Acridine Orange DNA Research Articles

Differentiating cancer cells reveal early large-scale genome regulation by pericentric domains

Finding out how cells prepare for fate change during differentiation commitment was our task. To address whether the constitutive pericentromere-associated domains (PADs) may be involved, we used a model system with known transcriptome data, MCF-7 breast cancer cells treated with the ErbB3 ligand heregulin (HRG), which induces differentiation and is used in the therapy of cancer. PAD-repressive heterochromatin (H3K9me3), centromere-associated-protein-specific, and active euchromatin (H3K4me3) antibodies, real-time PCR, acridine orange DNA structural test (AOT), and microscopic image analysis were applied. We found a two-step DNA unfolding after 15–20 and 60 min of HRG treatment, respectively. This behavior was consistent with biphasic activation of the early response genes (c-fos - fosL1/myc) and the timing of two transcriptome avalanches reported in the literature. In control, the average number of PADs negatively correlated with their size by scale-free distribution, and centromere clustering in turn correlated with PAD size, both indicating that PADs may create and modulate a suprachromosomal network by fusing and splitting a constant proportion of the constitutive heterochromatin. By 15 min of HRG treatment, the bursting unraveling of PADs from the nucleolus boundary occurred, coinciding with the first step of H3K4me3 chromatin unfolding, confirmed by AOT. The second step after 60 min of HRG treatment was associated with transcription of long noncoding RNA from PADs and peaking of fosL1/c-myc response. We hypothesize that the bursting of PAD clusters under a critical silencing threshold pushes the first transcription avalanche, whereas the destruction of the PAD network enables genome rewiring needed for differentiation repatterning, mediated by early response bivalent genes.

Analysis of age‐associated alternation of SCSA sperm DNA fragmentation index and semen characteristics of 1790 subfertile males in China

BackgroundIt has been identified that incidence of infertility was about 20% among couples worldwide, about 50% caused by male elements. However, conventional semen laboratory detections could not handle clinical needs, which led to more comprehensive parameters for male fertility evaluation. We aimed to investigate the clinical relationship of age‐linked changes and the sperm chromatin structure assay (SCSA) sperm DNA fragmentation index (DFI), and routine semen characteristics among subfertile Chinese males.Methods1790 clinical semen specimens were enrolled from February 2018 to October 2019. Clinical and laboratory data including routine semen analyses, sperm DFI, and sperm morphology were collected and showed age‐related alterations in semen parameters.ResultsOur results, displayed an increase in sperm DFI with age, were demonstrated in three age‐groups, particularly within the ≥35‐year cohort. There were positive and inverse correlations of sperm DFI with abnormal semen characteristics and with normal morphological parameters, respectively. Furthermore, age, sperm morphology, concentration, and progressive motility, immotile sperm percentage, semen volume, sperm survival, and high acridine orange DNA stainability (indicating immature forms) were found to be independent risk factors affecting sperm DNA integrity. Likewise, men aged ≥35 years had a higher sperm DFI than did normozoospermic men in the overall cohort. Routine semen characteristics, sperm DFI, and morphology tended to alter with age.ConclusionsThe SCSA sperm DFI showed the greatest clinical application in the assessment of male fertility in this study, which should help infertility clinics decide on reproductive options for the treatment of older infertile couples.

A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay

We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.

Over expression of glutamate cysteine ligase increases cellular resistance to H2O2‐induced DNA single‐strand breaks

Hydrogen peroxide (H2O2) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H2O2 reduction varies with cell and tissue type. The role of the GPx cofactor glutathione (GSH) in oxidative defense can be further understood by modulating its synthesis. The first and rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which has a catalytic subunit (Gclc) and a modifier subunit (Gclm). Using mouse hepatoma cells we evaluated the effects of GCL over expression on H2O2-induced changes in GSH and ssDNA break formation with the single cell gel electrophoresis assay (SCG or comet assay), and the acridine orange DNA unwinding flow cytometry assay (AO unwinding assay). Cells over expressing GCL had higher GSH content than control cells, and both SCG and AO unwinding assays revealed that cells over expressing GCL were significantly more resistant to H2O2-induced ssDNA break formation. Furthermore, using the AO unwinding assay, the prevalence of H2O2-induced breaks in different phases of the cell cycle was not different, and the degree of protection afforded by GCL over expression was also not cell cycle phase dependent. Our results support the hypothesis that GCL over expression enhanced GSH biosynthesis and protected cells from H2O2-induced DNA breaks. These results also suggest that genetic polymorphisms that affect GCL expression may be important determinants of oxidative DNA damage and cancer.

Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa

The formation of mature sperm involves the expression of numerous proteins during spermiogenesis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse consequences to the offspring. Adult male rats were given CPA in one of two schedules: (i) subchronic, 4 days - day 1 (100 mg kg(-1)) and days 2-4 (50 mg kg(-1) per day) or (ii) chronic - daily (6.0 mg kg(-1) per day). Animals were euthanized on days 14, 21 or 28. The effects of CPA on epididymal sperm chromatin structure were germ-cell-phase specific; mid-spermiogenic spermatids were most sensitive. The acridine orange DNA denaturation assay showed significant increases in susceptibility to denaturation (P < 0.01). Chromatin packaging assessment revealed 1,4-dithiothreitol-dependent chromomycin A3 DNA binding and less condensed, protamine-deficient sperm; the total thiol (P < 0.001) and protamine contents (P < 0.01), measured using monobromobimane and the HUP1N protamine 1 antibody, respectively, were reduced. The sperm basic proteome was also altered; proteins that were identified are involved in events during spermiogenesis and fertilization. Paternal exposure to CPA alters sperm chromatin structure, as well as the composition of sperm head basic proteins. We speculate that these changes underlie effects on fertilization and embryo development.

Electroejaculated baboon (Papio anubis) sperm requires a higher dosage of pentoxifylline to enhance motility.

Sperm collected by electroejaculation often show poor motility. The objective was to determine whether the addition of the phosphodiesterase inhibitor, pentoxifylline, would stimulate electroejaculated baboon sperm motility. Electroejaculation was performed on several occasions on a male baboon and sperm collected after familiarization. Pentoxifylline was tested at the standard concentration (1 mg/ml) and at twice the concentration. Sperm parameters were evaluated using a sperm motility analyzer, as well as acrosome and DNA integrity techniques. Sperm exposed to 2 mg/ml pentoxifylline had higher total motility when compared with the control and 1 mg/ml treatment. Rapid progression and velocities were higher after pentoxifylline. The acridine orange DNA normality test showed that over 90% of collected sperm had intact unfragmented DNA. About half the sperm population had normal morphology and intact acrosomes. A low percentage had cytoplasmic droplets. Sperm collected by rectal probe electroejaculation required a higher concentration (2 mg/ml) of pentoxifylline for enhanced total motility, rapid progression, and higher velocity. This suggested differences in membrane properties or phosphodisterase activity in electrojeaculated sperm. The electroejaculation procedure did not denature sperm DNA at the acridine orange assay level nor were the acrosomes disrupted. The present study also documented unique information on baboon kinematic parameters.

In situ detection of apoptotic nuclei in the substantia nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using terminal deoxynucleotidyl transferase labelling and acridine orange staining

The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to generate a dose-dependent cell death of dopaminergic nigral neurons in the C57Bl mouse. Mice were injected with a total cumulative dose of 150 mg/kg of MPTP delivered over five days and killed at different time points both during and after the toxin injections. Two independent histological methods were used to determine whether the dopaminergic nigral neurons died via an apoptotic mechanism. In situ end-labelling with terminal deoxynucleotidyl transferase was used on paraformaldehyde-fixed, serial, frozen sections to identify cells with double-stranded DNA breaks. Apoptotic cell death was found to be initiated within 72 h of the first injection of the neurotoxin and peaked 24 h after the final MPTP injection. The metachromatic fluorochrome, Acridine Orange, was used on alternate sections to provide structural confirmation of the nuclear chromatin “clumping” considered to be representative of apoptosis. Confocal laser imaging combined with deconvolution techniques was used to resolve the fluorescent signal emitted by the in situ Acridine Orange–DNA complexes. The number of Acridine Orange-stained nuclei demonstrating chromatin clumping was identical to that of the positive in situ end-labelled nuclei observed over a 25 day period. Based upon these two independent methods of assessing apoptosis in situ, we conclude that a 150 mg/kg dose of MPTP can elicit apoptotic cell death in nigral dopaminergic neurons of the C57Bl mouse.

Isolation and characterization of DNase-deficient mutants ofClostridium acetobutylicum

DNase activity ofClostridium acetobutylicum was found to exhibit a relatively broad, acidic pH optimum of 3.5–4.5. The enzyme could be completely inhibited by addition of 0.2M EDTA (final concentration), whereas heat treatment or addition of diethylpyrocarbonate proved to be unsuccessful. Activity measurements in various cell fractions indicated a localization of DNase at the outside of the cytoplasmic membrane. Maximal activity could be found in the stationary growth phase. With the acridine orange-DNA overlay method, three mutants (D-1, D-2, and D-1.1) could be isolated that were DNase-deficient. The mutation proved to be very stable (reversion frequency of 0.2%). All mutants were “leaky”; however, D-1.1 was less leaky than the other two.

Interaction of intravenously injected liposomes with mouse liver mitochondria. A fluorescence and electron microscopy study

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.

Evolution de l'azote kjeldahl et de la biomasse bacterienne le long d'une filiere de traitement d'eau potable

Evolution de l'azote kjeldahl et de la biomasse bacterienne le long d'une filiere de traitement d'eau potable

Denaturation of nucleic acids induced by intercalating agents. Biochemical and biophysical properties of acridine orange-DNA complexes.

At high binding densities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar- and base-composition) of nucleic acids.

Competitive binding studies of compounds that interact with DNA utilizing fluorescence polarization

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.

Visualization of drug-nucleic acid interactions at atomic resolution: V. Structure of two aminoacridine-dinucleoside monophosphate crystalline complexes, proflavine-5-iodocytidylyl (3′–5′) guanosine and acridine orange-5-iodocytidylyl (3′–5′) guanosine

Acridine orange and proflavine form complexes with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine. The acridine orange-iodoCpG † † Abbreviation used: iodoCpG, 5-iodocytidylyl(3′–5′)guanosine. crystals are monoclinic, space group P2 1, with unit cell dimensions a = 14.36 A ̊ , b = 19.64 A ̊ , c = 20.67 A ̊ , β = 102.5 °. The proflavine-iodoCpG crystals are monoclinic, space group C2, with unit cell dimensions a = 32.14 A ̊ , b = 22.23 A ̊ , c = 18.42 A ̊ , β = 123.3 °. Both structures have been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least-squares. Acridine orange forms an intercalative structure with iodoCpG in much the same manner as ethidium, ellipticine and 3,5,6,8-tetramethyl- N-methyl phenanthrolinium ( Jain et al., 1977,1979 ), except that the acridine nucleus lies asymmetrically in the intercalation site. This asymmetric intercalation is accompanied by a sliding of base-pairs upon the acridine nucleus and is similar to that observed with the 9-aminoacridine-iodoCpG asymmetric intercalative binding mode described in the previous papers ( Sakore et al., 1977,1979 ). Basepairs above and below the drug are separated by about 6.8 Å and are twisted about 10 °; this reflects the mixed sugar puckering pattern observed in the sugar-phospate chains: C3′ endo (3′–5′) C2′ endo (i.e. each cytidine residue has a C3′ endo sugar comformation, while each guanosine residue has a C2′ endo sugar conformation), alterations in glycosidic torsional angles and other small but significant conformational changes in the sugar-phosphate backbone. Proflavine, on the other hand, demonstrates symmetric intercalation with iodoCpG. Hydrogen bonds connect amino groups on proflavine with phosphate oxygen atoms on the dinucleotide. In contrast to the acridine orange structure, base-pairs above and below the intercalative proflavine molecule are twisted about 36 °. The altered magnitude of this angular twist reflects the sugar puckering pattern that is observed: C3′ endo (3′–5′) C3′ endo. Since proflavine is known to unwind DNA in much the same manner as ethidium and acridine orange ( Waring, 1970), one cannot use the information from this model system to understand how proflavine binds to DNA (it is possible, for example, that hydrogen bonding observed between proflavine and iodoCpG alters the intercalative geometry in this model system). Instead, we propose a model for proflavine-DNA binding in which proflavine lies asymmetrically in the intercalation site (characterized by the C3′ endo (3′–5′) C2′ endo mixed sugar puckering pattern) and forms only one hydrogen bond to a neighboring phosphate oxygen atom. Our model for proflavine-DNA binding, therefore, is very similar to our acridine orange-DNA binding model. We will describe these models in detail in this paper.

Comparison of the early chromatin reaction of the mouse thymocytes incubated with phytohemagglutinin and concanavalin A.

The preliminary activation of mouse thymocytes in vitro following exposure to the mitogens phytohemagglutinin and Concanavalin A, was compared. For this purpose the ratio of the fluorescence intensity for acridine orange-DNA complex at light wave length gamma = 590 nm to the fluorescence intensity of this complex at light wave length gamma = 530 nm was determined. It was shown that 100% of thymocytes showed an increase in chromatin activity under the influence of Con A within 15 minutes of incubation, but that following incubation with PHA an increase in activity occurred only in 25% of cells and was detectable only after 60 minutes of incubation. The results show that differences in the reactivity of thymocytes to PHA and Con A concern not only the late steps of activation such as RNA, protein and DNA synthesis, but also involve the earliest responses to the mitogen. The observed differences are discussed and correlated with the different degrees of maturity of the thymocytes.

Ternary complex of DNA with chymotrypsinogen A and acridine orange

Acridine orange was able to interact with the DNA of the soluble chymotrypsinogen-DNA complex to form a ternary complex. Nevertheless, chymotrypsinogen (which by itself did not interact with acridine orange) had an apparent inhibitory effect on binding of the dye to DNA. Visible absorption spectra of ternary mixtures, at both high and low dye: nucleotide ratios, closely resembled the spectra for corresponding acridine orange-DNA mixtures. Ternary complexes were isolated by either sucrose gradient sedimentation or Sephadex column chromatography although, in the presence of the protein, an apparent lesser amount of dye remained bound. After Sephadex chromatography, only dye bound by strong binding process I remained associated with DNA or with the protein-DNA complex, and both dye-DNA complexes were stable to rechromatography.

Simple methods for the orientation of DNA molecules in films suitable for optical studies.

AbstractThree methods are described for the preparation of oriented samples of DNA molecules in films. The methods involve simple equipment and produce films of good optical qualities, suitable for the determinations of dichroic spectra, for example. To illustrate the quality of the films the dichroism of DNA and of acridine orange–DNA complex are described. Evidence is given that native DNA structure is stabilized by poly‐(vinyl alcohol).

Differences in fluorescence spectra of acridine orange-DNA complexes related to DNA base composition.

Differences in fluorescence spectra of acridine orange-DNA complexes related to DNA base composition.

Kinetic studies on acridine orange-DNA interaction by fluorescence stopped-flow method

The fluorescence stopped-flow technique was applied to the kinetic investigation of acridine orange(AO)-DNA interaction. Increased sensitivity of detection has enabled one to study with much lower concentration range of dye (as low as 4 × 10 −8 M) than so far employed in the rapid reaction technique by light absorption measurement. The process of dimerization of AO on the outside of DNA and that of intercalation into base pairs have been separately observed.

Study on the binding nature of acridine orange to DNA by means of flow dichroism.

Abstract The binding nature of acridine orange to DNA was studied by means of equilibrium dialysis and flow dichroism with spectrophotometric analysis. The adsorption isotherm of the acridine orange-DNA system was obtained by means of equilibrium dialysis. From the experiment, the binding constant (K) and the fractional amount of the binding site to the total number of nucleotides (f) were estimated for native- and heat-denatured DNA. Under a shear gradient, the acridine orange-DNA complex showed a negative dichroism in visible region as well as in the ultraviolet region. This implies that acridine orange is oriented on DNA rather perpendicularly to its main axis, like base pairs. The magnitude of the dichroism of the complex at 500 mμ, near the visible absorption peak of acridine orange, was larger than each value of the complex and that of the DNA itself (260 mμ). The transition moment corresponding to the absorption at 500 mμ may be oriented in a more orderly manner and/or more nearly perpendicularly to the main axis of DNA than that corresponding to 260 mμ.

Fluorescence-excitation spectra of acridine orange-DNA and -RNA systems.

The absorption, fluorescence and fluorescence-excitation spectra were investigated in free AO, AO-DNA and AO-RNA systems. In the case of Complex I, AO seems to be metachromatically bound to the two kinds of binding sites, phosphates neighboring purine bases and those neighboring pyrimidine bases, and becomes non-fluorescent.