Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.     

Dual-channel microfluorometer-photometer based on standard block assemblies and units

The principles of construction and methods of practical realizing of double-wave microfluorometers-photometers for investigation of molecular processes in cells and cell populations are considered.     

Radioprotective effect of a herbal product soma

A study was undertaken to examine the functional activity of the synthetic apparatus of rat blood lymphocytes by fluorescence microspectroscopy and the metabolic indices of the animals during the use of the herbal product Soma in normalcy and in acute x-irradiation. It was shown that Soma in normalcy caused a reliable increase in the synthetic activity of the cells with respect to the reference value on the 13th and 20th days of administration. Prior use of Soma for a month with subsequent pause increased the radioresistance of the animal organism (scheme 1), whereas the use of Soma immediately after irradiation (scheme 2) had no appreciable radioprotective effect. The study showed the effect of Soma on enhancement of metabolism, which may play an important role in restoration of the homeostasis of the organism. The results obtained suggest the expediency of further investigation of the radioprotective properties of Soma using various concentrations and administration schemes.     

Microspectral studies of neuroendocrine regulation of gametogenesis in mollusks

An associated culture of nervous ganglia and gonads was used to study the gametogenesis neuroendocrine regulation in mollusks (Mytilus edulis L.). The state of oocytes was controlled by microspectral fluorescent analysis on slides stained with acridine orange. The object of investigations was Mytilus edulis L. collected in La Manch in April-May. A microspectrofluorimetrical investigation of samples was carried out in Pushchino. As a criterion of cell synthetic activity, the parameter alpha was used. Parameter alpha is the ratio of fluorescence intensities in the red (I640) and green (I530) regions of the visible spectrum: alpha = I640/I530. The results obtained showed that the nervous system realized the endocrine regulation of gametogenesis in mollusks. There was no normal development of oocytes in an isolated mantle culture. The development of oocytes in a tissue culture proceeds in a similar manner as in the gonads of the native organism in normal natural conditions only if the mantle tissue is associated either with all nervous ganglia or with the cerebral ganglion alone. It was found that the cerebral ganglion plays a key role in the neuroendocrine regulation of gametogenesis.     

Cooperative Interactions in Photosensitized Modification of DNA with Oligonucleotide-derived Binary Reagents

Quantitative characteristics of thermodynamic and kinetic cooperativity arising in the process of photomodification of a single-stranded DNA fragment with binary systems of oligonucleotide conjugates forming an active site on the target were studied. Oligonucleotides of the binary system were complementary to adjacent segments of the DNA target, and contained arylazide (X) and perylene (S) residues covalently attached to their terminal phosphates. Upon irradiation at the perylene absorption wavelength, the target was modified by the arylazide residue, which was activated owing to the contiguity with the sensitizing perylene group in the tandem complex. Basing on the kinetic data, the constants of association of both derivatives of oligonucleotides with the target were determined: K _x = 1.13 · 10⁶ M^–1, K _s = 1.49 · 10⁴ M^–1. It was determined that association of both oligonucleotides with the target proceeded with a positive cooperativity characterized by parameter = 45. The kinetic cooperativity parameter was found to be approximately equal to 200; this characterized the acceleration of target modification in complex with the binary reagent versus that in the absence of sensitizer.     

Use of fluorescence microspectral method for studying bone marrow EGFP+ cell transplantation in 5-fluorouracil-treated mice

In this paper the experimental results of bone marrow transplantation from C57BL/6-Tg(ACTB-EGFP)1Osb/J transgenic mice into C57BL/6 mice subjected to 5-fluorouracil treatment are represented. It has been shown that EGFP⁺ cell engraftment in bone marrow, spleen and thymus of host mice after 5-Fu treatment significantly increased. More long-term engraftment was recorded after transplantation between closely related donors and 5-fluorouracil treatment hosts. We have also obtained data on differences in the dynamics of EGFP⁺ cell engraftment in host investigated organs. To assess the effect of the donor’s bone marrow cells on the host immune system, functional activity of the synthetic apparatus (synthetic activity) of cells in bone marrow, spleen, thymus and blood have been investigated with fluorescence microspectral method. The results obtained allow of improving techniques for bone marrow transplantation without host irradiation in order to minimize the adverse effects.     

A new method of identifying a systems based on the minimal quadratic discrepancy criterion for biophysics problems

A wide range of biophysical systems are described by nonlinear dynamic models mathematically presented as a set of ordinary differential equations in the Cauchy explicit form: [formula: see text] Fij(X1(t),..,XN(t),t), (i = 1,...,N, j = 1,...,M), where Fij (X1(t), ..., XN(t), t) is a set of basis functions satisfying the Lipschitz condition. We investigate the problem of evaluation of model constants aij (the system identification) using experimental data about the time dependence of the dynamic parameters of the system Xi(t). A new method of system identification for the class of similar nonlinear dynamic models is proposed. It is shown that the problem of identifying an initial nonlinear model can be reduced to the solution of a system of linear equations for the matrix of the dynamic model constants [aj]i. It is proposed to determine the set of dynamic model constants aij using the criterion of minimal quadratic discrepancy for the time dependence of the set of dynamic parameters Xi(t). An important special case of the nonlinear model, the quadratic model, is considered. Test problems of identification using this method are presented for two nonlinear systems: the Van der Pol type multiparametric nonlinear oscillator and the strange attractor of Ressler, a widely known example of dynamic systems showing the stochastic behavior.     

Analysis of the temporal organization of the synthesis of RNA and protein in blood lymphocytes during the immune response of the body

The temporal organization of the synthesis of RNA and antibodies in immunocompetent cells has been investigated by fluorecsent spectroscopy. Two fluorochromes: acridine orange and 1-anilino-8-naphthalenesulfonate were used. Temporal changes in the mean values of the characteristic parameters alpha' and beta in blood lymphocytes stained with acridine orange and 1-anilino-8-naphthalenesulfonate in the immune reaction to the introduction of ovalbumin were obtained. It was shown that the maximal increase in RNA and protein synthesis in blood lymphocytes by a factor of 6-8 occurs after the fourth immunization. The simultaneous increase in RNA and protein synthesis in cells was indicative of cell activation. A general response of immunocompetent blood cells to the introduction of the antigen was observed. The results confirm the idea about the cooperative interaction between different lymphocyte subpopulations during the synthesis of antibodies.     

Effect of ionizing radiation on the synthetic activity of blood system cells in ground squirrels in different physiological states of animals

The functional (synthetic) activity of blood lymphocytes and bone marrow haemopoietic cells in ground squirrels during the annual cycle as well as in hibernating and awaken animals in winter have been studied by fluorescent microspectrometry. The effect of ionizing radiation on animals in different functional states of the hibernation-arousal bout was investigated too. It was shown that the synthetic activity (parameter alpha) in blood lymphocytes was minimal in hibernating state in winter and maximal in active euthermic spring animals, then slightly decreased in June and more considerably decreased in the prehibernating autumn period. In awake animals in winter, the values of parameter alpha reached the same values as in summer. The changes of parameter alpha in bone marrow haemopoietic cells were essentially the same: the minimal values were observed in the prehibernation autumn period and in awake animals in winter the alpha values were slightly higher than in active euthermic animals in summer. The maximal synthetic activity in bone marrow haemopoietic cells in active euthermic spring animals is due mainly to cells in G1-G2 phases of the cell cycle. The decrease of the synthetic activity in summer is a result of the cell transition from G2 to mitosis and transition of a part of cells to G0 When investigating the hibernation-arousal bout in ground squirrels in winter, during arousal, we found two stages considerably differing in both the values of parameter alpha in bone marrow haemopoietic cells and the number of blood cells. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of deep hibernation did not differ significantly from the state of non-irradiated hibernating animals. The negative effect of radiation appeared upon the arousal of these animals but it was expressed to a lesser degree in comparison with the animals irradiated in the active state. It was found that the acute irradiation of animals during arousal from hibernation in the second stage caused the most pronounced functional inactivation and cell death. The physiological state of ground squirrels subjected to ionizing irradiation at different phases of the hibernation-arousal bout plays a determining role in the changes of the qualitative and quantitative characteristics of blood system cells. Thus, the hypometabolic state of ground squirrels in hibernation is a factor of protection from the action of ionizing radiation on the organism and the immune system.