Effects of flavonoid-containing preparation Extralife on hydrogen peroxide production and functioning of mitochondrial ATP-dependent potassium channel.

Extralife, a Pentaphylloides fruticos extract, in concentrations of 0.005-10 µg/ml dose-dependently increased H2O2 production in rat heart mitochondria in the presence of respiration substrates. Extralife decreased ATP-induced accumulation of H2O2 related to inhibition of mitochondrial ATP-dependent potassium channel. This effect was observed only at low doses of the adaptogen (0.05-3 µg/ml). High doses of the substance (5-10 µg/ml) did not abolish ATP-dependent production of H2O2 and increased the rate of H2O2 generation by the mitochondria. We concluded that Extralife in trace concentrations could activate mitochondrial ATP-dependent potassium channel and decrease H2O2 accumulation in the mitochondria.

[Morphological changes of THP-1 tumor cells exposed to dopamine in vitro].

The effect of dopamine (DA) on the viability and morphology of cultured tumor THP-1 cells (human acute monocytic leukemia) was studied. DA in concentration of 10(-5) M had virtually no effect on the culture, while in concentration of 10(-4) M to 10(-3) M it stopped the growth and caused a sharp increase in cell death after 24 and 48 hours. Incubation with DA reduced the cell diameter, progressively increased their vacuolization and intensity of fluorescence after treatment by Falck method. Electron microscopical study has shown that cells exposed for 1 day to DA in the concentrations starting with 10(-4) M, demonstrated smoothing of their surface with the disappearance of microvilli and clasmatosis vesicles, actin filaments perforating the plasma membrane, the emergence of an increasingly dense network of filaments in the cytosole and karyoplasm and, finally, apoptotic cell death. It is suggested that the oncotherapeutic cellular target for DA is a cytosolic G-actin, which at a certain DA concentration, turns into filaments that damage the cells, break the cell cycle and cause cell death.

[Morphological bases of dopamine effect on HEP-2 tumor cells viability].

The purpose of the present investigation was to study the morpho-functional organization of a classical object of cytological research - cultured HEp-2 tumor cells, using dopamine as a penetrating agent, inducing the polymerization of cytosolic actin. It was demonstrated that dopamine introduced into the incubation medium reduced viability and caused morphological disturbances of cultured HEp-2 cells; these effects were proportional to dopamine concentrations (1.0 x 10(-4) M to 1.0 x 10(-3) M) and exposure duration (2 to 3 days). These cells, according to ultrastructural data, underwent fusion and lysis because of the appearance of actin filaments network in the loci of globular actin prevalence in control cells. Dopamine receptors had no effect on cytotoxic effect of dopamine. This was indicated by fluorescent microscopical evidence of dopamine penetration into experimental cells in the presence of haloperidol, as well as destruction of HEp-2 cells under the action of pyrimidinethione, similar to dopamine by characteristics, but lacking its own receptors. It is suggested that cytoplasmic target for dopamine is globular actin and that induced polymerization of this cytoskeletal protein caused injury to tumour cells.

Effect of dopamine on viability of BHK-21 cells.

We studied the effects of dopamine added to culture medium on survival of floating or adherent BHK-21 cells differing by organization of actin cytoskeleton. The viability of floating cells more drastically decreased with increasing dopamine concentration and duration of exposure than that of adherent cells. The cells worse adhered to the substrate and formed a monolayer. The formed monolayer degrades, cell borders become blurred, cells, polygonal in the control, are rounded. Preliminary blockade of dopamine receptors with haloperidol, inessential for cell survival and morphology, does not prevent the destructive effect of dopamine on the cells. Ultrastructural study revealed increased density of filamentous actin threads in deep compartments of cell cytoplasm after dopamine treatment, this increase being more pronounced in cells grown in suspension. Bearing in mind the polymerizing effect of dopamine on globular actin in vitro and the fact that the content of this protein in floating cells is higher than in adherent cells, we can conclude that the decrease in viability of BHK-21 cells is caused by interaction of dopamine with cytoplasmic globular actin.

[Effect of hypoxenum on bioenergetic processes in mitochondria and the activity of ATP-sensitive potassium channel].

The effect of hypoxenum on bioenergetic processes in heart and liver mitochondria of rats, connected with respiration, the generation of hydrogen peroxide, and the activity of ATP-sensitive K-channel ((mitoK)ATP) has been studied. It was shown that hypoxenum in the concentration range of 0.05-10 microg/ml stimulates respiration, increases the coupling in the respiratory chain, and enhances the formation of H2O2 and energy-dependent swelling associated with potassium transport in mitochondria. Hypoxenum removes the inhibitory effect of ATP on the energy-dependent swelling of mitochondria and partially reduces the accumulation of H2O2 in the presence of ATP. The role of antihypoxic and antioxidant action of hypoxenum associated with the activation of (mitoK)ATP is discussed.

[The influence of dopamine on the ultrastructure of BHK-21 cells].

The influence of dopamine on the haloperidol of BHK-21 cells being in suspension or attached to substrate was investigated. It was shown that the ultrastructural changes affected mainly the cellular loci enriched by the cytoskeleton actin such as intercellular desmosome-like contacts, microvilli and cortical layer or mesh just beneath the plasmatic membrane. The desmosome-like contacts were hypertrophied, their electron density was increased and fibrilar bridges appeared in specialized contacts. Many microvilli fused with each other and with plasma membrane of the neighboring cells, or, on the contrary, split up. Frequently, the membrane surface between microvilli and particularly their apical parts was seen to be pierced by thin thread, morphologically similar to actin filaments. The cytoplasmatic matrix onto ultrathin sections had blotched appearance and at the ultrastructural level was represented by numerous randomly oriented actin filaments. The effect of dopamine was more pronounced in the BHK-21 cells when being in suspension than in attached to the substrate ones, which presumably occurred due to known lesser differentiation of the cytoskeleton in the formers. Finally, it was established that the preliminary blockade of cellular D2 receptors with haloperidol neither affected the ultrastructure of BHK-21 cells nor prevented the following effect of dopamine. The data obtained suggest the direct interactions of dopamine with the actin cytoskeleton.

Combined vitamins Bl2b and C induce the glutathione depletion and the death of epidermoid human larynx carcinoma cells HEp-2.

The combination of hydroxocobalamin (vitamin B12b) and ascorbic acid (vitamin C) can cause the death of tumor cells at the concentrations of the components at which they are nontoxic when administered separately. This cytotoxic action on epidermoid human larynx carcinoma cells HEp-2 in vitro is shown to be due to the hydrogen peroxide generated by the combination of vitamins B12b and C. The drop in the glutathione level preceding cell death was found to be the result of combined action of the vitamins. It is supposed that the induction of cell death by combined action of vitamins B12b and C is connected to the damage of the cell redox system.

[Controlled cell cultivation. V. The effect of various rubbers on the isolated neurons of Limnaea stagnalis in culture]. / Upravliaemoe kul'tivirovanie kletok. V. Vliianie nekotorykh rezin na izolirovannye neirony Limnaea stagnalis v kul'ture.

Various domestic rubbers were tested for toxicity to cultured isolated neurons of Lymnaea stagnalis, the survival time of these cells, and their morphological differentiation being used as criteria. According to the effect towards the neurons, the tested materials could be divided into inert, low-toxic, and toxic ones. Type 52336/4 silicon rubber, the rubber of surgical gloves, and dry rubber of "b" type produce no unfavourable effect on isolated neurons. Stoppers of penicillin bottles, baby's dummies, and silicon-based BgO-1 "Germetic" paste are low-toxic rubbers. By contrast, silicon rubbers of 52336 and 14p-6 types, KLT-30 silicon paste and black rubber of HO-68-1 type decreased sharply the survival time of the neurons and depressed their morphological differentiation in culture.

[Regulation of the synthesis of total cellular proteins and monoclonal antibodies in a hybridoma cell culture]. / Reguliatsiia sinteza summarnykh kletochnykh belkov i monoklonal'nykh antitel v kul'ture gibridomnykh kletok.

A study was made of the regulation of total protein synthesis in cells of the mouse hybridoma producing monoclonal antibodies (McAb) against lambda phage, and in the course of hybridoma growth and at the change of fetal bovine serum (FBS) concentration. FBS strictly affected proliferation of hybridoma cells, the specific production of McAb per cell being unchanged. The rate of total cellular protein synthesis does depend on FBS concentration in the medium, whereas the rates of protein degradation and secretion do not. Evidence is presented that the reduction in the protein-synthesis rate, after the removal of FBS from the medium, is caused by a coordinated decrease in both the rate of protein synthesis initiation and the rates of polypeptide chain elongation and translation termination. The decrease in the protein synthesis rate at the stationary phase of cell growth was shown to be related to the three main factors: 1) a 15-25% decrease in ribosome content per cell; 2) a two-fold decrease of the ribosome portion involved in mRNA translation; 3) a 5 to 15% decrease in the rate of mRNA translation. Evidence is presented that the decrease in the portion of mRNA translating ribosomes is due to the decrease in the rate of protein synthesis initiation.

[Protein metabolism during the growth of hybridoma cells]. / Belkovyi metabolizm v protsesse rosta gibridomnykh kletok.

Protein metabolism during the growth of mouse hybridoma, producing monoclonal antibodies to phage lambda has been studied. The rate of protein accumulation Va at any time of growth was equal to that of protein synthesis Vs minus the degradative rate Vd and protein secretion rate Vsecr (Vn = Vs--Vd--Vsecr). Cellular protein metabolism was described in terms of the relationship 6.3% hr = 8.1%/hr--1%/hr for the middle of exponential growth phase, and in terms of the relationship 1.4%/hr = 2.9%/hr = 0.8%/hr--0.7%/hr for the early stationary phase. The results obtained enabled us to conclude that the synthesis of cellular proteins may be the main process regulating the protein metabolism during hybridoma cell growth.

[The proliferative characteristics of cells in culture during perfusion of the medium]. / Osobennosti proliferatsii kletok v kul'ture pri perfuzii sredy.

The proliferation of Chinese hamster fibroblasts (CHF) and NIH 3T3 cells was studied at different flow rates immediately above the cells. It is shown that there is a limiting density of saturation of the perfused culture that accounts for 1.7 x 10(6) - 2.0 x 10(6) cells/cm2 for NIH 3T3 cells, and for 6 x 10(6) - 7 x 10(6) cells/cm2 for CHF. The growth curves and the distribution of cells with respect to the phases of the cell cycle during cultivation with and without perfusion are presented. Based on the results obtained it is assumed that the limit of saturation density of perfused CHF culture is due to the mass transfer of the growth-inhibiting metabolites inside the multilayer structures. The assumption seems to hold true for NIH 3T3 cells, too, even though the multilayer character of growth of this culture is less pronounced than in CHF.

[Controlled cell culture. I. A modified perfusion chamber]. / Upravliaemoe kul'tivirovanie kletok. I. Modifitsirovannaia perfuzionnaia kamera.

The construction of a modified perfusion chamber is presented, which can be used for a prolonged cultivation of mammalian cells and tissues, for observation of the behavior of living cells as well as for the study of different effects on these cells. The chamber is made as non-demountable of optical glass, with a diffusive barrier separating the pericellular zone from that with a perfusion medium. The scheme of the equipment for cultivation of cells and tissues in this diffusion chamber on controlling the composition of nutrient medium and gas phase is given.

[Controlled cell cultivation. III. The flow-diffusion chamber]. / Upravliaemoe kul'tirovanie kletok. III. Protochno-diffuzionnaia kamera.

A perfuse chamber is described intended for a continuous culturing of cells and tissues, for in vitro observations and for the screening of biologically active substances. The chamber is dismountable. A ring-shaped partition of the chamber, separating the pericellular zone from the external flowing one, contains to diametrically opposite diffuse filters in the form of cylindrical stoppers made of porous glass. Depending on the angle of rotation of the diffuse filter axis with respect to the axis of pipe connections of the chamber, the wanted rate of material transfer between pericellular and flowing zones is achieved. Biological tests have shown that the survival time of isolated molluscan neurones of Limnaea stagnalis in this chamber considerably exceeds that in conventional perfusion chambers without partition. The chamber can be used in studies of embryogenesis, for incubating fertilized oocytes under conditions of controlled culturing.

[Controlled cell cultivation. IV. The cultivation of isolated neurons of the mollusc, Limnaea stagnalis, in a flow-diffusion chamber]. / Upravliaemoe kul'tirovanie kletok. IV. Kul'tivirovanie izolirovannykh neironov molliuska Limnaea stagnalis v protochno-diffuzionnoi kamere.

A possibility of the long-term cultivation of isolated neurons of adult molluscs Limnaea stagnalis in a flow-diffusion chamber has been shown. The construction of the chamber is described by the authors elsewhere; the diagram of the pefusion apparatus has been presented. In the process of cultivation, the nerve cells undergo morphological differentiation to form neuron nets on the glass that involve both individual neurons and their aggregates of 2 to 5 cells. By days 16-18 of cultivation, most of the neurons join together to form several large aggregates of 10-40 cells, connected to each other by thick nerve fibre bundles. A sharp decrease of survival time and the complete inhibition of morphological differentiation of neurons was observed when the diffusional exchange between the extracelluar space and the flowing nutrient medium was replaced in the chamber by the medium perfusion through the extracellular (=pericellular) space. The cause of this phenomenon is discussed.

[Use of the disordered total internal reflection effect to study the adhesion of animal cells to glass. I. Results of a theoretical analysis]. / Ispol'zovanie éffekta narushennogo polnogo vnutrennego otrazheniia dlia izucheniia adgezii kletok zhivotnykh k steklu. I. Rezul'taty teoreticheskogo analiza.

Theoretical analysis data have been presented on the effect of frustrated total reflection due to light scatterring on cells situated near the glass surface. A conclusion is made that using this effect it is possible to measure the index of refraction of the cytoplasm, to determine the distance between the glass and the cytoplasmic membrane of monolayer cells, and to study the kinetics of changes in the cell surface on contacting the glass.

[Use of the effect of impaired complete internal reflection for studying the adhesion of animal cells to glass. II. Kinetics of flattening of the cell surface facing the glass]. / Ispol'zovanie éffekta narushennogo polnogo vnutrennego otrazheniia dlia izucheniia adgezii kletok zhivotnykh k steklu. II. Izuchenie kinetiki uploshcheniia kletochnoi poverkhnosti, obrashchennoi k steklu.

The kinetics of form change (flattening) of Chinese hamster fibroblast cell surface facing the glass has been studied during attachment of fibroblasts to the glass using the effect of frustrated total reflection. Two stages of the process were shown to exist. The first one is characterized by a rapid (5 min) partial flattening and requires no serum in the medium. The second stage occurs only in the presence of serum in the medium, it lasts no longer than one hour and ends in a complete flattening. After the second stage is completed, spreaded cells appear.

[Use of the effect of impaired complete internal reflection for studying the adhesion of animal cells to glass. III. Measurement of the refractive index of hyaloplasm and determination of the distance between cell membrane surface and glass]. / Ispol'zovanie éffekta narushennogo polnogo vnutrennego otrazheniia dlia izucheniia adgezii kletok zhivotnykh k steklu. III. Izmerenie polazatelia prelomleniia gialoplazmy i otsenka rasstoianiia mezhdu poverkhnostnoi membranoi kletki i steklom.

The kinetics of form change (flattening) of Chinese hamster fibroblast cell surface facing the glass has been studied during attachment of fibroblasts to the glass using the effect of frustrated total reflection. Two stages of the process were shown to exist. The first one is characterized by a rapid (5 min) partial flattening and requires no serum in the medium. The second stage occurs only in the presence of serum in the medium, it lasts no longer than one hour and ends in a complete flattening. After the second stage is completed, spreaded cells appear.

[A flow diffusion chamber for research on cells in culture]. / Protochno-diffuzionnaia kamera dlia issledovaniia kletok v kul'ture.

A flow diffusion chamber designed for studying cells and tissues in culture is described. The chamber contains a plate with a great number of isolated holes, which enables one to perform the cultivation of cells at different distances from the porous membrane separating the cells from the perfused medium. An individual porous membrane can be placed above each hole. Evidence for the selective permeability of domestic membranes under the conditions of cell culture in chamber is presented. The chamber makes possible a simultaneous cultivation of a great number of various cultures with different conditions of mass exchange with common perfused medium, which contributes to intensification of studies.

[Growth of a cell culture on porous capillaries]. / Rost kul'tury kletok na poristykh kapilliarakh.

The growth of BHK-21 culture cells on the outer surface and in the clear space of capillaries made of phenylone or polyacrylnitrile was studied. The cultivation of cells in the clear space of the capillary extends their survival time and outlines possible ways of continuous cultivation of tissue cells which are cultivated in the attached state.

[Primary culture of epithelial secretory cells from venom gland of the common adder Vipera berus]. / Pervichnaia kul'tura kletok sekretornogo épiteliia iadovitoi zhelezy gadiuki obyknovennoi Vipera berus.

A primary culture of epithelial secretory cells from the venom gland of Vipera berus was obtained. The cells adhered to collagen 1 and to a mixture of adhesion proteins (Matrigel), proliferated and retained the features of differentiation. Electron microscopy demonstrated the presence of all ultrastructures typical of these cells in vivo, a full complex of intercellular junctions, and cellular membrane polarity. The immunohistochemistry confirmed the capacity of secretory cells to synthesize venom in culture. We have studied the role of carbochole, an agonist of M-cholinoreceptor, in the initiation of the secretory cycle in cells in vitro. We propose that M-cholinoreceptors may play an important role in the initiation of the secretory cycle in vivo.