[Change in localization of cellular vesicular apparatus during differentiation of myoblasts into myotubules in cell culture].

The study of changes in the intracellular processes during differentiation of myoblasts into myotubules is of great importance for understanding several fundamental problems of cell biology. At first, this concerns the spatial organization of vacuolar apparatus that reflects the alterations in the properties of cell membranes, cytoskeleton elements and dynamics of vesicular transport in the course of differentiation. The distribution of acidic membrane organelles (lysosomes, late endosomes, Golgi cisternae) during the myotubule formation was revealed. It was shown that perinuclear localization of acidic organelles in myoblasts was replaced by diffuse distribution of these structures in the whole volume of myotubules. Using lipophilic fluorescent dyes, RH 414 and di-8-ANEPPS, the process of formation and dynamics of endocytic vesicles in myoblasts and myotubules was investigated. In the present work, semiconductive nanocrystals, quantum dots (QDs), conjugated with TAT-peptide, which belongs to cell-penetrating peptides, were used to characterize nonspecific endocytosis. It was shown that QDs--TAT complexes penetrate myoblasts but do not penetrate myotubules even after 24 h incubation, which might be connected with plasma membrane changes during the process of skeletal muscle differentiation.

[Untargeted quantum dots in confocal microscopy of living cells].

The problem of non-specific binding of quantum dots (QDs) with cells is very important but not fully understood taking into account the possible application of QDs in medical and fundamental studies. The interactions of untargeted CdSe/ZnS QDs with isolated frog muscle fibres, HeLa cells and J774 cells were investigated. The observations were performed on living cells using laser confocal microscopy (Leica TCS SL). The QDs covered with polyethylenglycol without any functional reactive groups with emission maximum at 565 nm were used in the study. This type of QDs is suggested to prevent an interaction of QDs with biological molecules. It has been shown that QDs do not enter HeLa cells, T-system and sarcoplasm of skeletal muscle fibres. However, during long-term incubation J774 cells can uptake QDs. The data obtained has demonstrated the diversity of interactions of untargeted QDs with different cell types and are important for understanding of the problems of non-selective uptake and cytotoxicity of QDs.

[AO distribution and fluorescence spectra in myoblasts and single muscle fibres].

Using spectral scanning regime of Leica TCS SL confocal microscope, acridine orange (AO) fluorescence spectra in nuclei and cytoplasms of living myoblasts L6J1 and frog single muscle fibres have been studied. AO fluorescence spectra in salt solutions dependent on free AO concentrations and in AO complexes with DNA have also been obtained for comparison. Myoblasts nuclei fluoresced in green spectral region with maximum at approximately 530 nm (corresponding AO monomers fluorescence), nucleoli fluoresced most brightly. Nuclear chromatin fluoresced not uniformly in these cells. We saw similar to myoblasts AO emission in nucleoli and nuclei of frog single muscle fibres. The uniformed weak green fluorescence was observed for myoblast cytoplasm. As to the muscle fibres sarcoplasm, we saw also AO green fluorescence in A-discs. In myoblasts and muscle fibre cytoplasm we saw the fluorescent red, yellow and green granules which were acidic organelles. The comparison of AO fluorescence spectra in living cells with fluorescence spectra of different AO concentrations and complexes of AO with DNA in buffer solutions allows estimation of AO concentration in acidic granules which is of interest in the investigation of cellular organelles functions in the processes of intracellular transport, adaptation, apoptosis and a number of pathological conditions.

[DNA complexes with polycations used for delivery of DNA into cells].

The formation and physicochemical properties of high-molecular thymus and plasmid DNA complexes with synthetic polymers based on (dimethyl-amino)ethyl methacrylate (DMAEM), (diethyl-amino)ethyl methacrylate (DEAEM), and polyvinyl amine (PVA) were investigated in solutions of different ionic strength by low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The toxicity of complexes in T98G cells was studied. It was shown that, when the ratio of polycations to DNA charged groups concentration (N+/P) reaches values > 1, DNA condensation occurs. It is accompanied by increasing optical density of solutions. Changes in DNA size after condensation were estimated. Phase diagrams of systems DNA/polycation in the presence of NaCl were obtained. It was shown by MTT-analysis that DNA complexes with polycations in the range of concentrations used have low toxicity.

[Effect of alkaloid sanguinarine and a pharmaceutical preparation ukrain on modulation of vesicular membrane fusion and actin cytoskeleton of macrophages].

A study was made of modulations of lysosome-phagosome fusion process and of fibrillar actin content in mouse peritoneal macrophages by an antitumor alkaloid sanguinarine and a derivative drug Ukrain. In addition, effects of these substances on in vitro polymerization of monomeric globular actin from rabbit muscle were investigated. Sanguinarine and Ukrain stimulated lysosome-phagosome fusion and increased the content of polymerized fibrillar form of actin in mouse macrophages. Effects of these substances were enhanced at their higher concentrations. Both sanguinarine and Ukrain induced in vitro polymerization of globular actin from rabbit muscle. A possible role of sanguinarine and Ukrain in changing vesicular membrane states during intracellular membrane interaction in lysosome-phagosome fusion process was discussed. The influence of these substances on actin polymerization and actin cytoskeleton rearrangement was evaluated. It could be supposed that sanguinarine and Ukrain may alter intracellular membrane transport.

[Effects of cationic vectors complexed with plasmid DNA on the phagosome-lysosome fusion in murine peritoneal macrophages and J744 macrophages]. / Vliianie kationnykh vektorov transfektsii v komplekse s plazmidnoi DNK na sliianie lizosom s fagosomami v peritoneal'nykh makrofagakh myshi i v makrofagakh linii J774.

Polyethylenimine (PEI) and cationic polypeptides complexed with plasmid DNA are the most efficient nonviral vectors for gene therapy. It is believed that endocytosis is the major pathway for cell entering by PEI/DNA or cationic peptides/DNA complexes. Effects of plasmid DNA complexed with PEI, poly-L-lysine (PLL), poly-D-lysine (PDL) and polyarginine (PA) on the phagosome-lysosome fusion (P-LF) were studied in murine peritoneal macrophages and J774 macrophages. Cationic polypeptide PLL can be hydrolysed by cellular peptidases, but its stereoisomer, PDL, cannot be split by these enzymes. PEI, PDL, and PA have been shown to inhibit P-LF. PLL showed a low effect on the P-LF. On the basis of these studies, we assume that lysosomotropic agents able to change functions of lysosomes in the cell may affect transfection efficiency and thus be used for gene therapy.

[Involvement of SH-groups in the regulation of adenylate cyclase signal system by isoproterenol and serotonin in cultured murine fibroblasts L (subline LSM)]. / Uchastie SH-grupp v reguliatsii adenilattsiklaznoi signal'noi sistemy izoproterenolom i serotoninom v kul'ture fibroblastov myshi linii L (sulinii LSM).

The adenylyl cyclase system (ACS) plays a key role in transduction of a hormonal signal into eukaryotic cells. The functional activity of the system depends on SH-groups of proteins involved in the ACS: receptor, G-protein, and enzyme adenylyl cyclase (AC). We studied the influence of thiols and SH-blockers on the regulation of AC activity by nonhormonal (NaF and Gpp[NH]p) and hormonal (biogenic amines isoproterenol and serotonin) agents in homogenates of cultured murine fibroblasts of line L (subline LSM). In the presence of thiols 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM) the basal AC activity somewhat increased, whereas the stimulating effects of NaF, Gpp[NH]p, and hormones decreased. No potentiating action of Gpp[NH]p on hormonal effect in this case was found. The SH-blockers 25 mkM p-chloromercuribenzoic acid (CMBA) and 0.2 mM N-ethylmaleimide significantly inhibited both the basal AC activity and that stimulated by different agents. Thiols partially restored CMBA inhibited AC activity (in the case of N-ethylmaleimide restoring effects of thiols were insignificant). This, the ACS of murine fibroblasts of subline LSM is SH-sensitive. The forms of SH-groups in proteins involved in the ACS determine their functional activities and a possibility of transduction of the hormonal signal on the effector systems.

[Effect of polyamine synthesis inhibitors separately and in combination with epidermal growth factor on fusion of lysosomes with phagosomes and F-actin level in mouse peritoneal macrophages]. / Vliianie ingibitorov sinteza poliaminov razdel'no i v sochetanii s épidermal'nym faktorom rosta na sliianie lizosom s fagosomami i na soderzhanie F-aktina v peritoneal'nykh makrofagakh myshi.

Effects of polyamine (PA) synthesis inhibitors--alpha-difluoromethylornithinchloride (DFMO) and alpha-methylornithinchloride (MO)--separately or in combination with the epidermal growth factor (EGF)--on lysosome-phagosome fusion (P-LF) and F-actin content in murine peritoneal macrophages were studied using fluorescent dye Acridine orange for lysosome labelling, FITC-phalloidin for F-actin, and yeast cells as a target. DFMO and MO significantly inhibited P-LF and decreased F-actin content in murine peritoneal macrophages. A combination of DFMO and MO with EGF failed to inhibit P-LF or to decrease F-actin content in these cells. The results obtained with DFMO and MO suggested new cellular targets of their effects. These results may be extended to cancer research to provide a rationale for clinical trials using combinations of EGF with DFMO or MO.

[Effect of some DNA-intercalators and antioxidants on the phagosome- lysosome fusion and F-actin content in murine peritoneal macrophages]. / Vliianie nekotorykh DNK-interkaliatorov a antioksidantov na sliianie fagosom s lizosomami i na soderzhanie F-aktina v peritoneal'nykh makrofagakh myshi.

Effect of DNA-intercalators ethidium bromide (EB, 0.005 and 0.015 mM) and dimidium bromide (DB, 0.005 and 0.010 mM) and antioxidative compounds acetylsalicylic acid (ASA, 0.05 and 0.50 mM) and beta-carotene (0.01, 0.02, 0.05 mM) on the phagosome-lysosome (P-L) fusion and F-actin content in murine peritoneal macrophages were studied. EB, DB, ASA and beta-carotene were found to stimulate P-L fusion and the effect depending on the concentration of compounds tested. The strongest influence as evoked by 0.5 mM of ASA and 0.05 mM of beta-carotene. The compounds tested enhanced the F-actin content in macrophages, especially by the action of beta-carotene (0.05 mM). The obtained data indicate a correlation between P-L fusion stimulation and F-actin content under the influence of compounds tested in murine peritoneal macropheages.

[The effect of biologically active compounds on lysosome fusion with phagosomes and the F-actin content in mouse peritoneal macrophages and on the status of the lysosomal membranes in mouse hepatocytes]. / Vliianie biologicheski aktivnykh soedinenii na sliianie lizosom s fagosomami i soderzhanie F-aktina v peritoneal'nykh makrofagakh myshi i na sostoianie lizosomnykh membran gepatotsitov myshi.

Effects of biologically active compounds bilirubin (BR, 0.1 and 0.2 mM), chelerythrine (CR, 0.1 and 0.5 mM) and farmorubicin (FR, 0.6 and 6.0 mM) on the phagosome-lysosome fusion (P-LF) were studied using fluorescent dye acridine orange for lysosomal labelling and yeast cells as a target. To investigate mechanisms of these effects, changes in fluidity of lysosomal membranes from murine liver were studied by measuring of fluorescence intensity, lifetime and polarization of the fluorescent membrane probes: DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH [1-(4-triphenylamino)-6-phenyl-1,3,5-hexatriene] incorporated in isolated murine liver lysosomes. In order to characterize the induced cytoskeleton changes, the F-actin content in murine peritoneal macrophages was determined. It was found that all three compounds tested enhanced P-LF. Our results demonstrate that BR induces a decrease in DPH and TMA-DPH fluorescence polarization, FR increases DPH and TMA-DPH fluorescence polarization, and CR causes an increase in TMA-DPH fluorescence polarization in lysosomal membranes. All the three compounds increase F-actin content in peritoneal macrophages. Thus, the action of BR extended on P-LF is associated with increasing lysosomal membranes fluidity and cytoskeleton changes. The enhancement of P-LF under the action of FR and CR can be most likely explained by changes of cytoskeleton.

[The protein spectrum and thermostability of the lysosomal hydrolases in heat-resistant sublines of Chinese hamster cells]. / Izuchenie spektra belkov i teploustoichivosti lizosomal'nykh gidrolaz v termorezistentnykh subliniiakh kletok kitaiskogo khomiachka.

It has been shown by electrophoresis that the earlier obtained thermoresistant sublines of the CHO-K1 cell line do not accumulate heat shock proteins when cultured at 37 degrees C. The thermostability of two lysosomal proteins--acid lipase and acid phosphatase--were higher in the thermoresistant cells than in the parental cells, whereas no differences in thermostability of galactosidase were found between heat resistant and parental lines. Thus, it is concluded that changes in the level of conformational flexibility of protein molecules may be one of the mechanisms of cell adaptation to growth at higher temperatures.

[The role of phagocytic cells in the pathogenesis of idiopathic fibrosing alveolitis]. / Rol' fagotsitiruiushchikh kletok v patogeneze idiopaticheskogo fibroziruiushchego al'veolita.

The paper deals with the examination of the role of phagocytes--alveolar macrophages and neutrophils--and peripheral monocytes, in the pathogenesis of idiopathic fibrosing alveolitis (IFA). As the disease aggravates, activation of the absorbing capacity of monocyte-macrophagal cells corresponds to a sharp rise in the level of circulating immune complexes in the blood of IFA patients. Higher activities of elastase and collagenase are observed in the IFA patients' bronchial lavage fluid.