Peculiarities of Molecular Mechanisms Involved in Modification of the Structural and Molecular Organization of the Cell Genome.

Within the framework of the previously proposed model of structural organization of DNA that supplements the Watson-Crick model and is based on a mathematical regulation - Fibonacci sequence, we suppose the existence of nucleotides without nitrogenous base and acting as linkers connecting DNA subunits.

Fibonacci Sequence and Supramolecular Structure of DNA.

We proposed a new model of supramolecular DNA structure. Similar to the previously developed by us model of primary DNA structure [11-15], 3D structure of DNA molecule is assembled in accordance to a mathematic rule known as Fibonacci sequence. Unlike primary DNA structure, supramolecular 3D structure is assembled from complex moieties including a regular tetrahedron and a regular octahedron consisting of monomers, elements of the primary DNA structure. The moieties of the supramolecular DNA structure forming fragments of regular spatial lattice are bound via linker (joint) sequences of the DNA chain. The lattice perceives and transmits information signals over a considerable distance without acoustic aberrations. Linker sequences expand conformational space between lattice segments allowing their sliding relative to each other under the action of external forces. In this case, sliding is provided by stretching of the stacked linker sequences.

Changes in the Quantitative Composition of the Population in Delayed Periods After γ-Radiation Exposure of the Cells in Various Phases of S Period of the First Mitotic Cycle.

Using the autoradiographic method, we studied the kinetics of DNA synthesis over the mitotic cycle in mouse corneal epithelium cells in delayed periods after γ-irradiation in different points of the S phase of the first mitotic cycle. The index labeled cells during 1-3 periods of DNA synthesis most adequately reflects quantitative changes in the cell population composition after cell exposure during the first S period. The relative number of labeled S phase cells in the second mitotic cycle in experiments where the cells were irradiated in the S1 phase of the first S period was 4-fold lower than in experiments where the cells were exposed during S2 phase. This effect is determined by inhibition of the transcription factors activation. It seems that two territorially different sites of the genome controlling the regulatory stimuli and involved in modification of the quantitative composition of the population are responsible for changes in its quantitative balance.

The Mechanism of Adaptation of Breast Cancer Cells to Hypoxia: Role of AMPK/mTOR Signaling Pathway.

We studied the mechanisms of adaptation of human breast cancer cells MCF-7 to hypoxia and analyzed the role of AMPK/mTOR signaling pathway in the maintenance of cell proliferation under hypoxic conditions. It was found that long-term culturing (30 days or more) of MCF-7 cells under hypoxic conditions induced their partial adaptation to hypoxia. Cell adaptation to hypoxia was associated with attenuation of hypoxia-dependent AMPK induction with simultaneous constitutive activation of mTOR and Akt. These findings suggest that these proteins can be promising targets for targeted therapy of tumors developing under hypoxic conditions.

Delayed Consequences of Changes in DNA Synthesis in Cells Exposed to Single Irradiation at the End of S Phase of the Mitotic Cycle.

Kinetics of DNA synthesis throughout the mitotic cycle in mouse corneal epithelial cells after single γ-irradiation of cells (4 Gy) at the end of S phase was studied by the method of radioautography. It was found that single irradiation increased the duration of S phase due to reparation of damage in the cell at the expense of time that normally falls on g1 phase. During reparation, two parallel DNA synthesis processes occur in the damaged cells: de novo synthesis at the site of injury after excision of the damaged fragments (reparative synthesis) and supplementary synthesis during the repair period in the remaining undamaged genome competent for replication. During supplementary synthesis, repeats appear in DNA structure, which increases the amount of genetic material in the cell and affect S phase duration. All reparative processes take place in the cell population consisting of subpopulations of "differentiated", "resting", and "proliferating" cells. The changes in the proportions between the subpopulations under the influence of extreme factors can induce the appearance of metastatic cells in the population.

Reparation as a factor contributing to genetic variability of the biological system.

Kinetics of DNA synthesis in mitotic cycle of in mouse corneal epithelial cells after γ-irradiation in different S phase points was studied by the method of autoradiography. Normally, S phase of corneal epithelial cells consists of two phases (S1 and S2) separated by an interval without DNA synthesis. Each phase, in turn, includes two subphases with a pause in DNA synthesis. It was hypothesized that pauses in DNA synthesis, similar to that during presynthetic g1 phase, correspond to periods of cell preparation to the next stage of their development. After irradiation, reparation proceeds during pauses at different phases of the cell cycle. The mechanism of reparation induces cell genome rearrangement.

Dynamics of DNA synthesis disturbance and recovery after fractionated irradiation of S phase cells.

Using the autoradiographic method we studied the kinetics of DNA synthesis during themitotic cycle of mouse corneal epithelial cells after γ-irradiation in a dose of 2 Gy at different S-phase points. Normally, S phase in corneal epitheliocytes includes S1 and S2 phases separated by an interval during which DNA is not synthesized. Double exposure modifies the pattern of DNA synthesis in the cell due to reparation of injuries. The reparative processes in the cell are realized during the interval between the S1 and S2 phases and at the expense of g1 period of the mitotic cycle. If the cell has no time for reparation, the injuries are transferred into the class of "latent" injuries.

Comparative analysis of the kinetics of DNA synthesis after exposure during different phases of the cell cycle S period.

The kinetics of DNA synthesis in the mitotic cycle of mouse corneal epithelial cells was studied after a single γ-irradiation of cells in a dose of 4 Gy at different S-phase points. Normally, corneal epitheliocyte S phase consists of S1 and S2 phases separated by an interval during which no DNA is synthesized. The duration of each phase was lengthened after single irradiation due to reparation of injuries in the cells at the expense of the time normally occupied by g1 period of the mitotic cycle. The first event during reparation is excision of damaged complex from the DNA molecule; this complex consists of labeled daughter fragment and matrix site of DNA chain that was used for the synthesis of the daughter fragment. Presumably, the entire reparation process in the cell consists of two stages: "reparative" synthesis and "additional" synthesis. The reparative synthesis, in turn, includes two stages: de novo synthesis of matrix fragment in the DNA chain at the site of the gap formation and de novo synthesis of the daughter fragment after the synthesis of the new matrix fragment is over.

Effect of irradiation on DNA synthetic period of the mitotic cycle in cells.

Kinetics of DNA synthesis in mitotic cycle of mouse corneal epithelial cells after single γ-irradiation (4 Gy) at the end of S period was studied by the method of radioautography. Normally, S period of corneal epithelial cells consists of several stages separated by intervals without DNA synthesis. The estimated mean duration of the first (S(1)) and second (S(2)) phases of S period was 16 and 10 h, respectively, and the interval between them was 7 h. Single irradiation at the end of S period changed the duration of mitotic cycle periods: S(2) phase became 2.2-longer than S(1) phase and the duration of g(1) period decreased, because the time for reparation in irradiated cell increases at the expense of g(1) period. Shortening of g(1) period is a factor promoting the appearance of transformed cells.

[To the history of organisation and development of cardiac anesthesiology in the A. N. Bakulev Research Center of Cardiovascular Surgery of the Russian Academy of Medical Sciences: the start of activities (1956-1965)].

The authors present data on the development and introduction of anesthetic techniques during cardiac surgery at the Institute of Thoracic Surgery, USSR Academy of Medical Sciences, in 1956-1960 and after its reorganization to the Institute of Cardiovascular Surgery, USSR Academy of Medical Sciences, in 1961-1965. It is shown that in the years of introduction of closed operations on the heart, the methods of one- and many component inhalational anesthesia were mastered, its techniques were developed, anesthesia apparatuses and an anesthesia schedule were designed, cardiac anesthesiological studies were conducted, training of physicians from the country's regions was initiated, and the first guidelines for general anesthesia were published. In these years, the firm foundation was laid for the development of cardiac anesthesia. Later on the Institute developed and introduces all basic types of inhalational anesthesia during operations on the open heart under both extracorporeal circulation and hypothermia. The gained experience allowed the laboratory staff to defend several dissertations, to issue two monographs, and to analyze errors and risks of general anesthesia in patients with cardiovascular diseases at surgery.

[Transcobalamin II levels in blood plasma of children with acute leukemia]. / Soderzhanie transkobalamina II v plazme krovi u detei s ostrym leikozom.

Transcobalamin II (TcII) level was studied in plasma of 40 children with acute leukemia. TcII is a cobalamin-binding protein which mediated the cellular uptake of Cbl and interacted with surface membrane receptor of hemopoietic cells. Plasma TcII and cobalofilins were analysed by PAGE using 57Co-cyanocobalamin. In addition, the mature human placenta with high specificity and affinity to TcII receptors was applied for TcII plasma identification. As compared to control, significant difference of TcII activity in the plasma of children with ALL was noted. There were children with low and high TcII concentration vs. control (484 +/- 42 and 1166 +/- 62, p > 0.001). Therefore, it is necessary to assay individually all the biochemical parameters of Cbl-transport system of children with ALL for adequate metabolic correction.

Lipoxygenase pathway of arachidonic acid metabolism in growth control of tumor cells of different type.

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.

[The expression of plasmalemma transcobalamin-II receptors on human blood lymphocytes stimulated by mitogens]. / Expressiia retseptorov transkobalamina-II plazmalemmy limfotsitov krovi cheloveka, stimulirovannykh mitogenami.

The influence of mitogens on the expression of surface membrane TC-II receptors of human blood lymphocytes and internalization of (TS-II+57Co-CNCbl) complex into cytoplasm were investigated. Mature lymphocytes have a very small number of surface receptors to plasma TC-II but their expression is increased significantly by PHA or Con-A stimulation. CBl transport to cytoplasma is activated in definite sequence by two different mechanisms. Stimulated cells take free CBl without participation of TC-II in early hours of mitogen action (12-42 hrs) before maximal 3H-thymidine incorporation into DNA. On day 3 of cultivation, specific mechanism of CBl transport triggers and the number of lymphoblast receptors is increased manifold. Radioactive CBl enters cytoplasma due to interaction of TC-II-CN [57Co] CBl of the medium with surface membrane receptor of the cells. Thus, the definition of TC-II receptors as an important functional parameter may serve a marker of proliferating cells.

Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells.

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [14C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [3H]thymidine and biosynthesis of prostaglandin E2 (PGE2). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE2 production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE2 in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE2-dependent proliferation.