Activity of topoisomerase I and endonucleases in cells transfected by a ras oncogene

Activities of nuclear endonucleases and topoisomerase I were measured in rat fibroblasts which were at the stages of tumor transformation: control embryonal fibroblasts--CEF; cells immortalised by transfection of S1A segment of SA7 adenovirus--REF-1; intermedius cells transfected once by EJras oncogene--REF-1EJ; and cells transformed after the second transfection by the same oncogene--REF-2EJ. The topoisomerase I and Ca2+, Mg2+-dependent endonuclease was most decreased at the stage of immortalised cells, and the intermedius stage (REF-1EJ) was characterized by the lower activity of Ca2+, Mg2+-dependent endonuclease. The highest activity of Mn2+-dependent endonuclease is seen in REF-2EJ cells. In model experiments the ability of Ca2+, Mg2+-dependent endonuclease to split non-stochastically the EJras oncogene inserted into pBR322 plasmid was shown. The role of the investigated enzymes in the restriction of plasmid integration, cellular immortalisation and recombination of plasmids with chromosomes during cell transformation is discussed.     

A novel transposition system in Drosophila melanogaster depending on the Stalker mobile genetic element.

Crosses between the Drosophila melanogaster y2sc1waG strain or some of its derivatives and the FM4 strain yielded insertional mutagenesis with a frequency of 10(-3)-10(-4). The system differs in several respects from the known cases of hybrid dysgenesis: (i) it does not depend on the direction of a cross; (ii) destabilization continues for a long time after initial crosses; (iii) mutations may occur at different stages of development. The mutation in the yellow locus has been cloned and found to depend on insertion into the coding region of the gene of a novel mobile genetic element designated as Stalker. The sequencing of Stalker termini reveals 405 bp direct repeats (LTRs) and a target 3 bp duplication, as well as some other sequences typical of retrovirus-like retrotransposons. The number of Stalker copies per genome and chromosomal localization vary among D. melanogaster strains. Before crosses, the location of Stalker on chromosomes is fairly stable in a particular strain but thereafter numerous changes in Stalker distribution take place. Most novel substrains are internally heterogenous which is indicative of the continuing Stalker transposition. Other mobile elements tested do not move. Possibly, only Stalker is mobilized in the system. Many known and novel mutations have been obtained. Comparison of their genetic localization with Stalker distribution suggests that the majority of them have been induced by the Stalker insertion.     

Effect of cross linkers on the bacteriorhodopsin photocycle

A general behavior of bacteriorhodopsin in purple membranes from Halobacterium halobium has been observed upon modification resulting in cross-linking of carboxyl and lysine groups. The rise of the M-intermediate contained two components with approximately 50-50% intensity; its decay showed three components with approximately 25-50-25% intensity respectively in a pH range of 5-9. The significance of these remarkably similar data with respect to the proton translocation mechanism in bacteriorhodopsin is that chemical modification allows us to conclude that disturbing parts of the hypothetical "proton conducting chain" does not inhibit proton translocation.     

Directed mutagenesis of chromosomal genes of Escherichia coli in vivo: construction of RecA- and HtpR- mutants

The deletions in Escherichia coli chromosomal genes recA and htpR were constructed using the site-directed mutagenesis techniques. The obtained RecA- mutants are UV-sensitive and have a phenotype defective for the homologous DNA recombination. HtpR- mutant is temperature sensitive for growth and deficient in intracellular proteolysis. As a result a HtpR- mutant seems to be a preferable candidate for attempting to synthesize efficiently any alien protein in Escherichia coli cells.     

Stable shifts in systemic arterial pressure in a controlled experiment with feedback

On the bases of experiments with bio-control feed-back connection (automatically triggering electro-cutaneous, stimulation), a model evolved permitting to achieve stable directed changes in the levels of the systemic arterial pressure (AP) in curarized rabbits and cats. Two variants of reactions are possible: 1) gradual formation of a new raised or lowered level of AP with its pronounced retention after the end of learning, and 2) reaction of following type with directed rise in AP, which is observed only during learning. It is found that the stable shifts of the AP level are accompanied by changes in the periodicity of the third order AP waves, the heart rate showing no significant changes. It is assumed that the changes in the characteristics of the third order waves reflect the rearrangement of the mechanisms providing for homeostasis.     

Phospholipid metabolism in acetylcholine and substance P induced desensitization of guinea pig ileum smooth muscle

The amount and the rate of inorganic phosphate incorporation into mono-, di- and triphosphoinositides and into total phospholipids of ileum smooth muscle were studied at various time of exposure to physiological concentrations of acetylcholine and substance P. The data obtained show a phase character of the mediator influence on the phosphate incorporation into mono and triphosphoinositides-a stimulation initial period, an inhibition during the desensitization period and a return to the normal level after the mediator elimination.     

Dendrimers based lysine and their "starburst" polymeric derivatives: prospects of use in compacting DNA and in vitro delivery of genetic constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-NH2 (D1) and (Lys)8-(alpha, epsilon-Lys)4-(alpha, epsilon-Lys)2-(alpha, epsilon-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm)8-D1 and (pLys)n-D1, containing poly(N-vinylimidazole) and polylysine chains bound at a single point to the dendrimer amino groups. The conditions of dendrimer-plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on C2C12 mouse myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

The use of human fibroblasts grown on microcarriers for the formation of the connective tissue equivalent

Live equivalents of tissues, specifically those produced on the basis of fibroblasts and collagen gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the fibroblasts grown on microcarriers or such a connective tissue equivalent when the fibroblasts on microcarriers are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embedding the fibroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According to our results, the optimal time of use of the live tissue equivalent amounts to three--four days after embedding of fibroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with the gel.     

Bromoacyl analogues of phosphatidylcholine with intramolecular fluorescence quenching and their use as substrates for continuous monitoring of phospholipase A2 activity

Two new derivatives of phosphatidylcholine with intramolecular fluorescence quenching were obtained by substituting residues of pyrene butyric and bromine-containing fatty acids for acyl chains. The two compounds can be used for quantitative evaluation of catalytic activity of pancreatic phospholipase A2 in kinetic mode.