Ways of realizing apoptosis of human lymphocytes induced by UV-light and reactive oxygen species

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.     

An assessment of the degree of chromophore photo damage to the heme and globin components in UV-irradiated molecules and electrophoretic fractions of human carboxyhemoglobin

The contribution of hem and globin components of electrophoretic fractions of UV-irradiated human carboxyhemoglobin to photodestruction of the protein was studied. The changes observed are the result of summation of some processes unequal in intensity and direction that take place in microheterogeneous media of photomodified protein. Photosensitivity of hemoproteid in electrophoretic fraction depends on apoprotein condition, whereas the hem photoresistance cannot be the evidence of the photostability of the whole molecule.     

Study of the effects of laser irradiation (540 nm) on some components of blood enzyme antioxidant system

The influence of blood laser irradiation (lambda = 540 nm) in doses range 0.3-1.2 J/cm2 on the neutrophyles superoxide-dismutase and katalase activity has been studied. Correcting effect of laser light (0.6; 1.2 J/cm2) on the functional activity of studied enzymes has been found. The high degree of correlation (r = 0.6) between neutrophil superoxide dismutase and neutrophil catalase activity of the irradiated blood has been discovered. The close correlation between components of the antioxidant system and processes of active oxygen metabolite generation has been revealed. The scheme of free radical mechanisms of the laser irradiation influence on the several parts of the enzyme antioxidant system has been suggested.     

UV-sensitivity of hemoglobin dimers in free state and in valency hybrids: modification by serotonin]

Changes of oxygen-binding activity of hemoglobin dimers modified by the therapeutical doses of UV-light and serotonin in free state and in valency hybrids are analyzed. The prior role of photodissociation to dimers at the UV-radiation action on heme-protein molecules has been shown. It has been observed that the complex between hemoglobin and serotonin is formed in fields of alpha beta-dimers contacts.     

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe²⁺ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.     

Fragmentation of DNA lymphocytes the human in dynamics of development apoptosis, induced influence of UV-radiation and reactive oxygen species

It is established that UV-light (240-390 nm) in doses of 151, 1510 and 3020 J/m2 and reactive oxygen species and singlet oxygen induce DNA fragmentation lymphocytes cells of the human 20 h after influence. Using a method of DNA-comets it is revealed that DNA damages (single strand breaks) are found out right after UV-irradiations of lymphocytes in doses of 1510 and 3020 J/m2 and additions hydrogen peroxide in concentration of 10-6 mol/l (a comet of type C1) and reach a maximum through 6 h after influence on of cells UV-light and ROS (comets of types C2 and C3). Assumption about the leading part of a p53-dependent way in realization apoptosis human lymphocytes in the conditions of influence of UV-light and reactive oxygen species is put forward.     

Mathematical models of the oxygen-binding function of intact human hemoglobin and hemoglobin modified by UV-radiation in the presence of carbon oxide

The influence of carbon oxide and UV-radiation in doses of 151-453 J/m2 on the physiological properties of human oxyhemoglobin has been studied. Mathematical models of the oxygen-binding function of intact and modified hemoprotein have been developed. It has been found that saturation of human hemoglobin with oxygen obeys the logistic dependence. In the presence of carboxyhemoglobin, the oxygenation parameters change and saturation curves are described by the equations of degree dependence. It has been shown that UV light had the stimulating influence on the functional properties of human hemoglobin modified by carbon oxide if the concentration of carboxyform of the hemoprotein in solution was no higher than 10 per cent. The disturbance of the oxygen-binding ability of hemoglobin by the action of higher concentrations of carboxyhemoglobin was irreversible and was not corrected by UV-radiation.     

Parameters of oxygen-binding function of human hemoglobin modified by carbon oxide and UV-light

The parameters of oxygen-binding function of human hemoglobin, modified by carbon oxide and UV-radiation: the pressure of half-saturation with the ligand (P50), Hill's constant (n), and arterial-venous difference of HbO2 concentration in the sample were studied. The presence of carboxyform in blood in boundaries of admissible values (lower than 10 per cents) did not noticeably influence to the oxygenation parameters. Functional properties of hemoproteid were substantially modified in case of HbCO concentration increasing from 30 up to 80 percent. It has been discovered, that the leading mechanism of protection from hemic hypoxia in case of poisoning with CO is decreasing of degree of cooperative interactions and relative affinity of hemoglobin for ligands. The stimulating influence of UV-light to the functional properties of modified with carbon oxide human hemoglobin observed in case carboxyform hemoprotein concentration in solution was lower than 10 percent. The disturbance of oxygen-binding ability of hemoglobin at the influence of higher concentrations of Hb-CO was inconvertible and was not correct with UV-radiation.     

Activity of membrane-bound acetylcholinesterases in the presence of benzene alcohol and concanavalin A]

UV-sensitivity of membrane-bound acetylcholinesterases in the presence of agents, which selectively modify lipid phase and integral proteins of erythrocytic membranes (benzene alcohol and concanavalin A), has been studied. It has been determined, that UV-irradiation of human erythrocytic membranes within the range of wavelengths 240-390 and 300-400 nm leads to differently directed changes of enzymatic activity, which are caused by different number of membranous chromophores of UV-light and by their different nature. The scheme of process, causing the photomodification of membranous acetylcholinesterases, has been suggested. It takes into consideration a contribution of several structural components of membranes in these processes. Authors have made the conclusion about the important role of microenvironment in processes of acetylcholinesterases functioning and about the possibility of purposeful regulation of its UV-sensitivity by introduction of exogenous agents, which modify structural state of closest "neighbours" of enzyme.