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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.

The effect of human erythrocyte preincubation with acetylcholine or acridine orange on the process of their acid hemolysis has been investigated. It was found that degradation erythrocytes rate constant increase by 24.2 and 35.5% respectively.

Destruction of lambda phage DNA is studied under nanosecond pulse laser irradiation (lambda = 355 nm) of DNA-dye complexes in solution at 77K (dye--acridine orange, 8-methoxypsoralen, ethidium bromide). Free radicals induced by laser radiation are found to participate in DNA sugar-phosphate chain scission. It was observed that the quantity of DNA double-strand breaks correlated with that of the free radicals and that of oxygen influenced DNA laser destruction.

By means of polarization fluorescence microscopy changes in polarization degree of fluorescence of DNA-bound of Acridine orange in situ were observed. A marked decrease in polarization degree of chromatin fluorescence was found after the treatment of cell nuclei with 0.6 M NaCl. It shows that the removal of histone H1 from the chromatin results in destabilization of its structure. The data obtained show that the measurements of polarization degree of fluorescence allow to obtain relevant information about structural changes in DNA chromatin.

Changes in pH in rat brain synaptic vesicles (SV) were studied with the use of the fluorescent slightly basic dye acridine orange. The pH value in isolated SV was found to be acidic, which was confirmed by the ionophore sensitive accumulation of the dye. Addition of ATP provoked further acidification of the intravesicular medium. The acidification rate reached a maximum after dissipation of the existing H+ gradient seen during preincubation in the absence of ATP. The ATP-dependent acidification was eliminated by the protonophore carbonylcyanide m-chlorophenylhydrazone, H4Cl or the detergent triton X-199 (0.025%). Valinomycin inhibited the ATP-dependent translocation of H+ whatever the incubation medium (with KCl or NaCl). Dicyclohexylcarbodiimide, a known inhibitor of proton ATPases (100 microM) as well as ethylmaleimide (100 microM) completely blocked H+ translocation whereas oligomycin, a specific blocker of mitochondrial H+-ATPase, and ouabain did not influence that process. ATP induced H+ translocation only in the presence of Mn2+ or Mg2+ but not in the presence of Ca2+. The translocation of H+ was not affected by the replacement of univalent cations (KCl, NaCl or Cl), however, it was prevented completely upon replacement of the penetrating anion Cl- by the non-penetrating anion O2-4 or upon replacement of the salts by sucrose. It is concluded that the ATP-dependent translocation of H+ in SV is mediated via H+-ATPase which maintains the low pH value in SV.

Prematurely condensed chromosomes (PCC) have been obtained by polyethylene glycol (PEG) induced fusion in suspension of the Chinese hamster metaphase cultured cells with those in interphase. As alternative approach the PEG-fusion of the Chinese hamster asynchronous culture cells in monolayer with subsequent incubation in free medium was used. A comparative cytofluorimetric investigation of PCC and chromatin of the interphase nuclei of corresponding ploidy has shown some increase (up to 10%) of acridine orange and olivomycin binding with PCC chromatin. A similar slight increase in low molecular weight ligands binding with chromatin was also found in mitotic chromosomes. The data obtained confirm the opinion about the similarity of events taking place in chromatin during physiological mitosis and premature chromosome condensation. The cytochemical study of chromatin availability to low molecular weight ligands can be used as a criterion for judging on the properties of the artificially condensed chromatin.

A clear-cut difference between chromatin melting curves of cells from the patient with Down's disease and his mother and ones from healthy individuals and the proband's father was shown by fluorescence microscopy and acridine orange staining on human fibroblasts incubated in autologous serum. These data suggest the presence of an obvious genetic correlation between phenotypically healthy mother and her sick child. The identity of the chromatin structure of the patients detected both on lymphocytes and other type of cells, human fibroblasts, allows a suggestion that the phenomenon of the altered chromatin structure is typical generally of the given individual's tissues. Certain changes in the cell chromatin structure are mediated by the effect of autologous serum.

The interaction of serotonin and acridine orange dye with DNA isolated from bacterium Escherichia coli and the yeast Candida utilis has been analysed by spectrofluorimetric method. Using data on competitive binding to DNA of serotonin and acridine orange, known as DNA intercalator, a conclusion concerning the formation of intercalated complex between serotonin and DNA has been made. It is shown that for yeast DNA the constant of intercalated binding of serotonin is 3,5-fold smaller than for the bacterial one.

The cytofluorometric method was used to study changes occurring in the chromatin structure of lymphocytes during the first few hours following irradiation of lymphocytes with He-Ne-laser (lambda = 632.8 nm) of 28-112 J/m2. The changes were similar to those caused by PHA that is: the increase in acridine-orange binding to DNA during the first 45-90 min, its fall to the control level in 3-4 h and the subsequent increase.

Kinetics of Neutral red (NR) and Acridine orange (AO) uptake by cultured L cells (subline LSM) has been studied. It was found that the uptake of both NR and AO, with their constant concentrations in the medium was characterized as a two-phase process. During 2 hours, these cells concentrated as much as 90% of the total amount of NR and AO taken up during the whole incubation period. The segregation and accumulation of NR, AO as well as NH4Cl took place in lysosomes. NR and AO concentrations within the cells exceed by 600 and 400 times, respectively, those in the medium. NR, AO and NH4+ accumulation in cells resulted in inhibition of the activity of the following lysosomal hydrolases: cathepsins B and D, acid lipase, N-acetyl-beta,D-glucosaminidase, beta-galactosidase, acid phosphatase and galactosyltransferase, the latter being a marker of Golgi apparatus. The effect of lysosomal enzyme activity inhibition on the cell economy, and a possible role of lysosomotropic agents as regulators of the lysosomal apparatus functional activity are discussed.

A cytochemical method of chromatin study by means of nuclear staining with ethidium (bromide) is described. Dependence of stain binding by chromatin on ethidium concentration, ionic composition and buffer pH value has been analyzed. It is suggested that cells be stained in 2.10(-5) M solution of ethidium in 0.1 M tris-HCl buffer at pH 8.0 during 30 min. The fluorescence of nuclei stained with ethidium under conditions described is shown to reflect changes in physico-chemical properties of chromatin taking place in the course of its chemical modification and physiological activation in regenerating liver. The use of ethidium for chromatin cytochemistry allows to study chromatin properties in wide ranges of pH. Some other advantages of the method suggested over the commonly used method of acridine orange staining are discussed.