[The influenza viruses and their surface proteins impact on the metabolism of human blood vessel endothelium cells].

The modern influenza virus subtypes H3N2, H5N1, and H1N1 reduced the metabolism of the endothelial cells within the range from 20% to 60% (compared with control). The degree of the activity of the dehydrogenase reduction depended on the dose of virus and time of virus reproduction. HA and NA also actively reduced the metabolism of the cells ranging from 5% to 60%, depending on the concentration of the proteins and time of their impact on cells. Neuraminidase was more active than hemagglutinin in the MTT test (at concentration 50 microg protein/ml).

[Effect of different water-soluble forms of the fullerene C60 on the metabolic activity and ultra-structure of cells in culture].

In view of contradictory data on the toxicity of fullerenes for live organisms we studied the effect of water-soluble complexes of C60 with N-polyvivyl-pirrolidone (C60/PVP) and gamma-cyclodextrine (C60/gamma-CD) on MA-104 cells in culture. Both complexes proved to be non-toxic for cultured cells in the dark in wide range of concentrations. Both complexes provoke changes of cellular ultra-structure which reflect the enhancement of metabolic activity. At the same time only the exposition with the complex C60/PVP leads to the essential growth of number and size of mitochondria. However, the effect of two studied water-soluble forms of C60 under intensive UV-irradiation of cells proved to be opposite: C60/PVP had a cyto-protective action while C60/gamma-CD caused a significant growth of photo-toxicity. Possible reasons of the differences in the action of different forms of C60 on living organisms are discussed.

[Effect of polymer carrier origin and physical state on fullerene C60 phototoxicity in vitro].

Biological effects of water-soluble inclusion complexes of fullerene C60 with poly(vinyl pyrrolidone) (C60/PVP) and gamma-cyclodextrin (C60/g-CD) as well as solid C60 (C60-coated surface) on cell viability have been studied in vitro. It is established that both inclusion complexes (in a broad range of concentrations) and solid fullerene coatings are nontoxic in the dark for the cell of all lines tested. In contrast, under intense UV illumination, the C60/PVP complex reliably protected test cells from the UV radiation damage, whereas the C60/g-CD and fullerene-coated surface exhibited pronounced phototoxicity. Moreover, solid fullerene caused a photodynamic effect under irradiation with both UV and visible light. The radiation damage could be blocked by some antioxidants (e.g., hypoxen) and singlet-oxygen scavenger (sodium azide). This is evidence for the participation of 1O2 in phototoxicity manifestations. The results indicate that the biological properties of fullerene C60 in vitro depend on its aggregate state, form of solubilization, and, probably, the nature of solubilizing medium.

[Synthesis and biological activity of complexes of sulfonate-containing polyanions and gentamycin].

A series of copolymers of acrylamide (Am) with 2-acrylamido-2-methylpropanesulfonic acid (AmMPSA) and N-2-hydroxypropyl-methacrylamide (HPMA) with acrylic acid (AA) have been synthesized. Complexes of gentamycin in the base form with these polymers and dextran sulfate were obtained. It is established that both poly(AmMPCA) and its complex with gentamycin exhibit an antiviral activity in vitro against human A(H3N2) influenza virus and herpes simplex virus of type 1 (HSV-1). The HPMA-AA copolymer and its complex with gentamycin are only active against HSV-1. Complexes of gentamycin with the polyanions based on the Am derivatives and dextran sulfate showed a high level of antibacterial activity against four bacterial strains. All the polymers studied as well as their complexes with gentamycin demonstrated a low toxicity in vitro, which makes them promising for the creation of preparations with combined antiviral and antimicrobial properties.

[Antiviral action of some antioxidants/antihypoxants and their combinations with remantadine against human influenza A(H3N2) virus studied in in vitro models].

The possible antiviral activity of preparations with antioxidant and/or antihypoxant properties was studied on two in vitro models of influenza infection: (i) in cultures of chorio-allantoic membranes of chicken embryos and (ii) in MDCK cells. Preparations under study were hypoxene, reduced glutathione, dihydroquercetin, trolox, coenzyme Q10, and the enzymatic preparation of superoxide-dismutase (recsod). Preparations possessing combined antioxidant/antihypoxic and detoxicating properties (reduced glutathione and hypoxene) produced a significant antiviral effect and enhanced the antiviral effect of rimantadine. The antiviral effect of these preparations was manifested by a decrease in the production of viral particles and, to a more pronounced degree, by the inhibition of cytopathogenic action of virus on cultured cells, which was revealed in the tests for the activity of respiratory enzymes. In contrast to the compounds containing thio or sulfo groups, the antioxidants of "direct action" (free radical scavengers) - coenzyme Q 10, trolox, quercetin and the enzymatic preparation recsod did not show any pronounced protective effect and in some cases even enhanced the production of viral particles and decreased the antiviral action of rimantadine.

[Studying the effect of antioxidants and/or antihypoxants on cell cultures under conditions of cytotoxic action of rimantadine].

The effect of some preparations with antioxidant and/or antihypoxic properties was studied under the conditions of cytotoxic action of the antiviral drug rimantadine in an MDCK cell culture. The preparations under study were hypoxene, reduced and oxidized glutathione, metadoxil, trolox (water-soluble analog of vitamin E), dihydroquercetin, coenzyme Q, and recsod (superoxide dismutase enzyme preparation). The protective drug action on the model of cytotoxicity in vitro was observed for the preparations possessing a complex of antioxidant/antihypoxic and detoxicant properties: hypoxene, metadoxil, and reduced glutathione. The protective effect of preparations did not correlate with their direct antiradical activity with respect to the stable free radical DPPH. Some compounds of phenolic nature (trolox, coenzyme Q) and recsod enhanced the harmful effect of rimantadine on the culture cells under the conditions studied. A possible explanation of this fact could be the conversion, under certain conditions, of the effect of phenolic compounds from antioxidant to pro-oxidant, which is confirmed by some literature data. The results do not allow antioxidant preparations to be recommended for routine application as cytoprotectors during toxic stresses of different nature. The protective activity of such preparations should be estimated separately for each particular xenobiotic.

DNA-polycation complexes: effect of polycation structure on physico-chemical and biological properties.

The purpose of the study was to investigate the influence of cationic polymer structure on the formation of DNA-polycation complexes and their transfection activity. Primary, tertiary, and quaternary polyamines with molecular masses ranging from 8000 to 200,000 were investigated. DNA-cationic polymer interaction was characterized by low gradient viscometry, dynamic light scattering, circular dichroism, UV spectrometry, flow birefringence, DNA electrophoresis, and electron microscopy. Transfection activity of the complexes was evaluated by the expression of reporter gene (beta-galactosidase) and using synthetic FITC-labelled oligonucleotides. Complex formation was found to be dependent on the structure and molecular weight of the polymer and the ionic strength of the solution. Secondary DNA structure in complexes was not disrupted, and DNA was protected from protonation. Cell lines of different origin were used for testing of transfection activity of the complexes. The sensitivity of the cells to transfection was established to be highly dependent on the cell line. DNA-polycation complexes are non-toxic according to MTT. Polyallylamine, and polydimethylaminoethylmethacrylate were found to be the most promising polycations for gene delivery. Transfection efficacy of their complexes with DNA to T-98G cells reaches up to 90-100%. It was found that optimal molecular mass of polydimethylaminoethylmethacrylate is in the range of 8000-50,000 Da.

[Evaluation of metabolic parameters in vitro as a model for testing the cytotoxicity of antiviral drugs].

A new test system based on in vitro assessment of the cellular viability and/or metabolism is proposed, which offers an informative approach to the screening of antiviral drugs. Cytotoxic effects of the antiviral drugs rimantadine and polyrem were studied on the cultures of some mammalian cells. The results of short-term (2 h) exposures with the drugs tested were close to their LD50 values in mammals, which makes the proposed test system promising for the assessment of drug toxicity in vivo for the whole organism. A study of the state of cell metabolism after a long-term (48 h) exposure showed that the system of endocytosis is more sensitive than other indices with respect to antiviral drugs. Is was demonstrated on the cell level that the binding of drugs into polymeric complexes can decrease the degree of its cytotoxicity: the toxicity of polyrem (a polymeric complex of rimantadine) was lower as compared to that of the equimolar concentration of rimantadine or a mixture of rimantadine with a polymeric carrier.

[Effect of the bone cement on human fibroblast culture and possible ways of its correction by cytoprotector preparations]. / Osobennosti deistviia kostnogo tsementa na fibroblasty cheloveka v kul'ture i vozmozhnye puti ego korrektsii s pomoshch'iu tsitoprotektornykh preparatov.

The results of in vitro investigation are used to assess the effect of poly(methyl methacrylate) based bone cements (BCs) upon human fibroblast culture. The dose dependent cytotoxic effect of BCs (Polacris) was manifested by the development of oxidative stress and hypoxia (on the cell level) and by a decrease in the cell ability to multiplication. The drugs possessing antihypoxant (mafusol) and antioxidant (erysod) activity, introduced into the incubation medium in clinically adequate doses, significantly reduced the toxic effect of BCs. The maximum positive effect was observed upon a combined administration of both drugs.

[The study of the Ca2+ role in cytotoxic response of human cells in culture to the action of xenobiotics]. / Issledovanie uchastiia ionov kal'tsiia v toksicheskom deistvii ksenobiotikov na kletki cheloveka v kul'ture.

The role of Ca2+ in mechanisms of cell death, necrosis and apoptosis is diverse and generally recognized. The purpose of this work was to study Ca2+ participation in a cytotoxic response of human cultured cells in the presence of toxic concentrations of cationic antiseptic substance poly(hexamethylene guanidine), anionic surfactant SDS and monomeric methyl methacrylate (a component of bone cement applied in surgery). Human cell line U-937 grown in suspension was used for this study. A fluorescent probe chlortetracycline was used, as an indicator of Ca2+ transport through biologic membranes. Our results show that weakly toxic concentrations of xenobiotics under study, close to the minimum toxic doses, nearly always provoke a fair but statistically significant drop in Ca2+ binding by cells. At the same time, higher toxic doses lead to significant increase in Ca2+ influx. The latter event well compares with the majority of literary data, while the mentioned decrease in Ca2+ influx at low toxic concentrations of xenobiotics presumably correlates with the initial stage of acute cytotoxic response, accompanied by a metabolic activation and enhanced resistance of cells to injuring stimuli, demonstrated by the authors elsewhere. In parallel, a possible effect of Ca(2+)-channel antagonist nifedipine was explored under conditions of cytotoxic response of cell lines U-937, A-549 and human embryonic lung fibroblasts to poly(hexamethylene guanidine). Nifedipine (10 microM) was introduced in the incubation medium simultaneously with the toxic agent, and the cells were further maintained for 5 or 24 h in culture; their viability was monitored with the microtetrasolium test or by assessment of LDH leakage into the incubation medium. The effect of nifedipine proved to be dual, depending on the applied concentration of toxic agent: at low toxic concentrations the improvement of viability could be noticed, while at more pronounced toxic doses aggravation of viability was evident. From our point of view the explanation of this result could be the following. In weakly toxic conditions, as in intact cells, Ca2+ influx is brought about by specific mechanisms, mainly through Ca(2+)-channels, that is why nifedipine partly abolishes Ca(2+)-dependent cytotoxic response. At high concentrations, cell plasma membrane is directly damaged by toxic agent, Ca2+ enters cells mainly non-specifically, so that Ca2+ antagonist cannot protect cell injury. The reason of toxic effect aggravation by nifedipine in these conditions is still waiting for its explanation.

[Cytoprotective effect of antihypoxic and antioxidant preparations on cultured human cells in a model of toxic response]. / Tsitoprotektornoe deistvie antigipoksicheskikh i antioksidantnykh preparatov na kletki cheloveka v kyl'ture pri modelirovanii toksicheskogo otveta.

An oxidative stress is considered to be one of the major mechanisms of cytotoxicity. The purpose of present work was to study effects of some drugs with antihypoxic/antioxidant activity in cultured human lung embryonic fibroblasts under conditions of cytotoxic response, provoked by cationic or anionic antiseptics. The following preparations were under study: Mafusol (Na-fumarate), superoxide dismutase from human erythrocytes (SOD), cytochrome c, alpha-tocopherol and Thioctacid T (lipoate) which were applied at concentrations comparable with those, employed in clinical application. The combinations of the used drugs were also under study. The cytotoxic response was induced by an application of antiseptics into the cell incubation medium in 2-5 fold dilutions up to minimum toxic doses for 2-24 h. The drugs under study were introduced simultaneously with antiseptics. The maximum cytoprotective effect was revealed in the case of combination fumarate-alpha-tocopherol; the combination fumarate plus SOD being the second in effectiveness. When the drugs were introduced separately, the most effective proved to be fumarate, followed by vitamin E and cytochrome c. SOD and lipoate did not reveal any cytoprotective activity in our experimental conditions. The designed model of cytotoxicity in vitro can be considered as a prospective test-system for the screening of cytoprotective drugs and their combinations.

[The antimicrobial activity and cytotoxicity of anti-infective preparations in a human cell-culture model]. / Issledovanie protivomikrobnoi aktivnosti i tsitotoksichnosti antiinfektsionnykh preparatov na modeli kul'tury kletok cheloveka.

The complex evaluation of Polysept, a new antiseptic, by its antimicrobial activity with the determination of the minimal inhibiting concentration, its antiadhesive effect and its cytotoxic action with the determination of the minimal toxic dose is presented after preclinical trial, carried out on the culture of skin and lung fibroblasts of human embryo used as an experimental model. The presence of 50% human blood serum enhances the antimicrobial and antiadhesive effects and decreases the cytotoxic action of the antiseptic with respect to fibroblasts, which makes it a promising preparation not only for the treatment, but also for prophylaxis of wound infection.

[The effect of individual serum proteins and native blood serum on the adhesion of Staphylococcus aureus to cells in vitro]. / Vliianie otdel'nykh syvorotochnykh belkov i nativnoi syvorotki krovi na adgeziiu Staphylococcus aureus k kletkam in vitro.

Blood serum and its components were found to produce an antiadhesive effect which inhibited the attachment of S.aureus to cells HEp-2 and immortalized astrocytes. Normal and antistaphylococcal immunoglobulins exhibited the highest activity, inhibiting the process of bacterial adhesion in a serum-free medium. The antiadhesive activity level of native serum was considerably lower and constituted 4% of that of normal immunoglobulin and 85% of that of albumin. In spite of pronounced inhibiting action of normal immunoglobulin and albumin, their dissolution in serum did not increase its antiadhesive activity.

[Some biochemical indicators of the cytotoxic response of human fibroblasts cultured with natural and synthetic polycations]. / Nekotorye biokhimicheskie pokazateli tsitotoksicheskogo otveta fibroblastov cheloveka v kul'ture pod deistviem prirodnykh i sinteticheskikh polikationov.

Cationic antiseptics--catamine AB, polysept (polymeric derivative of chlorhexidine) as well as cationic protein protamine exhibited a pronounced cytotoxic effect on human skin and lung fibroblasts in cell culture. Their effect was accompanied by augmentation of lipid peroxidation products and by inhibition of DT-diaphorase, LDH, ATPase and glutathione reductase. Introduction of alpha-tocopherol into the cultural medium normalized the rate of lipid peroxidation but did not remove the inhibitory effect on activity of oxidoreductase studied. Blood serum proteins immunoglobulins and albumin diminished significantly the cytotoxic effect of cationic preparations contributing to restoration of all the parameters studied to control values; this phenomenon appears to occur due to nonspecific membrane protective and antioxidation effects of the blood serum proteins.

[A comparative analysis of the biological activity of an acid-extractable brain protein and fibroblast growth factors]. / Sravnitel'nyi analiz biologicheskoi aktivnosti mozgovogo kislotoékstragiruemogo belka i faktory rosta fibroblastov.

An acid-soluble protein was isolated from bovine brain tissue using a combination of 5% HClO4 acidic extraction, cation-exchange chromatography and heparin-Sepharose affinity chromatography. This preparation had a biological activity similar to that of the fibroblast growth factor (FGF). It has stimulated neurite outgrowth in organotypic culture of chicken embryo dorsal root ganglia and also has stimulated a proliferation of human embryonic fibroblasts in the culture. The biological activity of isolated preparation was totally inhibited by anti-bFGF antibodies, while anti-aFGF antibodies had no effect. The bFGF showed a neurite stimulating activity in the organotypic culture that was completely abolished by the anti-bFGF, IgG, while the aFGF was inactive. This data, as well as cationic properties of isolated protein and its heparin-binding ability, enable us to postulate a high degree of homology of the brain acid-soluble protein and the bFGF. At the same time the relations of the isolated growth factor and other brain-derived heparin-binding growth factors are yet to be established.

[Secretory activity of pancreatic acinar cells at different times of the day, and the sequence of secretory granule maturation]. / Sekretornaia aktivnost' atsinoznykh kletok podzheludochnoi zhelezy v raznoe vremia sutok i posledovatel'nost' protsessov sozrevaniia sekretornykh granul.

Certain parameters of secretory activity of the pancreatic acinus as a system consisting of separate cells were verified at the electron microscopic level. Thirty male Wistar rats were investigated; they were sacrificed by one every 49 min for 3 days. Volumetric portion of the condensing vacuoles, zymogen granules and all granules of the secrete within the limits of the cytoplasm zone, where these granules were present, as well as number of the granules and an average volume of separate condensing vacuoles and zymogen granules were estimated. The results of measurements were smoothed out by the method for calculating the sliding averages in order to expel frequent oscillations with the period up to 5 hours. Changes in the exported proteins reserves at various phases of the secretory cycle were obtained by fluctuations in the secreted granule numbers and their individual volumes. After an ecologically adequate feeding in the evening, the number of the condensing vacuoles increased, and then, in 1-1.5 h did the number of the zymogen granules. Increase in the cell synthesizing activity also resulted in increasing average volume of separate condencing vacoules and zymogen granules. The rhythmicity of different parameters characterizing the secretory process was coordinated, that demonstrated certain synchronization of individual secretory cycles in separate cells, nevertheless, it was not complete, since for synchronization of the whole population of the acinar cells at the stage of the zymogen granules maturation it was necessary 0.5-1 hour.