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The ultracytochemical acridine orange(AO) method has been employed to demonstrate DNA template activity in the developing cerebellar cortex in rats and its alteration after transplacental administration of ethylnitrosourea (ENU). Electron microscopic studies revealed that AO binds to DNA exclusively within the active extended euchromatin portion of the cell nucleus of the proliferating external granular layer. No or only a few AO reaction products were visible in the non-proliferating cell nuclei of the internal granular layer and of Purkinje cells. With increasing cellular differentiation of granule cells in the cerebellar cortex the number of AO positive cells as well as the number of AO reaction products per cell showed a progressive decrease. A single i.p. injection of 50 mg/kg of ENU in rats on day 15 to 21 of gestation resulted in occasional cell damage to the proliferating external granule cells of developing cerebellar cortex at 4 and 7 days after birth. Damaged cells of the external granular layer were found to have no or only a small number of AO reaction products in their nuclei. In further development from 14 to 28 days the cerebellar cortex of offspring from ENU-treated mother showed no abnormality and exhibited no definite alteration in distribution pattern of AO reaction products.

We describe protocols for the fractionation of isolated hepatocytes into eight sub-populations using centrifugal elutriation. The distribution of fluorescein isothiocyanate and acridine orange in hepatocytes prepared from livers pre-perfused with one of these dyes is described and used as an indicator of acinar zone derivation for each population. The cytochrome P-450 content and response to induction by 3-methylcholanthrene and phenobarbitone; the distribution of lactate dehydrogenase, glucose-6-phosphatase, pyruvate kinase and tyrosine aminotransferase activities in the sub-populations is also reported. A marked asymmetry of distribution in all these activities was observed. On the basis of putative zone derivations (based on data of fluorescent dye distribution) of eight factors studied, the distributions of six were consistent with the sub-populations being derived from different acinar zones. Two major discrepancies were noted however, the distribution of pyruvate kinase activity and the response of the sub-populations to phenobarbitone. We conclude from this study that while a metabolic heterogeneity was revealed in the sub-populations generated, further characterisation is required to determine whether acinar zone separation has occurred and if so to what extent.

Fluorescence quenching of acridine orange was used to characterize the generation and collapse of pH gradients by the Na+/H+ antiporter of brush border membrane vesicles prepared from rabbit renal cortex. Quenching was observed when acridine orange, a weak base, was taken up to an acidic intravesicular space. Na+/H+ exchange was examined with both Na+ uptake and efflux studies. Acridine orange fluorescence quenching demonstrated the cation specificity of the Na+/H+ antiporter (i.e., sodium and lithium) and was inhibited by amiloride. Parallel studies with nigericin, a K+/H+ antiporter, demonstrated that acridine orange responded very rapidly to pH gradients. Therefore, acridine orange equilibration was not rate limiting in our studies of the Na+/H+ antiporter. Initial rate measurements were made to obtain kinetic parameters for the Na+/H+ antiporter. In sodium influx studies, the half-maximal rate of acridine orange fluorescence change was obtained with an external sodium concentration of 13.3 +/- 0.5 mM.

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.

Uptake and release of acridine orange (AO), a fluorescent weak base that accumulates in acidic spaces, were studied in perfused frog gastric mucosa. Tissue was mounted between two flow-through chambers and loaded with AO on the mucosal side. AO washout and acid secretion rate were monitored simultaneously by a flow-through fluorescence detector and a pH-stat, respectively. Data were displayed on a computer screen, stored, and analyzed. AO, in concentrations as high as 0.02 mM, does not affect the acid secretion rate. Nonlinear least-squares analysis of AO washout curves resolved two exponential components: a faster component associated mainly with AO washout from the chamber and a slower component reflecting primarily AO washout from the tissue. The slower exponential declines more slowly at higher concentrations and/or longer duration of AO loading, whereas the faster exponential is unaffected. AO washout is unaffected by the level of the steady-state acid secretion rate. Nitrite inhibits the acid secretion rate but does not affect the AO washout. When nitrite is removed, acid secretion rate and fluorescence (AO concentration in the mucosal medium) increase simultaneously and transiently. The net amount of AO released from the tissue is proportional to the net amount of acid released. Stimulation by secretagogue in basally secreting tissue causes synchronous transient increases in acid secretion rate and fluorescence. We conclude that accumulation of AO provides evidence for the existence of an intracellular storage pool of free protons within the transporting epithelium.

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.

The binding of acridine orange and ethidium bromide to rat liver chromatin increases by 30% one hour after partial hepatectomy, returns to the control level by the second hour and increases again by the sixth hour. The changes described were found in investigations carried out on whole cells, on isolated nuclei, and on chromatin preparation in vitro. Increased ligand binding disappears after the treatment of the one-hour chromatin with a 0.3 M NaCl solution, but such a treatment does not change the binding of ligands to chromatin obtained six hours after hepatectomy. The one-hour chromatin is characterized by elongation of distances between individual nucleosomes whereas the two-hour chromatin is the same as in control.

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.

The thermodynamic binding parameters for intracellular heparin-acridine orange (AO) complexes were determined for Furth murine mastocytoma cells and were found to agree with 1) results from binding studies on heparin-AO complexes in solution, and 2) with biochemical analyses of the cells. The cells exhibited cooperative binding with a binding constant of 1.18 x 10(6) M-1. The cooperative binding constant of heparin-AO in 1 mM buffer was found to be 1.13 x 10(6) M-1. The addition of 1 mM NaCl to heparin-AO system in vitro detectably decreased the cooperative binding constant. Low ionic strength is the only condition in solution under which the cell and solution binding constants are equal. The cells have an average of 1.2 x 10(-14) mol of AO binding sites per cell. Using the biochemically measured heparin content per cell and the amount of AO bound by heparin in solution, 8 x 10(15) mol of sites/cell can be attributed to heparin. The remaining cellular binding sites (4 x 10(-15) mol of sites per cell) are essentially all accounted for by AO binding to DNA, the amount of which is calculated from its previously determined thermodynamic binding parameters. A theoretical isotherm, calculated from the binding parameters of both heparin-AO in solution and DNA-AO complexes in situ, agreed closely with the isotherm experimentally determined for the Furth mastocytoma cells. Ligand-binding analysis yields a binding constant, which may aid in identification of cellular bipolymers, and the number of ligand binding sites per cell. The latter is a measure of the amount of a given intracellular biopolymer present.

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.

We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+ transport specific activity was shown by column chromatograpy to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+ transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 15 mM), but was relatively insensitive to cation replacement as long as Cl- was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol greater than potassium gluconate greater than KCl) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+ transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl- salts and KNO3. Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+ transport activity similar to that of the coated vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Two hypotheses have recently been proposed for the thiocyanate inhibition of gastric acid secretion--a protonophore mechanism and an uncoupling model. The mechanistic aspects for the latter scheme have been examined on the following basis: capability of generating verifiable predictions, supporting evidence that is unambiguous, and compatibility with experimental realities. Gastric microsomes bind 5 nmol of SCN-/mg, and a "pure" and highly active fraction of H+,K+-ATPase prepared from gastric microsomes binds about 15 nmol of SCN-/mg. The affinity of SCN- binding to gastric microsomes changes from 10 to 25 mM in the presence of 20 mM K+ suggesting competition between K+ and SCN-. Potassium also displaces the bound SCN- from "pure" H+,K+-ATPase with a Ki of about 25 mM. Of the cations tested--Tl+, K+, Rb+, Cs+, NH4+, Na+, and Li+--Tl+ was the most effective in displacing bound SCN- while Na+ and Li+ were without effect. The effects of anions such as Cl-, NO3-, and gluconate were found to be nonspecific and absolutely dependent on K+ as cocation. Sulfate and OCN-, on the other hand, showed an ability to displace SCN- as both K+ and Na+ salts. For SO4(-2) the K+ form was much more effective than the Na+ salt. Besides these antagonistic effects of K+ and congeners with the H+,K+-ATPase-bound SCN-, a competition between K+ and SCN- was also observed at the level of gastric K+-stimulated pNPPase reaction. The effects of SCN- and two other unrelated anions, F- and NO2-, on artificial delta pH across the microsomal vesicles exhibited a lack of appreciable change up to 5 mM and a small (about 13%) reduction between 10 and 20 mM. However, a combination of CCCP and nigericin or valinomycin completely abolished the delta pH under identical conditions. The present data in conjunction with other reports suggest that the proton impediment model best explains the gastric antisecretory effects of SCN-.

The purpose of this study was to examine the distribution pattern of acridine orange (AO) binding to DNA in transformed cells and derived tumor cells. Two cell clones from hamster embryonic fibroblasts (HEF) transformed by herpes simplex virus type 2, tumor cells derived from the transformed cell clones, and normal HEF in culture were processed for ultracytochemistry. Uptake studies and autoradiography using [3H] thymidine and [3H]-uridine were also performed. Ultrastructural examination revealed that AO became bound to DNA exclusively within the euchromatin portion of the cell nucleus. The percentages of AO-positive cell nuclei were 18.0% and 15.5% in transformed cell clones and 21.3% and 18.3% in the derived tumor cells, but only 2. 7% in the HEF. The average amounts of AO-chromatin interaction products increased in the transformed and tumor cells. Labeling indices of [3H] thymidine were 28.9% and 33.2% in both the transformed cell clones, 32.4% and 36.9% in their tumor cells, and 3.7% in the HEF. Euchromatin/heterochromatin (EU/HEF) ratios were found to be higher in AO-positive nuclei than in AO-negative nuclei in each cell line. The increase in the number of AO-positive nuclei and in the EU/HET ratios may represent a basic reaction of cell nuclei in transformation and tumorigenesis.

Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.

The presence of membrane vesicles containing an ATP-driven H+ pump was demonstrated in rat kidney cortex homogenate using the delta pH-sensitive dye acridine orange (AO). These vesicles were purified by differential and Percoll density gradient centrifugation. ATP-driven H+ uptake was about 20-fold enriched compared with the homogenate. Determination of marker enzyme activities indicated that these vesicles do not originate from brush border and basolateral membranes, lysosomes, endoplasmic reticulum, mitochondria, Golgi membranes, or red blood cells. The identity with brush border membranes was further excluded by the absence of Na+-H+ exchange. Renal cortical endocytotic vesicles that had taken up horseradish peroxidase or fluorescein isothiocyanate-labeled dextran (FITC-dextran) after injection of these substances into rats in vivo comigrated with the H+ pump activity on the Percoll gradient. Similar characteristics of the H+ pump demonstrated by the AO method and by fluorescence changes of in vivo trapped FITC-dextran proved the identity of H+ pump-containing vesicles with endocytotic vesicles. ATP-driven H+ uptake into endocytotic vesicles was stimulated by Cl- and weakly inhibited by oligomycin. N-ethylmaleimide, dicyclohexylcarbodiimide, and Dio-9 were stronger inhibitors. Histochemical studies revealed that horseradish peroxidase-filled endocytotic vesicles are localized in the apical region of proximal tubule cells. An H+ pump with similar characteristics, but much lower activity, was found in brush border membranes, basolateral membranes, and mitochondria isolated by standard techniques, suggesting a possible contamination of these preparations with endocytotic vesicles.

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.

Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme.

The title compound is now undergoing Phase I clinical trials as an experimental antitumor agent. Fluorescent polarization was used to determine the extent of inhibition of the binding of acridine orange to DNA. Displacement of 50% of the acridine orange was achieved by 0.21 microM of the title compound, 0.32 microM daunorubicin, 0.41 microM doxorubicin, 0.61 microM dactinomycin, and 7.8 microM cis-platinum. Binding inhibition was essentially equivalent with natural DNAs (calf thymus and Micrococcus luteus) and synthetic polymers [poly d(A):d(T) and poly d(G):d(C)] of widely differing adenine plus thymidine content.

The ultracytochemical acridine orange (AO) method has been employed to demonstrate DNA template activity within untreated human brain tumors. A total of 16 biopsies from brain tumors removed for frozen section examination were investigated. Ultrastructural examination revealed that AO binds to DNA exclusively within the extended euchromatin portion of the cell nucleus of brain tumors. The percentage of AO positive cells varied from 6.0 to 48.6% in various types of brain tumors. A few AO reaction products were visible in the nuclei of the endothelial cells and in the pericytes of the capillaries within neoplastic tissue. The present results suggested that untreated human brain tumors exhibit de-repression of DNA template normally repressed in the adult state. This altered DNA templates may be related to abnormal cell proliferation and differentiation diverted from the normal state.

Na+-stimulated H+ movement and H+-stimulated Na+ uptake were studied in basolateral (blLPM) and canalicular (cLPM) rat liver membrane vesicles. H+ movement was monitored with the fluorescent amine acridine orange; 22Na uptake was assayed by a rapid Millipore filtration technique. In blLPM, inwardly directed Na+ gradients stimulated H+ efflux and outwardly directed Na+ gradients stimulated proton influx. Outwardly directed proton gradients (pH in 5.9/pH out 7.9) stimulated initial 22Na uptake rates 5- to 10-fold over pH-equilibrated conditions (pH in 7.9/pH out 7.9). Conversely, inwardly directed proton gradients (pH in 7.9/pH out 5.9) inhibited 22Na uptake. pH-dependent 22Na uptake was inhibited by amiloride and harmaline but not by other transport inhibitors, bumetanide, furosemide, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and acetazolamide. Lithium also inhibited H+-stimulated 22Na uptake. Although a component of pH-stimulated 22Na uptake appeared to be dependent on membrane potential, this electrogenic component was amiloride insensitive. Proton gradient-stimulated 22Na uptake in blLPM was saturable, with a Km of 5.4 mM and a Vmax of 14 nmol . min-1 . mg prot-1. In contrast, in cLPM, no Na+ gradient-stimulated proton movement and no pH-dependent Na+ uptake occurred. These findings establish an electroneutral Na-H antiport in blLPM but not cLPM in rat liver. The polarity of this exchanger supports a model of bile formation that is dependent, in part, on canalicular HCO-3 and/or OH- excretion.

Fluorescence flow cytometry was used to generate DNA histograms of acridine orange stained leukemic cell populations in G0-G1 phase of the cell cycle. Complexes of the intercalating agent, acridine orange, with double-stranded DNA in situ, emit green fluorescence upon excitation with blue laser light. The histograms were evaluated by first determining the standard deviation of the fluorescence intensity relative to the mean channel of fluorescence, i.e., the coefficient of variation, and then dividing the coefficient of variation of a patient's sample by that of a control sample (rCV). The mean rCV of cell populations of acute lymphoblastic leukemia (31 patients) differed significantly from that of nonlymphoblastic leukemia (21 patients). When cells were treated with a solution of citric acid and magnesium sulfate prior to their staining with acridine orange, the mean rCV of cell populations of acute lymphoblastic leukemia increased while that of acute nonlymphoblastic leukemia decreased compared to their respective pretreatment values. The mean difference of rCVs between untreated and treated cells (rCVD) within each disease category was statistically significant. A logistic regression model, based on rCVD, confirmed the conventional classification of acute lymphoblastic leukemia and acute nonlymphoblastic leukemia cells in 90% of the cases.

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 purpose of this study was to examine the effects of actinomycin D on localization of acridine orange (AO) binding to DNA in rat astrocytoma C6 cells and to discuss briefly the significance of AO chromatin interaction products. Actinomycin D markedly inhibited 3H-uridine incorporation into RNA and the percentage of AO positive cells was reduced to approximately 40% of that of the untreated control cells, whereas no distinct decrease of 3H-thymidine incorporation was induced by actinomycin D. Electron microscopic radioautography combined with the AO ultracytochemistry revealed that silver grains indicating binding of 3H-actinomycin D are located mostly over the euchromatin portion near the segregated nucleolus and heterochromatin and that no or only a few AO chromatin complex was found in nuclei labeled heavily with 3H-actinomycin D. These results of the present study together with the results of the previous studies seem to indicate that AO might selectively bind to active or derepressed DNA template sites for DNA dependent RNA polymerase in the euchromatin portion of the cell nucleus.

Mouse lymphocytes were activated by a mitogenic dose of concanavalin A and analysed by flow cytometry to monitor the increases of mitochondrial activity (using rhodamine 123 as probe) and of lysosomal activity (using euchrysin [acridine orange] as probe). Cyclosporin A-treated lymphocytes were not capable of responding to concanavalin A in the same way as untreated lymphocytes: both the increased uptakes of rhodamine 123 by mitochondria and of acridine orange by lysosomes were strongly diminished, though not abolished. Cyclosporin may thus interfere at a step of activation prior or concurrent to those early changes of lymphocyte physiology. It looks like that it allows mitogen-activated cells to go through part of the mitochondrial maturation which precedes initiation of nuclear DNA synthesis, after which the cells remain blocked at that incomplete maturation level.

A procedure was devised for fractionating crude thyroid lysosomal particles (P750-15,000) by self-forming density gradient centrifugation with colloidal silica. Two discrete particle-containing peaks were observed, based on 131I-labeling and acid phosphatase activity: a heavy peak (density, 1.11-1.12) and a light peak (density, 1.05). Ultrastructural analysis revealed that the heavy peak consisted almost entirely of lysosomes, whereas the light peak represented a heterogeneous mixture of small vesicles and fragments of other intracellular organelles. In thyroids removed from rats 30 min after 131I injection, almost all of the 131I was present in the low density peak. This 131I appeared on sucrose density gradient centrifugation as a 19S peak, and it was almost completely insoluble in trichloroacetic acid. This was interpreted as indicating that the low density peak contained pinocytotic vesicles. In thyroids removed 4 days after 131I injection, the radioactivity appeared largely in the high density peak. Both the trichloroacetic acid solubility and the pattern on sucrose density gradient centrifugation indicated that the [131I] thyroglobulin had undergone extensive proteolysis. Thyroglobulin proteolytic activity was found primarily in the high density particles and to only a small extent in the low density particles. Studies performed at intervals after 131I injection combined with double labeling (131I and 125I) experiments provided evidence that radioactivity was transferred from the low density to the high density particles. Heterogeneity existed within the dense peak, related to the degree of thyroglobulin degradation, as it was observed that thyroid lysosomes become denser with increasing proteolysis of thyroglobulin. The acid phosphatase in the low density particles could be distinguished from that in the high density (lysosomal) particles by its elution pattern on Sephadex G-200 column chromatography, its response to freezing and thawing, and its reactivity with p-nitrophenylphosphate. It was concluded, therefore, that the acid phosphatase in the low density fraction was derived from prolysosomal structures such as vesiculated Golgi-endoplasmic reticulum-lysosomes. The prolysosomal acid phosphatase associated with the low density fraction appeared to be a large membrane-bound molecule which could be transformed into lysosomal acid phosphatase by incubation at pH 5.0.

Megamitochondria, resulting from cuprizone feeding of Swiss ICR mice, were fluorescent in hepatocytes after the intravenous injection to mice of a liposome-encapsulated acridine orange-DNA complex (AO-DNA). Flow cytofluorimetric analysis of isolated megamitochondria showed that the proportion of liposome-encapsulated AO-DNA which localized in megamitochondria increased from 0.02% of the dose injected per liver cell at 3 min after injection to an average of 0.34% at 1 h after injection. Megamitochondria showed negligible fluorescence by fluorescence activated cell sorting (FACS) analysis when free AO-DNA was intravenously injected. Transmission electron micrographs of mouse liver tissue after intravenous injection of liposomes encapsulating iron dextran showed an association of the liposomes with megamitochondria which appeared identical to liposome association with normal mitochondria. These results support and extend our earlier observation that a fraction of the liposomes injected intravenously into mice associate with mitochondria in the liver, and possibly deliver their aqueous contents there.

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.

Lysosomes of living human fibroblasts, SV40-transformed rat fibroblasts and human CMV-transformed hamster fibroblasts were examined by fluorescence microscopy after pretreatment with acridine orange at a supravital concentrations (5 x 10(6) M). Dye uptake by human primary fibroblast lysosomes was considerable and independent of the age of the cultures. In the transformed cultures, cytoplasmic granular red fluorescence indicating lysosomal acridine orange uptake could not be observed in part of the cells; cells showing no cytoplasmic granular fluorescence appeared as early as after 48 hr incubation and were growing in dependence on the age of the culture. Staining of living cells by acridine orange solutions at supravital concentration is a practicable method for the examination of functional changes of lysosomes.

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.

The influence that changes in proton distribution have on the fluorescence of acridine orange was examined using negatively charged liposomes. Our results indicate that at least two mechanisms are involved: distribution of the probe between the internal aqueous phase of the liposomes and the outside medium, and binding of the probe to the liposome membranes.

Spectral properties of acridine orange (AO) alone or in complexes with natural and synthetic nucleic acids of various base composition have been studied in aqueous solutions by absorption and fluorescence spectroscopy. The dimerization constant and absorption spectra of the dye in monomeric and dimeric form were established; dimerization of AO resulted in quenching of its fluorescence. Complexes of the dye with synthetic nucleic acids differed in the degree of enhancement of fluorescence quantum yield, varying between 1.42 to 2.38 fold as compared to AO monomer; these differences, however, were not base-dependent. Affinity of the dye to natural and synthetic polymers was studied and analyzed using McGhee-von Hippel model of polymer-ligand interactions. Because the sterical requirement for intercalative binding assumes interaction of dye monomer, the correction for AO dimerization was made in all calculations. All studied DNAs (natural and synthetic ones, the latter being homopolymer pairs or alternating copolymers of A,T or G,C or I,C base composition) had similar intrinsic association constants (KI = 5 X 10(4) - 1 X 10(5), M-1) and binding site size (n = 2.0-2.4 b.p.). The exception was poly(dA).poly(dT), having KI = 1.2 X 10(4) and n = 19.3 b.p. The results of KI measurement for calf thymus DNA and AO in different sodium ion concentration were in good agreement with predictions of the counterion condensation theory. The intercalation of AO into DNA is discussed in view of recent theoretical models of DNA-ligand interactions.

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.

Several metals are known mutagens and carcinogens. These metals effectively displace acridine orange from DNA when measured by fluorescence polarization. Displacement of 50% of the acridine orange is obtained with less than 0.5 mM concentrations of lead, manganese, cobalt, zinc, cadmium, nickel, iron, copper, and cis-platinum. In contrast, greater than 80 mM concentrations of lithium, sodium, and potassium are required to displace an equivalent amount of acridine orange from calf thymus DNA. Although cis-platinum shows the best DNA reactivity in this assay, the interaction between this metal and DNA does not occur immediately, as it does for the other metals tested. These results indicate the acridine orange displacement assay provides a relative measure of the interaction of metals with DNA, and this DNA reactivity shows a positive correlation with mutagenic/carcinogenic potential.

The purpose of this study was to examine effects of aphidicolin and alpha-amanitin on visualization of acridine orange (AO) binding to DNA in rat astrocytoma C6 cells and to discuss briefly the significance of AO chromatin interaction products. Aphidicolin inhibited DNA synthesis but percentage of AO positive cells was approximately 60% of that of the untreated control cells. alpha-Amanitin caused a slight inhibition of RNA synthesis and 3H-uridine incorporation in the treated cells was about 64% of that of the untreated cells, whereas a distinct decrease of the number of AO positive cell nuclei was observed. The results suggest that activity of RNA polymerase II and mRNA synthesis is mainly concerned in visualization of AO chromatin interaction products.

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.

We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.

Concanavalin A (conA) inhibits the degradation of [3H]leucine-labeled cellular proteins of human lymphocytes. The lectin also stimulates the migration of non-histone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H]actinomycin D [(3H]AD). Decreased protein breakdown and increased migration of NHP are parallel events, i.e. both changes occur as a function of the lectin concentration and display a similar time course, suggesting that these events could be related. Similar effects are observed with fluoride, chloroquine and iodoacetate: these agents simultaneously decrease proteolysis and increase the nuclear level of NHP, associated with increased cellular [3H]AD binding. Fractionation of the acidic NHP according to pH 2.5-6.5 shows that proteins with a high degree of degradation in unstimulated cells correspond to proteins with a high degree of migration in conA-stimulated cells. A similar correlation was observed in fluoride-treated lymphocytes. conA, fluoride and iodoacetate decrease cellular [3H]chloroquine [(3H]CQ) accumulation, indicating a lysosomotropic effect. These and previously reported data suggest, but do not prove that conA inhibits degradation of cellular proteins via the lysosomal pathway. Ammonium chloride, methylamine and sodium azide also inhibit proteolysis and increase cellular [3H]AD binding; however, their effects are weak. On the basis of these observations it appears that lysosomal degradation and migration of NHP to the nucleus are linked; however, the mechanism of the linkage is unknown.

The radioactive amine [14C]methylamine is accumulated to a great extent by eggs, with kinetics that are dependent upon temperature (Q10 = 5) and sensitive to metabolic inhibitors. Efflux of [14C]methylamine from eggs (preloaded with tracer concentrations) is increased immediately after fertilization or NH4Cl activation. Fluorescent amines (9-aminoacridine (9AA) and acridine orange (AO)) are concentrated in small intracellular compartments, presumably vesicles. The possible role of these vesicles in the accumulation of amines by sea urchin eggs and the activation of the metabolism that ensues is discussed.

The increase in the fluorescence polarization of acridine orange upon binding to DNA molecules is used as the basis of a competitive method to study the interaction of a variety of fluorescent and non-fluorescent compounds with DNA. Test compounds that interact with DNA inhibit both the binding of acridine orange to DNA and the accompanying increase in fluorescence polarization. Actinomycin D exhibits a dose-dependent inhibition of acridine orange-DNA binding with Micrococcus luteus DNA, calf thymus DNA, and poly(dG-dC); no detectable inhibition of acridine orange intercalation into poly(dA-dT) is observed. In contrast, proflavine shows similar acridine orange inhibition for poly(dA-dT), calf thymus DNA and M. luteus DNA.

By using the 'acidotropic' vital dye, Acridine Orange, we have found that the presynaptic terminals of rod and cone photoreceptors in retinas of Rana pipiens maintain a low pH relative to the surrounding medium through an energy dependent mechanism. When this pH is raised, by exposing the retinas to weak bases like ammonium chloride, the terminals exhibit large, membrane-delimited compartments, many of which accumulate endocytic tracers. This effect is partly reversed when the weak bases are removed. We infer that among the acidified structures within the terminals are endocytic compartments with at least some of the characteristics of the endosomes that participate in receptor-mediated endocytosis in other cell types. One role of these structures in the terminals may be in the recycling of synaptic vesicles.

The uptake of the fluorescent, lysosomotropic weak base acridine orange (AO) by living cells in culture was studied by flow cytofluorometry. A mouse myeloma cell line (SP 2/0), growing in suspension, and an anchorage-dependent human malignant glioma cell line (U-251 MG), brought into suspension by trypsinization, were used. The consequences of trypsinization were also studied using static cytofluorometry. The lysosomal accumulation of AO by myeloma cells growing in suspension was found to be only moderately affected by starvation (i.e. incubation without medium change) for a period of up to five days. Trypsinization of the glioma cells after staining with AO caused pronounced release of the fluorescent dye while trypsinization before staining with AO did not significantly change the average lysosomal concentration of AO. We did, however, notice certain side effects of trypsinization in the form of both increased cellular green fluorescence and greater intercellular variability that reduce the validity of data obtained from cells detached by routine trypsinization. In conclusion, the condition of the lysosomal vacuome of living cultured cells growing in suspension may be studied by flow cytofluorometry after vital staining with the lysosomotropic weak base AO. Anchorage-dependent trypsinized cells, however, yield unsatisfactory results when examined in a flow cytofluorometer system and are better studied while still attached to their substratum, using static cytofluorometry.

The use of the supravital mitochondrial-specific dye Rhodamine 123 (Rh 123) in combination with flow cytometry permits the monitoring of the changes in the mitochondrial transmembrane potential, reflecting the overall mitochondrial activity of the living cell. While this probe appears to be a potent tool for these studies, it also exhibits an important limit in the interpretation of the results: it cannot distinguish between an increase in mitochondrial activity without biogenesis and a modification of mitochondrial content. 10-n-Nonyl Acridine Orange chloride (NAO) constitutes another mitochondrial specific fluorochrome. In contrast with Rh 123, NAO accumulation in the cell does not seem to be driven by the proton-motrice force but does seem to be related to specific interactions with mitochondrial membrane proteins and/or lipids. In this work, the cytotoxicity of NAO, the kinetics of cellular uptake and the release of the dye have been determined using flow cytometry. The use of several ionophores or mitochondrial inhibitors has confirmed the independence of NAO uptake regarding mitochondrial transmembrane potential. NAO was also used to examine the changes in the mitochondrial compartment during the transfer of articular chondrocytes from cartilage to the culture conditions, where Rh 123 evidenced changes in mitochondrial activity and/or biogenesis, in order to know whether the use of probes with different specificity allows one to distinguish between mitochondrial activity and biogenesis.

The nematode Caenorhabditis elegans contains autofluorescent lipofuscin granules, located exclusively in the 32-34 intestinal cells. Using epifluorescence microscopy on live adult animals, we have shown that fluorescent-labeled exogenous probes are taken up by endocytosis and accumulate within the granules. Macromolecular solutes such as proteins and dextran appear to be taken up by fluid-phase pinocytosis. There is no phagocytosis of latex particles with diameter greater than or equal to 0.25 micron. The granules concentrate the lysosomotropic weak base acridine orange, indicating that they have an acidic internal milieu. These observations imply that the lipofuscin granules in the intestinal cells are secondary lysosomes which remain active recipients of endocytosed materials.

The development and hatching of Ancylostoma ceylanicum and Ancylostoma tubaeforme eggs in a range of potentially inimical solutions has been examined using time-lapse video micro-recording techniques. In all compounds tested the larvae developed to the active pretzel stage. The inception of activity resulted in an increase in shell permeability to water and small molecules. In anthelmintics of molecular weight (Mr) 400 development continued normally until about the time that control eggs hatched, when there was a marked decrease in larval activity and eclosion did not occur. The addition of a low Mr surface-active agent potentiated the effect of the anthelmintic and caused larval activity to be lost earlier. The calcium inhibitor lanthanum chloride affected larval development rather than eclosion. Large Mr compounds did not affect either development or hatching and the results suggest that the egg-shell may act as a form of sieve, allowing the essential oxygen to reach the developing larva while excluding large potentially dangerous molecules. No appreciable differences were found between the two species of hookworm examined.

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 microM in about 10 minutes at 37 degrees C. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 microM of the Ca++-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++-containing physiological saline, the addition of A23187 (at a final concentration of 8 microM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 microM A23187 in Ca++-containing saline completely abolished NH4+-sensitive dye uptake; less A23187 (0.5 microM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a delta pH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)-H+ exchange.