Presence Of Acridine Orange Research Articles

Spectroscopic, calorimetric and cytotoxicity studies on the combined binding of daunorubicin and acridine orange to a DNA tetrahedron

Chemotherapy–phototherapy (CTPT) combination drugs co-loaded by targeted DNA nanostructures can achieve controlled drug delivery, reduce toxic side effects and overcome multidrug resistance. Herein, we constructed and characterized a DNA tetrahedral nanostructure (MUC1-TD) linked with the targeting aptamer MUC1. The interaction of daunorubicin (DAU)/acridine orange (AO) alone and in combination with MUC1-TD and the influence of the interaction on the cytotoxicity of the drugs were evaluated. Potassium ferrocyanide quenching analysis and DNA melting temperature assays were used to demonstrate the intercalative binding of DAU/AO to MUC1-TD. The interactions of DAU and/or AO with MUC1-TD were analyzed by fluorescence spectroscopy and differential scanning calorimetry. The number of binding sites, binding constant, entropy and enthalpy changes of the binding process were obtained. The binding strength and binding sites of DAU were higher than those of AO. The presence of AO in the ternary system weakened the binding of DAU to MUC1-TD. In vitro cytotoxicity studies demonstrated that the loading of MUC1-TD augmented the inhibitory effects of DAU and AO and the synergistic cytotoxic effects of DAU + AO on MCF-7 cells and MCF-7/ADR cells. Cell uptake studies showed that the loading of MUC1-TD was beneficial in promoting the apoptosis of MCF-7/ADR cells due to its enhanced targeting to the nucleus. This study has important guiding significance for the combined application of DAU and AO co-loaded by DNA nanostructures to overcome multidrug resistance.

Photocatalytic hydrogen evolution by degradation of organic pollutants over quantum dots doped nitrogen carbide

Semiconductor photocatalysts are of great importance for addressing current environmental and energy crises. In this study, we developed a simple exfoliation-sonication route to fabricate nitrogen carbide quantum dots (CNQDs) doped nitrogen carbide nanosheet (CNS) composite photocatalysts which were employed to produce hydrogen and degrade organic pollutants (methyl orange, acridine orange, aniline, and phenol) synchronously under visible light irradiation. The presence of acridine orange and aniline enhanced the hydrogen evolution efficiency from 8.8 mmol g−1 h−1 to 32.1 and 11.7 mmol g−1 h−1, respectively. On the contrary, methyl orange and phenol with the same concentration inhibited hydrogen evolution. Based on the proton chain and energy band analyses, the synchronous mechanism of photocatalytic hydrogen evolution and organic pollutant degradation on CNQDs/CNS was also proposed. On one side, the oxygen-containing functional groups on the surface of CNQDs and the surrounded water molecules constructed proton chains, increasing the combination probability between protons and photo-generated electrons. On the other side, the heterojunction of CNQDs/CNS induced the separation of photo-generated electron-hole pairs. The photo-generated electrons migrate to CNQDs, on which the protons were transformed into hydrogen molecules, while the holes migrated to CNS where the organic pollutants were oxidized synchronously.

Multi-spectroscopic and molecular modeling studies to reveal the interaction between propyl acridone and calf thymus DNA in the presence of histone H1: binary and ternary approaches

DNA is the primary target of many anticancer drugs involved in important intercellular processes, especially in transcriptional regulation, and histone is known to inhibit gene expression. Small molecules can bind to histone-DNA and impair the cell division, growth, inhibition, and apoptosis in cancer cells. In this research, the interaction of a histone H1-calf thymus DNA (ct DNA) complex and propyl acridone (PA) was investigated in Tris-HCl buffer, pH 6.8, using multi-spectroscopic, viscosity, and molecular modeling techniques. The Stern Volmer plot of the (H1-ct DNA) PA complex demonstrated two sets of binding sites with various binding affinities at three different temperatures. Thermodynamic parameters (ΔH° < 0 and ΔS° < 0) indicated that hydrogen bonds and van der Waals forces played the main roles in the binding of the drug to H1-ct DNA. The interaction between PA and ct DNA as well as (H1-ct-DNA) in the presence of acridine orange and ethidium bromide showed two different interaction behaviors in ternary systems. According to results from UV absorption spectroscopy and melting temperature (Tm) measurements, the binding mode of PA with ct DNA and the (H1-ct DNA) complex was indicative of an intercalative binding for the binary system and of both intercalative with groove binding with molecular fraction for the ternary system. Furthermore, the PA-induced detectable changes in the circular dichroism spectrum of ct DNA as well as changes in its viscosity. All of the experimental results proved that the intercalative binding between PA and ct DNA as well as the (H1-ct DNA) complex as binary and ternary systems must be predominant. The results obtained from experimental data were in good agreement with molecular modeling with regard to the determination of the binding site of PA to ct DNA in the absence and presence of histone H1.

Experimental and theoretical investigation effect of flavonols antioxidants on DNA damage

A new electrochemical biosensor was developed to demonstrate the effect of Acridine Orange (AO) on DNA damage. Then, the biosensor was used to check the inhibitors effect of three flavonols antioxidants (myricetin, fisetin and kaempferol) on DNA damage. Acridine Orange (AO) was used as a damaging agent because it shows a high affinity to nucleic acid and stretch of the double helical structure of DNA. Decreasing on the oxidation signals of adenine and guanine (in the DNA) in the presence of AO were used as probes to study the antioxidants power, using DNA-modified screen printed graphene electrode (DNA/SPGE). The results of our study showed that the DNA-biosensor could be suitable biosensor to investigate the inhibitors ability of the flavonols antioxidants on the DNA damage. The linear dependency was detected in the two regions in the ranges of 1.0–15.0 and 15.0–500.0 pmol L−1. The detection limit was found 0.5 pmol L−1 and 0.6 pmol L−1 for guanine and adenine, respectively. To confirm the electrochemical results, Uv–Vis and fluorescence spectroscopic methods were used too. Finally molecular dynamic (MD) simulation was performed on the structure of DNA in a water box to study any interaction between the antioxidant, AO and DNA.

Persistent, Toxin-Antitoxin System-Independent, Tetracycline Resistance-Encoding Plasmid from a Dairy Enterococcus faecium Isolate

A tetracycline-resistant (Tet(r)) dairy Enterococcus faecium isolate designated M7M2 was found to carry both tet(M) and tet(L) genes on a 19.6-kb plasmid. After consecutive transfer in the absence of tetracycline, the resistance-encoding plasmid persisted in 99% of the progenies. DNA sequence analysis revealed that the 19.6-kb plasmid contained 28 open reading frames (ORFs), including a tet(M)-tet(L)-mob gene cluster, as well as a 10.6-kb backbone highly homologous (99.9%) to the reported plasmid pRE25, but without an identified toxin-antitoxin (TA) plasmid stabilization system. The derived backbone plasmid without the Tet(r) determinants exhibited a 100% retention rate in the presence of acridine orange, suggesting the presence of a TA-independent plasmid stabilization mechanism, with its impact on the persistence of a broad spectrum of resistance-encoding traits still to be elucidated. The tet(M)-tet(L) gene cluster from M7M2 was functional and transmissible and led to acquired resistance in Enterococcus faecalis OG1RF by electroporation and in Streptococcus mutans UA159 by natural transformation. Southern hybridization showed that both the tet(M) and tet(L) genes were integrated into the chromosome of S. mutans UA159, while the whole plasmid was transferred to and retained in E. faecalis OG1RF. Quantitative real-time reverse transcription-PCR (RT-PCR) indicated tetracycline-induced transcription of both the tet(M) and tet(L) genes of pM7M2. The results indicated that multiple mechanisms might have contributed to the persistence of antibiotic resistance-encoding genes and that the plasmids pM7M2, pIP816, and pRE25 are likely correlated evolutionarily.

Acridine orange-induced signal enhancement effect of tyrosinase-immobilized carbon-felt-based flow biosensor for highly sensitive detection of monophenolic compounds

Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at -0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.

Cathodic Stripping Voltammetric Behavior of Cytosine in Presence of Acridine Orange at the Static Mercury Drop Electrode

Cathodic adsorptive electrochemical behavior of cytosine in the presence of acridine orange (DNA‐intercalating dye) at the static mercury drop electrode was investigated. A 0.001 M NaOH solution containing 2 ppm of acridine orange was found to be the optimal supporting electrolyte. Other optimum experimental conditions were found to be as follows: the initial potential at 0.00 V, linear n, and the potential scan rate at 50 mV s−1. The analytical response was found to be linear in a range of cystosine concentrations from 0.30 ppm to 1.1 ppm. For a direct response, the detection limit was 1.2 ppb. The presence of ssDNA and the interference of some trace metals were also investigated.

Acidification of the parasitophorous vacuole containing Toxoplasma gondii in the presence of hydroxyurea

Toxoplasma gondii multiplies within parasitophorous vacuole that is not recognized by the primary no oxidative defense of host cells, mainly represented by the fusion with acidic organelles. Recent studies have already shown that hydroxyurea arrested the intracellular parasites leading to its destruction. In the present work we investigated the cellular mechanism involved in the destruction of intracellular Toxoplasma gondii. Fluorescent vital stains were used in order to observe possible acidification of parasitophorous vacuole-containing Toxoplasma gondii in presence of hydroxyurea. Vero cells infected with tachyzoites were treated with hydroxyurea for 12, 24 or 48 hours. Fluorescence, indicative of acidification, was observed in the parasitophorous vacuole when the cultures were incubated in presence of acridine orange. LysoTracker red was used in order to determine whether lysosomes were involved in the acidification process. An intense fluorescence was observed after 12 and 24 hours of incubation with hydroxyurea, achieving it is highly intensity after 48 hours of treatment. Ultrastructural cytochemistry for localization of the acid phosphatase lysosomal enzyme was realized. Treated infected cultures showed reaction product in vesicles fusing with vacuole or associated with intravacuolar parasites. These results suggest that fusion with lysosomes and acidification of parasitophorous vacuole leads to parasites destruction in the presence pf hydroxyurea.

Cathodic Adsorptive Stripping Voltammetry of Cytosine and Guanine in Presence of Acridine Orange

A cathodic adsorptive electrochemical behavior of cytosine and guanine in the presence of acridine orange (DNA‐intercalating dye) at the static mercury drop electrode was investigated. Optimum experimental conditions were found to be the use of a 0.001 M NaOH solution with an acridine orange concentration of 2 ppm, an initial potential of −0.0 V, a scan rate of 10 mV s−1, and a linear scan mode. The more convenient relation to measuring the cytosine and guanine in the presence of acridine orange is 1:1:2. A new approach for improving the selectivity in analysis using copper and zinc as masking agents also was investigated. Cyclic voltammetry was used to characterize the interfacial and redox behavior.

Acridine Orange Sensitization and DNA Inhibition Effects on the Cyclic Voltammetry of Naphthoquinones Using Bare and DNA Modified Gold Electrodes

An ion pair mechanism is used to characterize the inhibition by DNA, and sensitization by acridine orange (AO) of the peak currents in the cyclic voltammetry (CV) of naphthoquinones with differing ionic substituents. Gold electrodes modified with adsorbed synthetic ′5-thiol-(GC)10 double strand DNA oligomers are used to study the redox activity of ferricyanide, 1,2-naphthoquinone-4-sulfonic acid sodium salt (NQS), 4-amino-1,2-naphthoquinone (ANQ), or 1,2-naphthoquinone (NQ) in the presence of AO. Parallel studies are performed using bare gold electrodes with double strand calf thymus DNA (CT-DNA) in the bulk solution. AO is known to bind intercalatively to DNA and is seen to sensitize the redox activity of ferricyanide or NQS with DNA either adsorbed on the electrode or present in the electrolyte. AO otherwise does not sensitize the peak currents in the CV of either ANQ or NQ regardless of the presence of DNA, nor does the DNA inhibit their CV, as it does the AO sensitized CV of the anions in the presence of DNA. The electroanalysis is done in pH 7 phosphate buffer with the solute and calf thymus double strand DNA base pair concentrations in the 10–3 molar range, and AO concentrations in the 10–4 molar range. The anion selective electrolytic sensitization combined with the intercalative property of AO may be applied to either the design of DNA based biosensors for the detection of anions, or DNA sensitive biosensors using AO as a marker.

DNA—Dye Fluorescence Enhancement Based on Shifting the Dimer—Monomer Equilibrium of Fluorescent Dye

In this paper, the investigation of DNA–dye fluorescence enhancement based on shifting the dimer–monomer equilibrium of a fluorescent dye, acridine orange (AO), is reported. Formation of a virtually nonfluorescent dimeric dye, acridine orange homodimer (AOAO), induced by the pre-micellar aggregation of an anionic surfactant, sodium dodecyl sulfate (SDS), was observed. The possibility of using the in situ formed AOAO as a fluorescent probe for nucleic acids and polynucleotides was studied. The results showed that a nearly 1000-fold fluorescence enhancement was observed upon addition of calf thymus DNA (CT DNA). The fluorescence enhancement effect of DNA was thought to be based on the DNA modulated shift of the dimmer monomer equilibrium of AO in the anionic surfactant solution. Intercalation of the monomer in DNA caused the dissociation of AOAO and led to a very high fluorescence enhancement. It seemed that the dimeric dye molecules acted as a source of monomer molecules ready for interacting with nucleic acids and, at the same time, decreased the inherent fluorescence of monomer molecules, which proved to be unfavorable to the detection of fluorescence enhancement. A linear dependence of fluorescence intensity on CT DNA concentration over a range from 7.8 ng/mL to 10.0 g/mL, in the presence of AO at a concentration of 1.65 × 10−6mol/L and of SDS at a concentration of 8.0 × 10−4 mol/L, allowed sensitive quantitation of CT DNA in a conventional fluorometer. Calibration graphs for yeast RNA and polynucleotides, such as poly A, poly U, and poly I, were also obtained.

Production of C 2-symmetrical phenazines by some actinomycetes

The C 2-symmetrical phenazines produced by Microbispora and Streptosporangium were identified as 1,6-phenazinediol-5,10-di- N-oxide (φ d-dioxide) and 1,6-phenazinediol (φ-diol). Both oxidation of φ-diol to φ d-dioxide and reduction of φ d-dioxide to φ-diol were detected with M. amethystogenes and M. parva. No albino mutants lacking φ-diol-oxidation ability were isolated from cultures of Microbispora incubated in the presence of acridine orange, or those incubated at sublethal temperatures.

CALCIUM UPTAKE IN SEALED PLASMALEMMA VESICLES ISOLATED FROM POST HARVEST CaCl2-TREATED SOUR CHERRIES

Calcium chloride (CaCl2) enhances turgidity and quality of postharvest sour cherry, Prunus cerasus L., fruit. Mechanisms by which plasma membrane (PM) ATPase maintains the electrochemical gradient in cell turgor were studied in isolated PM vesicles isolated from tapwater-, CaCl2- and chelated amino acid-calcium-treated Montmorency sour cherry fruit. Electron microscopy and periodic-chromicphosphotungstic acid staining indicated 85-90% closed PM vesicles. Protein activity associated with the PM was four times higher in both Ca treatments than in untreated cherries. ATPase activity was insensitive to NO3 and NaN3, but inhibited by vanadate, indicating absence or low levels of tonoplast and mitochondrial ATPases. PM vesicles exhibited a pH jump in the presence of acridine orange (A493-530nm). Cherry fruit appeared to have a PM ATPase similar to that of other plant species. Generation of a positive membrane potential across the PM was dependent upon ATP.

ペスト菌の夾膜形成能と細胞内夾膜抗原産生能が各々異なったプラスミドによって支配されている可能性

Variants which lacked capsular envelopes on their cell surface were isolated from the culture of a highly virulent Yreka strain of Yersinia pestis grown in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at incompletely growth-inhibitory concentrations. The variant could be divided into two types on the basis of the presence and the absence of intracellular envelope antigen. Both types of the variants lacked the 13 megadaltone (Md) plasmid. Thus, it may well be said that the 13 Md plasmid would play some decisive role in extracellular envelope formation and no concern with the synthesis of intracellular antigen. It was clarified that these characters were carried by each different gene. The intracellular envelope antigen synthesis could not be correlated with other plasmids isolated from Yreka strain, i.e., 7, 23, 44 and 59 Md plasmids. On the other hand, further treatment of the intracellular-positive variant with the above inhibitors resulted in the occurrence of the antigen-deficient type variant at a rate of 1-2%. The high frequency appearance of the variant by the plasmid-depleting agents might indicate possible presence of some yet unknown plasmid responsive to the intracellular synthesis of envelope antigen.

Trehalose accumulates in Saccharomyces cerevisiae during exposure to agents that induce heat shock response

The storage disaccharide, trehalose, is accumulated in yeast during a temperature shift from 30 to 45°C. The response peaks at 90 min and is transient since levels of trehalose decline rapidly in cells returned to 30°C. Storage of trehalose is inhibited when cells are incubated in the presence of acridine orange or ethidium bromide prior to and during temperature shift, suggesting a requirement for de novo RNA synthesis. Accumulation of trehalose occurs when cells are exposed to either ethanol, copper sulphate or hydrogen peroxide at 30°C, indicating that the phenomenon may be a general response to physiological stress. Parallels are drawn between the trehalose accumulation response and the heat shock response in yeast.

Involvement of a plasmid in growth on and dispersion of crude oil by Acinetobacter calcoaceticus RA57.

A crude-oil-degrading Acinetobacter species, Acinetobacter calcoaceticus RA57, was isolated by standard enrichment culture techniques on the basis of its ability to utilize the oily sludge found in the vicinity of a local gas station. Strain RA57 was found to contain four plasmids: pSR1 (5.1 kilobases [kb]), pSR2 (5.4 kb), pSR3 (10.5 kb), and pSR4 (20 kb). Both supercoiled and open circular forms of the first three plasmids were identified by two-dimensional gel electrophoresis. Restriction endonuclease analysis of pSR4 demonstrated that the plasmid contained a circular map. Colonies were isolated at random after growth in the presence of acridine orange and found to fall into two categories: (i) those which had lost the ability to grow on and disperse crude oil in liquid culture and concurrently were cured of pSR4 and (ii) those which retained the ability to both grow on and disperse crude oil and which contained pSR4. Strains from the first class continued to grow on hydrocarbon vapors, indicating that the defect associated with the curing of pSR4 was related to the physical interaction of the cells with the hydrocarbon substrate, rather than to its metabolism. No differences in either adherence to hydrocarbons or production of extracellular emulsifying activity were found between the two classes of mutants. In growth experiments on crude oil in mixed culture with strains which either contained or lacked pSR4, no sparing of the growth defect was observed. The results are consistent with the possibility that pSR4 encodes a factor(s) which is tightly associated with the cell surface.

Salt Tolerance in Suspension Cultures of Sugar Beet

Cell suspension cultures of sugar beet were grown at various salinities (0-200 millimolar NaCl). Their tolerance to Na(+) was comparable to that of the intact plant. Tonoplast vesicles were prepared by sucrose density gradient centrifugation of microsomal membranes and shown to be highly purified. The vesicles were subjected to a pH jump in the presence of acridine orange and the rate of recovery of fluorescence after addition of Na(+) was used as a measure of Na(+)-dependent H(+) efflux. In the presence of K(+) and valinomycin, the Na(+)/H(+) antiport showed saturation kinetics. Increasing Na(+) in the growth medium did not change the apparent K(m) for Na(+), but increased V(max) to about twice the control value, suggesting a specific induction of antiport synthesis by salt.

Plasmid-like properties of the four virulence-associated factors of Yersinia pestis.

The virulence-associated factors of Yersinia pestis, which determine the abilities to produce pesticin I (Pst+), capsular fraction I antigen (Fra+), V and W antigen complex (Vwa+) and a cell-surface component for adsorption of exogenous pigments (Pgm+), were independently eliminated by cultivation of the cells in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at a subinhibitory concentration. A virulent Y. pestis strain, Yreka, harbored at least five extrachromosomal DNA molecules of different sizes. In these molecules, a novel 13-megadalton DNA which was cured concomitantly with the elimination of the Fra factor was found, in addition to the known species of 7 and 44 megadaltons which were lost with the conversions to Pst- and Vwa-, respectively. Although the conversion to Pgm- could not be correlated with the lack of any proper extra-chromosomal DNA, the factor was transmitted to Pgm- cells with the aid of self-conjugative RP4 plasmid. The cells acquiring the Pgm factor regained virulence for mice.

Selection and Characterization of Chlorella Mutants Deficient in Amino Acid Transport

Six amino acids are transported at high rates across the plasmalemma of Chlorella vulgaris only after the induction of two specific transport systems. Induction is achieved either by pretreatment with glucose, glucose analogs, or by nitrogen starvation. Mutants for these transport systems were obtained after incubation of Chlorella cells in the presence of acridine orange or ethidium bromide, followed by a selection procedure using the toxic amino acid analogs l-canavanine (for l-arginine), and l-azetidine-2-carboxylic acid (for l-proline). Mutants isolated by this method had lost their ability to induce the corresponding transport system. Double mutants deficient in transport of both these amino acids still possess the general amino acid transport system, a third system which was described previously. Evidence for additional amino acid transport systems in Chlorella is discussed.

Ultrastructural modifications of proteoglycans coincident with mineralization in local regions of rat growth plate.

We carried out histochemical and electron-microscopic studies to examine the relationship between mineral deposition and changes in the organization of proteoglycans during mineralization in the cartilaginous growth plate of the rat. To preserve the distribution and organization of proteoglycans in the extracellular matrix and to stain proteoglycans, acridine orange was included in the solutions that were used for fixation and demineralization. In undecalcified sections, mineral crystals that initially appeared at the level of the penultimate hypertrophic chondrocyte grew into dense, spherical mineral clusters, roughly one micrometer in diameter, in the longitudinal septa of the lowermost hypertrophic zone. Following the removal of mineral with EDTA in the presence of acridine orange, proteoglycan structures with a characteristic rosette-like architecture were revealed within the matrix of the longitudinal septa of the lower hypertrophic zone. The location of these rosette-like proteoglycan structures was identical to that of the dense, spherical mineral clusters in the undemineralized controls, and they were remarkably similar in morphology and size. In the electron micrographs stained with uranyl acetate and lead citrate, the greatly increased density of the rosette-like structures and large diameter of the proteoglycan structures located in mineral clusters contrasted sharply with the slender strands of more faintly stained proteoglycan that was diffusely distributed throughout most of the extracellular matrix. X-ray microprobe analysis for sulphur confirmed the existence of proteoglycans in the rosette-like structures and demonstrated that the concentration of proteoglycans was selectively increased in these regions. The cores of the metaphyseal calcified cartilage also exhibited rosette-like proteoglycan structures, which before demineralization were totally obscured by the diffuse, dense homogeneous deposition of mineral. Without stabilization of the proteoglycans in the extracellular matrix with acridine orange, the rosette-like proteoglycan structures could not be demonstrated. These results clearly indicate that there is a selective increase in the concentration of proteoglycans in exactly the same regions where mineral clusters are formed in the hypertrophic zone of the cartilaginous growth plate.