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Use of the 295- to 300-nanometer circular dichroism through of ribonucleic acid to study helix winding: effect of acridine orange.

Acridine orange decreases the amplitude of the 295-nm circular dichroism (CD) trough of ribosomal ribonucleic acid (rRNA) where the trough has been related to coil character. Since acridine orange is known from earlier work to intercalate between base pairs of nucleic acids, causing an unwinding of the coil, and our studies show a decrease in the 295-nm CD trough, it appears that CD measurements may be used to observe relative unwinding of rRNA. Under similar solution conditions, melting temperatures with acridine orange indicate no significant change in the stabilization of rRNA structure by acridine orange. Hypochromicity studies show no increase in the percent base pairing in rRNA when 0.1 M tris(hydroxymethyl)aminomethane (pH 7.6) with 1.35 M KCl is used. These results indicate that CD changes in the amplitude of the 295-nm trough of rRNA are related to helix winding in rRNA.

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Gliosis and glioma distinguished by acridine orange.

Acridine orange fluorochrome of nucleic acids was applied to sections of cerebral tissue from 20 patients showing acute or chronic reactive gliosis. The results were compared with the findings in 39 well differentiated and malignant astrocytomas. The orange cytoplasmic fluorescence of ribonucleic acid is lacking in reactive astrocytes of all ages including gemistocytes, but is uniformly present in astrocytoma cells. Acridine orange is a useful supplementary stain for distinguishing between astrocytosis and astrocytoma, particularly for small cerebral biopsies showing scattered or diffusely infiltrating pleomorphic glial cells.

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A fast and easy fluorescent counterstaining method for neuroanatomical studies by using Acridine Orange.

Acridine Orange is commonly used as a fluorescent counterstain in fluorescent tract tracing techniques. Here we describe a method in which the substitution of the standard washing solutions (i.e., 0.9% saline) for a diluted solution of Acridine Orange (0.001%) during the perfusion of the animal before fixation provides a fluorescent counterstaining compatible with Fast Blue fluorescent retrograde labeling. In contrast to other fluorescent counterstaining methods, this procedure minimizes the diminution in the fluorescence of the tracer during the handling of sections.

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Cytoplasmic RNA in nervous system tumours in children: a fluorochromic histochemical study using acridine orange.

Acridine orange was used as a fluorochromic histochemical stain of nucleic acids, applied to 78 neoplasms of the central and peripheral nervous systems of 60 children. Some cases were compared with 5 adults and 4 other cases of chronic reactive gemistocytic gliosis. Opposite concentration gradients of cytoplasmic ribonucleic acid (RNA) was demonstrated in tumours of the neuronal/neuroectodermal series, and those of the glial/neuroepithelial series. Minimal AO-RNA fluorescence was seen in 8 cerebellar medulloblastomas and in a retinoblastoma; strong AO-RNA fluorescence occurred in one cerebellar medulloblastoma and in 3 primitive neuroectodemal tumours of the cerebral cortex. Intermediate intensity of fluorescence was found in neuroblastomas, and strong fluorescence was shown in well differentiated ganglioneuroma cells and in cells of chromaffin tumours. Among glial tumours, by contrast, the most anaplastic cells displayed the most RNA fluorescence, while better differentiated astrocytoma cells showed much less. Gradients also were found within some astrocytomas, corresponding to zones of relative anaplasia. Minimal or no fluorescence was detected in reactive gemistocytes or in oligodendroglioma cells. Ependymomas were weakly fluorescent and choroid plexus papillomas showed more fluorescence, similar to the findings in normal ependyma and choroid plexus. Several non-neuroepithelial tumours of the nervous system and Schwannomas also were studied. The acridine orange technique applied to either frozen or paraffin sections of nervous system tumours, has value as an adjunct in the diagnosis and grading of these neoplasms and perhaps in distinguishing reactive gliosis from benign astrocytoma.

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Curing of the killer character of Saccharomyces cerevisiae with acridine orange.

Acridine orange, an intercalating dye usually employed in the curing of bacterial plasmids, was tested for its ability to cure K1 and K2 killer strains (laboratory and wine strains). The results showed a high curing percentage of the killer character. This was demonstrated by the loss of M1 or M2 dsRNAs (responsible for toxin production and resistance to it) and because the meiotic products exhibited non-Mendelian segregation. The curing percentages varied, depending on the strain but not on the killer type, and showed similar efficiency as compared with other known curing agents.

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Increased accessibility of bases in DNA upon binding of acridine orange.

Acridine orange (AO) forms 1:1 complexes with dsDNA which are insoluble in aqueous media, exhibit red luminescence, have minimal green luminescence and resemble complexes of AO with ss nucleic acids. During formation and/or dissociation of these complexes, accessibility of DNA bases to two conformational probes, formaldehyde and diethyl pyrocarbonate is increased, suggesting that the base pairing is destroyed and DNA at least partially denatured. Adriamycin and Ellipticine, but not Ethidium Bromide exert similar destabilizing effects. The results confirm our earlier predictions based on thermodynamic calculations that the double helix undergoes destabilization upon binding an intercalator characterized by high cooperativity in interaction with ss nucleic acids. Thus, the highly cooperative ligand binding to ss sections during the "breathing" of the polymer may progressively destabilize the adjacent ds structure.

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Total tumor cell elimination with minimum damage to normal tissues in musculoskeletal sarcomas following photodynamic therapy with acridine orange.

Acridine orange (AO) has unique biological actions enabling tumor visualization (fluorovisualization) and a strong cytocidal effect (photodynamic therapy: AO-PDT) under illumination with blue light. Accordingly, in this study, we attempted to develop a new surgical technique for total tumor cell elimination using these photodynamic reactions with AO in a mouse osteosarcoma model. The results showed that local tumor recurrence was significantly inhibited (23%) in the group treated with curettage under fluorovisualization and AO-PDT, compared to that (80%) in the control group treated with curettage alone under ordinary light. Therefore, we concluded that the combination of curettage under fluorovisualization and AO-PDT may be useful for total tumor cell elimination with minimum damage to normal tissue in musculoskeletal sarcomas.

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Renal basolateral membrane SO4/HCO3 transporter characterized by fluorescent acridine orange.

Acridine orange (AO), a pH-sensitive fluorescent indicator, was used to study the characteristics of SO4/HCO3 transport in basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex. The BLMV preparation containing a low buffer concentration and preloaded with 25 mM HCO3 was mixed with buffer containing AO and SO4 in the absence of an initial pH gradient. SO4 influx tended to drive HCO3 efflux, causing intravesicular accumulation of AO and fluorescence quenching. There was no AO quenching in the absence of HCO3 with or without an external SO4 gradient. 100 microM 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) inhibited the fluorescence quenching completely. 25 and 100 mM external Cl did not cause AO quenching. There was no effect of pH (6.5-8.0) on SO4/HCO3 transport. The Kd for SO4 was 8.2 mM. A positive inwardly directed diffusion potential (K(in) = 5 mM, K(out) = 100 mM with valinomycin) did not exert any effect on the SO4/HCO3 transport, indicating that the transport process is insensitive to voltage. The Ki for DBDS inhibition of SO4/HCO3 transport was 2.3 microM.

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Aggregation of acridine orange: crystal structure of acridine orange tetrachlorozincate 2C17H19N3-2HCl-ZnCl2-CH3COOH.

The crystal structure of the biological stain, "acridine orange," has been determined. This compound, when crystallized from ethanol, is shown to be a zinc chloride double salt of acridine orange, containing, in addition, acetic acid of crystallization. These additional components are residuals from the method of preparation of acridine orange. This complex, 2 acridine orange-2HCl-ZnCl2-CH3COOH, (2C17H19N3-2HCl-ZnCl2-CH3COOH) crystallizes in the monoclinic space group P21, a = 9.965 (2), b = 21.507 (6), c = 9.645 (2) A, beta = 113.98 degrees (2), V = 1888.7 (8) A3, FW = 800.0, Z = 2, DX = 1.41 g-cm-3, Dobs = 1.43 (9) g-cm-3. Three-dimensional diffraction data were collected with CuKalpha radiation, and the structure refined to R = 0.065 for 1885 observed reflections. In the crystal structure hydrogen bonds are formed, via the protonated nitrogen atom of the central rings of two acridine orange cations, to two chloride ions in a ZnCl42- tetrahedral grouping. These two acridine orange molecules are stacked in parallel planes, approximately 3.4 A apart, with the long axes of the ring systems inclined at 26.5 to each other. Thus an apparent dimerization of the acridine, orange is facilitated by the anions present, resulting in the complex studied. The two -N(CH3)2 groups of each acridine orange molecule are not protonated in this crystalline form. The mode of molecular packing found here may be relevant to models for the external stacking of acridine orange around a DNA molecule. The importance of removing any zinc salt from acridine orange preparations prior to aggregation studies is stressed.

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Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe.

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I(-), ClO(3) (-), NO(3) (-), Br(-), or SCN(-), acridine orange reported lower pH gradients than either quinacrine or [(14)C]methylamine. Acridine orange, but not quinacrine, reduced [(14)C]methylamine accumulation when NO(3) (-) was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO(3) (-) increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24 degrees C than at 12 degrees C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H(+), possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO(3) (-)/H(+) symport involved in the export of NO(3) (-) from the vacuole is probably an artefact caused by acridine orange.

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Study on the formation and depolymerization of acridine orange dimer in acridine orange-sodium dodecyl benzene sulfonate-protein system.

Experiment indicates that the fluorescence of acridine orange (AO) can be greatly quenched by anionic surfactant sodium dodecyl benzene sulfonate (SDBS), but when protein is added into the AO-SDBS system, the fluorescence intensity of the latter is enhanced. It is considered that SDBS can promote the formation of AO dimer, resulting in the quenching of the fluorescence of AO. When bovine serum albumin (BSA) is added into AO-SDBS system, BSA and SDBS can interact and form negative micelle-like cluster complex with "aromatic ring stacking," which destroys the formation conditions of AO dimer and makes some AO dimers turn into monomer, resulting in the fluorescence enhancement of AO-SDBS system. Whereas the positive AO and residual AO dimer are dissolved in the negative BSA-SDBS cluster through electrostatic and hydrophobic forces and form a large association. Here, the fluorescence enhancement of AO-SDBS is considered to originate from the hydrophobic microenvironment provided by BSA and SDBS, the depolymerization of AO dimer and intermolecular energy transfer between BSA and AO.

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Interactions of acridine orange with nucleic acids. Properties of complexes of acridine orange with single stranded ribonucleic acid.

Interactions between acridine orange (AO) and nucleic acids (calf thymus DNA, and homoribo- and homodeoxyribo-polynucleotides) were studied in solutions containing ethanol as a cosolvent. Light absorption, scattering and luminescence were measured as a function of AO concentration at different dye/phosphate (D/P) ratios, and the data were analyzed using the McGhee-von Hippel probabilistic model of the polymer-ligand interactions. The absorption spectra of AO complexes with four homoribopolymers are presented. The intrinsic association constants and cooperativity coefficients of the formation of the complexes were calculated. The effects of ethanol (up to 35%, v/v) on these interactions were concentration dependent and may be extrapolated to zero concentration of this cosolvent. The possibility of destabilization of the double helix of nucleic acids by AO at high D/P ratios is discussed in light of the available thermodynamic data.

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[The luminescence of blood leukocytes, stained with the fluorochrome acridine orange, from surgical patients in the dynamics of emotional, anesthesiological and surgical stress].

Luminescence of blood leukocytes of surgical patients during treatment was studied using orange acridine. Luminescence intensity at 640 nm wave length (I640) was the most informative. It characterized amount of single-stranded nucleic acids, synthesized in cell due to previous activation of protein-synthesizing system and hence cell functional activity, while in lymphocytes these changes exceeded by duration and amplitude those in neutrophils. During premedication (emotional stress) I640 was minimal, during anesthetization it developed, and sharply raised during traumatic period of surgery and after it, as reflection of immune system participation in reparative regeneration.

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Differentiation of the various stages of Blastocystis hominis by acridine orange staining.

Acridine orange staining differentiates the cystic and the central body forms of Blastocystis hominis and offers a very convenient and easy method to observe the internal structure of the parasite. Acridine orange stains the nuclei and the central body of the rounded vacuolar forms of the parasite bright and dull green, respectively. The colour changes to yellow and then to flaming red-orange when the rounded central body forms of the parasite become cystic.

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[Distribution of RNA during neuronal migration in developing normal and dysplastic brains in man. Acridine orange study].

Acridine orange, a fluorochrome stain of nucleic acids, was used to study neuronal maturation in human brains during development. The central nervous system in 15 normal fetuses and neonates of 16 to 44 weeks gestational age were examined, as well as 24 dysplastic brains exhibiting abnormal cellular migrations. The increase in cytoplasmic RNA of neurons coincides with the onset of neurotransmitter synthesis, temporally synchronized to begin after migration in the cerebral hemispheres but before migration of the external granular layer of the cerebellum. The presence or absence of orange fluorescence in heterotopic nerve cells serves as a marker of the state of maturity by indicating whether the cell was still migrating at the time of death or had already arrived at its definitive site in the brain, whether normal or abnormal. Furthermore, the type of neurotransmitter produced by a given neuron influences ribosomal concentration and hence the intensity of orange RNA fluorescence. Neurons that secrete peptides, acetylcholine, or monoamines show stronger fluorescence than do those synthesizing simple amino acids as transmitters. Transitory transmitters of developing brain may account for stronger fluorescence in certain neurons of the fetus than in the adult, an example being the granule cells of the cerebellar cortex. Acridine orange is a useful supplementary tool in pediatric neuropathology.

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Microabscesses in gastric biopsies shown by acridine orange staining.

Acridine orange staining of endoscopic biopsies of gastric mucosa was used in 70 patients with various upper gastrointestinal symptoms to identify Campylobacter pylori abscess formations with polymorphonuclear leucocyte infiltration. In comparison with cultures, the staining test proved to be a rapid and reliable test particularly in outpatient clinics as results are available in 6-8 min, thus enabling any necessary treatment to start immediately.

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