An NMR study of the conformational mobility of steroid estrogen 7α-methyl-8α analogues

All the signals in the ¹H and ¹³C NMR spectra of some analogues of 7α-methyl-8α-and 6-oxa-8α-steroid estrogens were completely assigned. Considering the values of nuclear Overhauser effect and vicinal coupling constants, these steroids were shown to exhibit a fast, on the NMR time scale, conformational equilibrium arising due to the inversion of ring B. The conformer populations were obtained from a comparison of the experimental and theoretical values of the dihedral angles and the interproton distances. This conformational equilibrium was shown to depend on the nature of atom in position 6: for the 7α-methyl-6-oxa-8α analogues of the steroid estrogens, the population of the conformer with the pseudoaxial orientation of the 7α-methyl group was observed to be decreased compared with the 7α-methyl-8α analogue.     

OmpC-like porin from outer membrane of Yersinia enterocolitica: Molecular structure and functional activity

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the “warm” variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the “cold” variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short “periplasmic” and longer “extracellular” loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the “extracellular” loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.     

Molecular cloning, isolation, and properties of chaperone Skp from Yersinia pseudotuberculosis

The skp gene of Yersinia pseudotuberculosis was expressed without its signal sequence in Escherichia coli BL21(DE3) cells. The recombinant protein Skp accumulated in soluble form in the cytoplasm of the producer strain. The protein was isolated and characterized: the molecular weight, isoelectric point, N-terminal amino acid sequence (20 amino acid residues), and the content of the secondary structure elements were determined. Using cross-linking stabilization and high-mass MALDI-TOF mass spectrometric analysis, it was found that rSkp forms a stable homotrimer in solution and interacts with human IgG. Three-dimensional models of the Skp trimer and its complexes with Fc- and Fab-fragments of human IgG1 were constructed by computer modeling.     

Monitoring the seasonal dynamics of ecologically tolerable levels for water regime, temperature, and pH in water objects of the Lower Don River

The feasibility of ecological standardization of physicochemical factors that do not refer to concentrations of polluting substances (water regime, water temperature, and pH) is confirmed. The opportunity of standardization is provided with the approach developed by the authors, which is based on joint analysis of the data of biological and physicochemical monitoring. Additional opportunities provided by the method used for the analysis are connected with calculating the admissible influence values that vary in time. The seasonal dynamics of the obtained ecologically tolerable levels (ETL) for factors disturbing the well being of biological indicators is investigated. The water sample points are analyzed concerning their ecological well being or ill being depending on observance or nonobservance of ETL values.     

Prooxidant and cytotoxic action of N-acetylcysteine and glutathione in combinations with vitamin B12b

Prooxidant and cytotoxic effects of thiols N-acetylcysteine (NAC) and glutathione (GSH) were studied in combinations with vitamin B_12b. Both GSH and NAC at physiological doses when combined with B_12b were shown to cause initiation of apoptosis. It was established that the prooxidant action of NAC (or GSH) combined with B_12b, i.e., generation and accumulation of hydrogen peroxide in culture medium, led to intractellular oxidative stress and cell redox imbalance. These effects are completely prevented by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine do not suppress the H₂O₂ accumulation in culture medium, but inhibit cell death induced by NAC combined with B_12b or by GSH combined with B_12b. Therefore, the thiols GSH or NAC in combination with vitamin B_12b reveal prooxidant properties and induce, with participation of intracellular iron, apoptotic HEp-2 cell death.     

Simulation of mechanoelectrical coupling in cardiomyocytes under normal and abnormal conditions

Mathematical models of the electromechanical function of cardiomyocytes and muscle duplexes, the simplest mechanically inhomogeneous myocardial systems, are developed. Using these models, the contribution of mechanoelectrical feedbacks to the contractive activity of the myocardium in normal and abnormal conditions is studied. In particular, the influence of the mechanical conditions of contraction on the shape and duration of the action potentials is reproduced and interpreted. In this context, different types of mechanical heterogeneity of the myocardium are analyzed. It is established that this heterogeneity can play a positive or negative role depending on the distribution of heterogeneous properties and on the order the elements of the system are activated. Using the same models, the contribution of mechanical factors to arrhythmogenesis under calcium overload of cardiomyocytes due to the weakening of the sodium-potassium pump is studied. Methods for correction of the contractive activity of cardiomyocytes in the case of such abnormalities are outlined.     

Investigation of plasma characteristics in an unbalanced magnetron sputtering system

Results are presented from experimental studies of a magnetron sputtering system for different configurations of the magnetic field above the cathode surface. The current-voltage characteristics of a magnetron discharge at different working gas pressures (0.08–0.3 Pa) and currents in the unbalancing coil were studied. The production and transport of charge carriers in a magnetron discharge with an unbalanced magnetic field was investigated by means of probe measurements of plasma characteristics and ion energies in the region between the substrate and the magnetic trap at the cathode surface. The radial distributions of the ion current density, plasma potential, and floating potential in the unbalanced operating mode are found to have pronounced extrema at the magnetron axis. It is shown that the plasma density near the substrate can be increased considerably when the axial magnetic field is high enough to efficiently confine plasma electrons and prevent their escape to the chamber wall.     

Recombinant phospholipase A<Subscript>1</Subscript> of the outer membrane of psychrotrophic <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>: Expression,purification, and characterization

The pldA gene encoding membrane-bound phospholipase A₁ of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A₁ (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A₁ was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A₁ were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A₁ from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.     

Effects of pH on structural and functional properties of porin from the outer membrane of Yersinia pseudotuberculosis. II. Characterization of pH-induced conformational intermediates of yersinin

pH-Induced intermediates of Omp F-like porin from the outer membrane of Yersinia pseudotuberculosis (yersinin) were characterized by fluorescence and fluorescent probe spectroscopy and circular dichroism. The most dramatic changes in the intrinsic fluorescence of the protein induced by pH titration correlated with different conformational states of the porin molecule. pH-induced conformational transitions of yersinin can be described in terms of a three-state model: (1) disordering of porin associates and formation of porin trimers structurally similar to the native protein; (2) unfolding of individual porin domains followed by cooperative dissociation of trimers into monomers; (3) formation of two loosely structured forms of monomer intermediates. It is assumed that one of these monomeric forms (at pH 3.0) corresponds to the molten-globule state of porin with native secondary structure, while the other one (at 2.0) represents a partly denatured (misfolded) monomer, which retains no more than 50% of the regular secondary structure. The putative mechanism of low pH-induced β-barrel unfolding is discussed in terms of a theoretical model of yersinin spatial structure.