Interactions of microsomal cytochrome P-450 with spin-labeled substrate analogs

The iodine-containing stable iminoxyl radicals with various distances between the N-O-group and the iodine atom are proposed to be used to study the structure of the active center of the microsomal cytochrome P-450. The radicals used induce changes in the optical spectra of the Fe3+ ion located in the active center of the enzyme, as in the case of type 1 substrates and inhibit essentially the microsomal oxidation of cytochrome P-450 substrates of type 1 and 2. This inhibition is neither due to suppression of the NADPH-cytochrome c reductase activity nor to cytochrome P-450 conversion to cytochrome P-420. Cytochrome P-450 substrates (aminopyrine) protect the enzyme against the radical-induced inactivation. The iodine-containing radicals are covalently bound to cytochrome P-450 in the vicinity of active center. The values of dissociation constants for the reversible enzyme-radical constants and the rate constants for the monomolecular transformation in the complex, k, were determined. The EPR method was used to detect the coupling between Fe3+ and the radical located in the active center of cytochrome P-450. The saturation curves of radical SPR spectra at 77 degrees K were employed to determine the contribution of Fe3+ to the relaxation time, T1, of the radicals covalently bound to cytochrome P-450 and to estimate the distances between the Fe3+ ion and the N-O-group of these radicals in the enzyme active center.     

Interaction of the MvaI restriction enzyme with synthetic DNA fragments

The cleavage of synthetic DNA duplexes by the restriction endonuclease MvaI has been studied. The main result of the cleavage experiments is that MvaI cleaves unmodified duplexes in two single strand scissions in separate events and that the two strands are cleaved at significantly different rates. One strand nicks within the recognition site do not affect the cleavage. Furthermore, neither a pyrophosphate internucleotide bond modification in one strand nor the absence of one phosphate group at the central dA-residue of the recognition site do inhibit the cleavage of the second strand.     

Tissue culture study of unit activity of the rat cerebral cortex

Spontaneous unit activity and morphological characteristics of visual cortical neurons from young rats aged 1 and 2 days were investigated during long-term culture (up to 34 days) of explants in vitro. Three types of spontaneous unit activity were found: single spikes, volleys, and grouped discharges. The types of spontaneous activity were found to depend on the duration of cultivation of the brain tissue. The discharge of single spikes, characteristic of neurons during the first 7–15 days in culture, subsequently was replaced by grouped discharges or volleys of spikes. The changes in unit activity were shown to coincide in time with morphological maturation of the synapses. In experiments in which strychnine (10 µg/ml) was added to the culture medium, a marked increase was observed in the mean frequency of spontaneous unit activity, accompanied by the appearance of discrete series of volley-type discharges. The genesis of spontaneous cortical unit activity is discussed on the basis of these findings.     

Synthesis and properties of DNA-duplexes, containing 9-(1'-hydroxy-2'-(hydroxymethyl)ethoxy)methylguanine]

Phosphoramidite derivative of 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) is synthesized which allows one to introduce point modifications in any position of the chemically prepared oligonucleotide chain. Oligonucleotides with 5'-terminal glG can be used in chemical ligation promoted by cyanogen bromide. The modified oligonucleotide duplexes were characterized by melting curves and CD spectra.     

Cleavage by restriction endonucleases MvaI and EcoRII of substrates modified in amino groups of heterocyclic bases

14-membered DNA-duplexes containing modified nucleoside residues, viz 4-N-methyldeoxycytidine (m4dC), 6-N-methyldeoxyadenosine (m6dA) or deoxyinosine (dI), in only one strand of the recognition site (CCA/TGG) of MvaI and EcoRII endonucleases were synthesized. It was shown that MvaI and EcoRII endonucleases interact with the exocyclic amino groups of the external dC residues and of the central dA residue of the recognition site exposed into the DNA major groove. These endonucleases which are isochizomers were found to possess different mechanisms of substrate cleavage. The ability of MvaI endonuclease to hydrolyze only unmodified strand of methylated duplexes allows one to make site-directed single-strand nicks in double-stranded DNA. Elimination of the 2-NH2-group located in the minor groove of DNA by substituting dI for dG had little, if any, effect on the hydrolytic activity of EcoRII and MvaI endonucleases.     

Learning of animals during adaptation to altitude and its relations with serotonin metabolism in the brain

The influence of high altitude (3 200 m) on learning was studied on 104 non-linear male rats weighing 120 to 140 g, along with biochemical analysis of serotonin content (5-HT) and noradrenaline (NA) in brain structures. A drastic deterioration in the animals' learning has been established in conditions of high altitude, both with alimentary and pain reinforcement attended with a considerable suppression of the 5-HT and NA brain systems activity. Systematic administration of 5-HTP resulting in an enhanced serotonin level in the cortex and the caudal part of the brainstem, improved the learning process, regardless of the emotional sign of the reinforcing stimulus. The prospect, is being substantiated, of evolving methods preventing pathological implications of external influences of high altitudes on the organism by means of pharmacological actions on monoamines' metabolism.     

Prokaryotic DNA Methyltransferases: The Structure and the Mechanism of Interaction with DNA

The review considers current views on the function of DNA methyltransferases (MTases) that belong to prokaryotic type II restriction–modification systems. A commonly accepted classification of MTases is described along with their primary and tertiary structures and molecular mechanisms of their specific interaction with DNA (including methylation). MTase inhibitors are also considered. Special emphasis is placed on the flipping of the target heterocyclic base out of the double helix and on the methods employed in its analysis. Base flipping is a fundamentally new type of DNA conformational changes and is also of importance in the case of other DNA-operating enzymes. MTases show unique sequence homology, and are similar in structure of functional centers and in the mechanism of methylation. These data contribute to the understanding of the general biological significance of methylation, since prokaryotic and eukaryotic MTases are structurally and functionally similar.     

The channels model of nuclear matrix structure

The specificity of eukaryotic DNA organization into loops fixed to the nuclear matrix/chromosomal scaffold has been studied for more than fifteen years. The results and conclusions of different authors remain, however, controversial. Recently, we have elaborated a new approach to the study of chromosomal DNA loops. Instead of characterizing loop basements (nuclear matrix DNA), we have concentrated our efforts on the characterization of individual loops after their excision by DNA topoisomerase II-mediated DNA cleavage at matrix attachment sites. In this review the results of applying this mapping approach are compared with the results and conclusions from studies of nuclear matrix DNA. An attempt is also made to reconsider all data about the specificity of DNA interactions with the nuclear matrix and to suggest a model of spatial organization of the eukaryotic genome which resolves apparent contradictions between these data.