[The recording of ATP in the erythrocytes using luciferase injected into the cells]. / Registratsiia ATF v éritrotsitakh s pomoshch'iu vvedennoi v kletki liutsiferazy.

The luciferase preparation obtained from fireflies Luciola mingrelica has entrapped into the human erythrocytes by means of reversible osmotic lysis. The addition of luciferin to such erythrocytes leads to the appearance of luminescence, conditioned by the entrance of luciferin into the cells. Luciferin is uniformly distributed between cells and external medium. Luciferin transport through the erythrocyte membrane is a result of simple diffusion. Values of rate constant of luciferin transport through the membrane lie between 0.009-0.021 l/s 1 cells for erythrocytes of different donors. The maximum luminescence intensity increases monotonously with rise of temperature and luciferin concentration. The dependence of the maximum luminescence intensity on luciferin concentration is described by Michaelis kinetics. Obtained in different experiments, values of luciferase Michaelis constant for luciferin inside erythrocytes lie between 4.1-21.5 microM. Luminescence intensity of the luciferase containing erythrocytes depends on the intracellular ATP concentration. Under the same luciferin concentration the correlation of luminescence intensities of control erythrocytes with normal ATP level and erythrocytes depleted without glucose is near to correlation of their ATP concentrations. After the addition of glucose to the depleted erythrocytes their ATP concentration rises and luminescence intensity approaches to the level of control erythrocytes. Luciferase entrapment permit one to control rapid ATP concentration changes in the erythrocytes.

[Dynamics of proliferating cell populations during exposure to periodic phase-specific factors]. / Dinamika populiatsii proliferiruiushchikh kletok pri periodicheskom fazospetsificheskom vozdeistvii.

Dynamics of the numbers of population of proliferating cells under periodic phase-specific cytotoxic effect with and without blocking action was studied on the basis of a mathematical model. It has been shown that at real values of the model parameters after the beginning of the effects the population number exponentially depends of time. Dependence of the population number dynamics on integral parameters of the cell cycle and the exposure regime was studied. It has been shown that at certain periods a resonance decrease of the damage of the population cells must be observed. The values of the periods corresponding to the resonance decrease of the damage are determined mainly by the mean duration of the cell cycle and the time of blocking action, at small duration of the blocking action they are approximately multiple to the average time of the cell cycle. The theoretical predictions are proved experimentally in the experiments on determining the relationship between the damage of small intestine epithelium and mouse survival and the period of repeated periodic injections of S-phase-specific cytotoxic agent hydroxyurea. A distinct resonance increase of mouse survival and decreased damage of the epithelium were observed under the injections of oxyurea with the periods near to the mean and doubled mean time of the cell cycle of crypt enterocytes. The results obtained not only support the correctness of theoretical predictions, but make it possible to estimate the parameters of the stem cell cycle of mouse small intestine epithelium. They also show that this approach can be used for reducing aftereffects of chemotherapy by means of phase-specific agents.

[The resonance nature of the dependence of epithelial lesions of the small intestine in mice on the interval between injections of an S-phase-specific agent--hydroxyurea]. / Rezonansnyi kharakter zavisimosti porazheniia épiteliia tonkoi kishki myshei ot intervala mezhdu in''ektsiiami S-fazospetsificheskogo agenta--oksimocheviny.

The dependence of the injury of murine small intestinal epithelium on the interval between multiple regular injections of hydroxyurea (HU) was investigated. Mice were injected 8 times with HU (5 mg per injection) in different experimental groups of animals, and the interval between injections varied from 6 to 19 hours. With the intervals between the injections close to 8 or 16.5 hours the resonance decrease of the injury was observed whereas the intervals of 6, 12 and 19 hours corresponded to maximum injury.

[Resonance nature of the relation of mouse survival to the interval between administrations of the S-specific agent--hydroxyurea]. / Rezonansnyi kharakter zavisimosti vyzhivaemosti myshei ot intervala mezhdu vvedeniiami S-fazospetsificheskogo agenta--oksimocheviny.

The dependence of murine survival on the intervals between periodic hydroxyurea (HU) injections was studied. The single dose of HU comprised 250 mg per kg. Intervals between injections varied from 5 to 19 hours while their number changed from 6 to 9 in different experiments. A resonant increase in the survival was observed under HU administered every 8-9 or 16 1/2 hours.

[Metabolic changes leading to oxidative lysis of erythrocytes maintained in a normal state in vitro]. / Metabolicheskie izmeneniia, vedushchie k okislitel'nomu lizisu éritrotsitov, podderzhivaemykh v normal'nom sostoianii in vitro.

It was shown that in vitro oxidative hemolysis of human erythrocytes occurs as a result of a great increase in membrane permeability to cations leading to osmotic damage of the cells. Infusion at a steady rate with a solution of tert-butylhydroperoxide in an erythrocyte suspension resulted in a rapid fall of the reduced glutathione level down to 0, when the rate of infusion exceeded the maximal rate of pentose phosphate pathway. Under these conditions the potassium ions liberation from the erythrocytes began with the drop of the reduced glutathione level down to zero, and the hemoglobin liberation - at the moment when more than 60% of potassium ions were liberated from the erythrocytes. The kinetics of potassium ion liberation remained unchanged in anisotonic media, but hemoglobin liberation from the erythrocytes greatly increased in hypotonic media as compared with isotonic ones. The kinetics of K+ and hemoglobin liberation were correlated only with lipid peroxidation but not with the oxidation of protein SH-groups.

[Relation between the toxic killing of mouse small intestine enterocytes and the interval between the administration of the S-specific agent hydroxyurea]. / Zavisimost' toksicheskoi gibeli énterotsitov tonkoi kishki myshei ot intervala mezhdu vvedeniiami S-fazovo-spetsificheskogo agenta-oksimocheviny.

The dependence of small intestinal lesions on the interval between repeated regular injections of hydroxyurea was studied. The doses of 1 g/kg and 0.2 g/kg were used. To activate the enterocyte proliferation the mice were pre-irradiated at 2 Gy. 3-4 hours after the last hydroxyurea injection the two distinct minimum points of epithelial lesions were observed at interinjection intervals of 9 and 16.5 hours. The result was dose-independent.

[Optimization of the exposure of normal and transformed cell populations to phase-specific agents]. / Optimizatsiia vozdeistviia na normal'nye i transformirovannye populiatsii kletok fazospetsificheskimi agentami.

Dynamic behavior of stem cells population of the "critical" tissue (normal population) and tumor cell population under periodic treatment with a phase-specific cytotoxic agent was considered. The results were used for optimization of anticancer chemotherapy. The schedules of treatment were found which provide a maximum rate of tumor-cell elimination for any given rate of the normal population size decrease. If the mean generation times of normal and tumor populations differ (which was stated for many tumors), usage of the optimal period markedly increases the selectivity of therapy, while application of other periods can result in selective elimination of the normal population. Problems concerned with practical realization of the proposed regimes are discussed.

[Permeability of human erythrocytes to asparagine]. / Pronitsaemost' chelovecheskikh éritrotsitov dlia asparagina.

Asparagine is able to penetrate into human erythrocytes from the external medium. The dependence of the asparagine transport rate on its concentration can be described by the Michaelis-Menten equation with parameters: Km = 2.50 mM, V = 0.24 mmol/l cells per hour. Loading of erythrocytes with asparaginase does not influence their permeability to asparagine. Aspartate is accumulated inside these erythrocytes during incubation with asparagine, thus reflecting rapid transformation of penetrating asparagine by entrapped asparaginase.

[The 2,3-diphosphoglycerate shunt and stabilization of the ATP level in mammalian erythrocytes]. / 2,3-difosfoglitseratnyi shunt i stabilizatsiia urovnia ATP v éritrotsitakh mlekopitaiushchikh.

The mechanisms of regulation of energy metabolism in erythrocytes of various mammalian species were investigated. In native erythrocytes of man, sheep, cow, dog and mouse the dependencies of the rates of glucose uptake on ATP concentration (i.e., regulatory parameters of glycolysis) were measured. These parameters plotted in normalized coordinates are not species-specific (invariant). The dependence of the rate of ATP-consuming processes on ATP concentration has been studied for the first time in intact mammalian erythrocytes. This dependence was found to be linear only in the species, in whose erythrocytes the activity of 2,3-diphosphoglycerate shunt is practically zero. In all species under study, the stabilization of ATP level is provided for mainly by the hexokinase-phosphofructokinase system. A comparison of regulatory mechanisms of energy metabolism in mammalian (sheep, cow) erythrocytes, in which the 2,3-diphosphoglycerate shunt is absent, with human and animal erythrocytes, in which this pathway is active, points to the important role of the 2,3-diphosphoglycerate shunt in regulation of energy conversion in erythrocytes. This shunt operates as an additional stabilizer protecting the cell from extremal influences.

[Changes in the regulatory characteristics of glycolysis in erythrocytes during storage of donor blood]. / Izmeneniia reguliatornoi kharakteristiki glikoliza v éritrotsitakh pri khranenii donorskoi krovi.

Changes in the glycolysis regulatory pattern (dependence of the glycolysis rate on ATP concentration) in erythrocytes were studied during donor blood storage using glucose-citrate hemoconservant 7b. During the first two weeks of storage the glycolysis regulatory pattern in the erythrocytes remained practically unchanged being represented by a bell-shaped curve similar to that found in fresh erythrocytes. At the same time, the physiological point of glycolysis shifted towards the maximal level on the curve. When the physiological point reached the maximal value, the shape of the curve began to change. The maxima on the curve became less distinct and shifted down and to the left from the initial position. These changes occurred within two-four weeks of storage. In some cases at the latest steps of storage the maxima on the glycolysis regulatory pattern disappeared leading to the monotony of the curve. The changes observed occurred in blood of different donors at various periods of storage. The nature of the changes observed and their influence on the erythrocyte viability are discussed.

[Relation of the survival of mouse hematopoietic stem cells to the interval between periodic injections of hydroxyurea]. / Zavisimost' vyzhivaniia stvolovykh krovetvornykh kletok mysh ot intervala mezhdu periodicheskimi vvedeniiami oksimocheviny.

The dependence was studied between the survival of proliferating hemopoietic stem cells (CFUs) on the interval between regular multiple injections of hydroxyurea (HU). Before HU injection mice were irradiated in a dose of 200 rad to trigger the proliferation of CFUs. The dependence obtained is resonance in character, with a maximum being attained during injections made with a 12-hour interval. Comparison with the mathematical model indicates that interval corresponding with the maximal survival should correlate with the mean time of the generation of proliferating CFUs.

[Steady-state dependence of the methemoglobin reduction rate on its concentration in intact human erythrocytes]. / Statsionarnaia zavisimost' skorosti vosstanovleniia metgemoglobina ot ego kontsentratsii v intaktnykh éritrotsitakh cheloveka.

The rate of methemoglobin reduction by the methemoglobin reductase system of intact human erythrocytes was measured as a rate of pyruvate formation in a quasi-steady state. Various methemoglobin concentrations (up to 100%) were generated by sodium nitrite additions. The steady state methemoglobin levels were maintained by infusion of a nitrite solution at a rate of 2.8 mmol/h/l cells. The rate of pyruvate formation was proportional to the steady state methemoglobin concentration in the range from the physiological value to 100%, the maximal value being as high as 500 mumol/h/l cells. It was found that the rate of CO2 output by the erythrocytes markedly increased in the presence of 8 mM sodium nitrite, reaching up to about 40% of the possible maximal value.

[Effect of glycolysis on the metabolism of adenylates in human erythrocytes]. / Vliianie glikoliza na metabolizm adenilatov v éritrotsitakh cheloveka.

The ATP content in human erythrocytes depleted without glucose falls down to half of the initial value within 2-3 hours and reaches practically zero within more than 10 hours. The ADP content increases 2-3-fold during the 1st hour after depletion and then slowly decreases. The AMP content increases 10-fold during several hours, but the rate of this process constantly decreases. The adenylate pool decreases at a constant rate ranging from 0.13 to 0.25 mmol/l cell. h; this is accompanied by accumulation of IMP. Addition of glucose to depleted erythrocytes results in partial recovery of the ATP level within 1-2 hours. The sooner glucose addition after the depletion, the greater the recovery. Simultaneously the ADP and AMP levels drastically decrease to new constant values. The decline of the adenylate pool ceases and the rate of IMP accumulation increases. Normally, the [ATP]/adenylate pool ratio lies within the small interval 0.85-0.94 irrespective of significant individual differences in the absolute values of [ATP]. This ratio is decreased during depletion and restored to the initial value after glucose addition. The mass-action ratio of the adenylate kinase reaction changes greatly during depletion and restoration of erythrocyte ATP.

[Regulatory characteristics of glycolysis in erythrocytes of sheep]. / Issledovanie reguliatornoi kharakteristiki glikoliza v eritrotsitakh barana.

The regulatory properties of glycolysis in sheep erythrocytes (i. e. dependence of the rate of glucose uptake on ATP concentration) were investigated. A decrease of ATP concentration in the erythrocytes by arsenate causes a sharp rise in the rate of glycolysis, the maximal rate being reached at ATP concentration of about 70% of the original one. Further decrease of ATP lowers the rate of glycolysis; no steady-state rates of energy metabolism are observed within this concentration range. The parameters of glycolysis measured in erythrocytes of various species coincide when the rates of glycolysis are normalized in terms of its maximal value and that of ATP -- in terms of its value at which the maximal rate is reached. The identities of regulatory properties of glycolysis take place even upon two-fold differences in the absolute values of rates and concentrations.

[Regulation of erythrocyte energy metabolism. Dependence of glycolysis characteristics on donor individual parameters]. / Reguliatsiia énergeticheskogo metabolizma éritrotsita. Zavisimost' kharakteristiki glikoliza ot individual'nykh parametrov krovi.

Approximate analytical expression is obtained for the dependence of erythrocyte glycolytic rate on ATP concentration and donor individual parameters: activation of hexokinase and phosphofructokinase, some kinetic constants and total concentration of adenine nucleotides in the cell.