Role of rapid movement of spin labels in interpreting EPR spectra for spin-labelled macromolecules

The method of spin labeling was used to monitor quick movements of side residues in protein monocrystals. The EPR spectra of monocrystals of spin-labeled lysozyme at different orientations of the tetrahonal crystal relative to the direction of the magnetic field were interpreted using the molecular dynamics method. A simple model was proposed, which enables one to calculate the trajectory of movements of the spin label by the molecular dynamic method over a relatively short period of time. The entire "frozen" protein molecule and a "defrozen" spin-labeled amino acid residue were considered in the framework of the model. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was constructed, and the parameters of force potentials for the atoms of the protein molecule and the spin label were specified. It follows from the calculations that the protein environment sterically hinders the range of eventual angular reorientations of the reporter NO-group of nitroxyl incorporated into the spin label, thereby affecting the shape of the EPR spectrum. However, the scatter in the positions of the reporter group in the angular space turned out to correspond to the Gauss distribution. Using the atomic coordinates of the spin label, obtained in a chosen time interval by the method of molecular dynamics, and taking into account the distribution of the states of the spin label in the ensemble of spin-labeled macromolecules in the crystal, we simulated the EPR spectra of monocrystals of spin-labeled lysozyme. The theoretical EPR spectra coincide well with the experimental.     

Accumulation of 90Sr in field crops in conditions of radioactive pollution of agricultural lands

In the field experiment the influence of manure, peat, lake silt and their combinations on the uptake of 90Sr by barley, corn and potatoes was studied. Saturation of soil with organic substance, the decrease in soil acidity resulted in the lower accumulation of 90Sr by plants. In the beginning of the experiment, the specific activity of barley grain was from 75 to 132 Bq/kg. After three years of application of fertilizers the content of 90Sr in the barley grain decreased to 39 Bq/kg. There were no increase in 90Sr accumulation by the basic field crops with time.     

Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.     

Cellular basis of transition between sleep and electrographic seizures

Epileptic seizures mainly develop during slow-wave sleep. Our experiments, using multi-site, extra- and intracellular recordings, show a transformation without discontinuity from sleep patterns to seizures. The cerebral cortex is the minimal substrate of paroxysms with spike-wave complexes at ~3 Hz. Simultaneously, thalamocortical neurons are steadily inhibited and cannot relay signals from the outside world to cortex. This may explain the unconsciousness during certain types of epilepsy.     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins. 2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis

The capacity of a previously described plasmid vector pAZ to deliver bioactive proteins to targets in vivo has been studied. This vector molecule has a strong constitutive promoter, is extremely stable in cells of vaccinal S. choleraesuis strain, and encodes the synthesis of marker protein beta-galactosidase which helps monitor the vector's fate in the host. The gene encoding hepatitis B virus core antigen (HBcAg) has been inserted into pAZ under its constitutive promoter. The resultant recombinant plasmid p19-24 has been used to transform Enterobacteriaceae (E. coli and S. choleraesuis) cells. Transformed cells produce immunologically active HBcAg. p19-24 was stable in S. choleraesuis cells during their culturing and during this strain persistence in mice. Triple oral immunization of rabbits in a dose of 1 x 10(9) S. choleraesuis cells TC177 induced the production of virus-specific antibodies. Successful transformation of cells of another vaccinal strain S. abortus ovis by this plasmid extends the potentialities of the vector. The results demonstrate good prospects of using pAZ vector for the construction of live oral vaccines.     

Structural polymorphism of oligo(dC) with mixed alpha,beta-anomeric backbone

Oligonucleotides with mixed alpha,beta-anomeric backbone have been proposed recently for the recognition of random DNA sequence via new triplex motif (Doronina and Behr, Chem. Soc. Reviews 26, 63-71 (1997)). In the present work we examined alpha- and beta- anomers of cytidine as possible candidates to recognize AT and TA base pairs of the double stranded DNA. The binding properties of beta-oligo(dC) were studied on a series of synthetic oligodeoxynucleotides by UV absorbtion spectroscopy, measurements of bound EtBr fluorescence polarization, circular dichroism (CD) and non-denaturing gel electrophoresis. The UV thermal denaturation, polarization studies and CD experiments with three stranded oligonucleotide 5'-((dCalpha) (dCbeta))5-L-(dAT)5-L-(dAT)5 (L = triethyleneglycol linker) and other oligonucleotide models showed that the formation of semiprotonated oligocytidilic complexes takes place at low temperatures and neutral pH, rather than folding of the clip into intramolecular triplex. The low-temperature transition was observed in denaturation profiles of any oligonucleotide containing beta- or mixed alpha,beta- cytidine stretches at the concentration of 1 microM. Self-association of alpha,beta-oligo(dC) was additionally confirmed by the appearance of two CD bands (at 290 and 265 nm) characteristic of CC+ base pairs. Despite the effective ability of alpha,beta-oligo(dC) to form self-associates, we succeeded in targeting 30-bp AT containing random DNA duplex by a 30-nt alpha,beta-oligocytidilate as evidenced by non-denaturing gel electrophoresis. A complete binding of the duplex was observed at a 5-fold excess of the third strand at 15 degrees C. Along with the formation of the three-stranded complex, self-association of mixed backbone oligo(dC) strands occurred.     

Cytotoxic derivatives of (22R,23R)-22,23-dihydroxystigmastane

(22R,23R)-22,23-dihydroxystigmast-4-en-3-one, (22R,23R)-22,23-dihydroxystigmast-4-en-3,6-dione, (22R,23R)-3beta,5alpha,6beta,22,23-pentahydroxystigmastane, (22R,23R)-5alpha,6alpha-oxido-3beta,22,23-trihydroxystigmastane, (22R,23R)-5beta,6beta-oxido-3beta,22,23-trihydroxystigmastane, and (22R,23R)-3beta,6beta,22,23-tetrahydroxystigmast-4-ene were synthesized. Their cytotoxicities were comparatively studied using the MCF-7 line of carcinoma cells of human mammary gland and cells of human hepatoma of the Hep G2 line.     

Plasma method for processing spent nuclear fuel

Plasma methods for processing spent nuclear fuel are analyzed. It is shown that, by ICR heating in a nonuniform magnetic field, the energy of the heated ash ions can be increased substantially, while nuclear fuel ions can be kept cold. Two methods for extracting heated ash ions from a cold plasma flow are considered, specifically, that by increasing the ion gyroradius and that due to ion drift in a curved magnetic field. It is found that the required degree of separation of ash and fuel ions can be achieved in systems with quite moderate parameters.     

New delta8(14)-ketosterols with an oxygenated side chain

New analogues of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3beta-hydroxy-24-methyl-22,23-oxido-5alpha-cholest-8(14)-en-15-ones and (22RS,23xi,24S)-24-methyl-5alpha-cholesta-3beta,22,23-triol-15-one] were synthesized from (22E,24S)-3beta-acetoxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC50 0.9 +/- 0.2 and 0.7 +/- 0.2 microM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC50 4.0 +/- 0.5 microM), (22E,24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one (IC50 3.1 +/- 0.4 microM), and the 3beta,22,23-triol synthesized (IC50 6.0 +/- 1.0 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.