced by 26 various phenol and diphenol derivatives, including industrial and natural antioxidants (ionol, bis-phenol 2246, alpha-tocopherol), on final product yields of radiation-induced free-radical processes involving peroxyl, alkyl, alpha-hydroxyalkyl and alpha,beta-dihydroxyalkyl radicals has been performed. Ionol and bis-phenol 2246 have been shown to be more effective than alpha-tocopherol or diphenol derivatives in suppressing hydrocarbon oxidation processes. At the same time, alpha-tocopherol and its water-soluble analogues, as well as diphenol-based substances, are more effective than phenol derivatives in regulating various homolytic processes involving carbon-centered radicals. This fact can be accounted for by taking into consideration the contribution to formation of the final product set and the respective yields made by semiquinone radicals and compounds with quinoid structure arising in the course of homolytic transformations in systems containing diphenol derivatives.     

Osmotically induced membrane tension modulates membrane permeabilization by class L amphipathic helical peptides: nucleation model of defect formation.

The mechanism of action of lytic peptides on membranes is widely studied and is important in view of potential medical applications. Previously (I. V. Polozov, A. I. Polozova, E. M. Tytler, G. M. Anantharamaiah, J. P. Segrest, G. A. Woolley, and R. M., Biochemistry, 36:9237--9245) we analyzed the mechanism of membrane permeabilization by 18L, the archetype lytic peptide featuring the class L amphipathic alpha-helix, according to the classification of Segrest et al. (J. P. Segrest, G. de Loof, J. G. Dohlman, C. G. Brouillette, and G. M. Anantharamaiah, 1990, Proteins, 8:103--117). We concluded that the 18L peptide destabilizes membranes, leading to a transient formation of large defects that result in contents leakage and, in the presence of bilayer-bilayer contact, could lead to vesicle fusion. Here we report that this defect formation is strongly enhanced by the membrane tension induced by osmotic swelling of vesicles. Even below standard leakage-inducing peptide/lipid ratios, membrane resistance to osmotic tension drops from hundreds to tens of milliosmoles. The actual decrease is dependent on the peptide/lipid ratio and on the type of lipid. We propose that under membrane tension a peptidic pore serves as a nucleation site for the transient formation of a lipidic pore. The tension is released upon pore expansion with inclusion of more peptides and lipids into the pore lining. This tension modulation of leakage was observed for other class L peptides (mastoparan, K18L) and thus may be of general applicability for the action of membrane active lytic peptides.     

Inhibition of the Peroxidase-Catalyzed Oxidation of Chromogenic Substrates by Alkyl-Substituted Diatomic Phenols

A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates--2,2-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3,5,5-tetramethylbenzidine--inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20°C in 0.015 M phosphate–citrate buffer (pH 6.0) containing organic cosolvents (0–10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (K _i), the duration of the lag period in the oxidation product formation (), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate–diatomic phenol pair. Various substrate–diatomic phenol pairs exhibited K _i values within the range of 11–240 M andfvalues from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine–substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate–substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food.     

Distribution of rat embryonal fibroblasts through cell cycle phases in the presence of inhibitors of active oxygen species formation and N-acetylcysteine

Intracellular level of reactive oxygen species (ROS) and distribution of primary rat embryo fibroblast throughout the cell cycle have been studied. Serum-starved cells were activated by the addition of 10% serum to the culture medium in the presence on N-acetyl-cystein (NAC) and ROS-inhibitors, diphenileniodonium (DPI) and rothenone. It has been shown that serum starvation could block the cells at the G1/S boundary. Activation of serum-starved cells by the addition of serum reactivated the cell cycle and caused cell progression into S phase, which was paralleled with the increase in the intracellular level of ROS. Effects of NAC, PAI and rothenone, similar to that of serum starvation, blocked cell progression into S phase and decreased ROS formation due to the action of serum growth factors. The antiproliferative effect of NAC is discussed.     

Ultrasonic DNA Cleavage: An Analysis of the Conformational-Dynamic Features of Regulatory Genome Regions and Sequencing Errors

This review is dedicated to the memory of Dr. Sergei L’vovich Grokhovsky, who was a distinguished scientists and experimenter; it briefly describes one aspect of his multifaceted research activity. Gro-khovsky was the first to describe the specificity of ultrasound-induced cleavage of internucleotide bonds in double-stranded B-DNA. The discovery of this new physicochemical phenomenon made it possible to associate the cleavage effects with conformational and dynamic features of B-DNA nucleotide sequences and provided a new basis for bioinformatics genome analyses. Certain conformational dynamic specifics are characteristic of functional DNA regions; this makes it possible to formulate and solve a number of problems of epigenetics and carcinogenesis.