Acridine Orange Treatment Research Articles

Perbandingan Pengaruh Agen Plasmid Curing Acriflavine dan Acridine Orange (AO) terhadap Laju Plasmid Curing dengan Kuantifikasi Fluoresensi Ekspresi Protein GFP pada Bakteri Escherichia Coli PRSET-emGFP

Plasmid curing is an activity to remove plasmids in a bacterium. Plasmid itself is a structure that can carry genes that are beneficial for bacteria, such as making the bacteria resistant to antibiotics. Acridine Orange (AO) and Acriflavine are intercalating agents that function to break down the super helical shape of plasmid DNA and then form circular or open linear plasmid DNA which is used to carry out Plasmid Curing in this study and then compare its effect on the rate of plasmid curing by fluorescence quantification of GFP protein expression. on Escherichia coli PRSET-emGFP bacteria. The results obtained from this experiment were the rate of plasmid loss per generation obtained by measuring the activity of the GFP reporter on the recombinant E. coli model given the curing agent Acridine Orange was 9.52% for the AO 50 treatment, 44.2% for the AO 100, 10.4% for the AO 50 treatment. Akriflavina 250, and 0.836% for Acriflavine 125. Therefore, it can be concluded that the Acridine Orange treatment was more effective than the Acriflavine treatment against the pRSET-EmGFP recombinant E. coli curing plasmid.

Drug resistance profile of biofilm forming Pseudomonas aeruginosa isolated from aquatic environment in South Eastern Nigeria

This study was carried out to determine drug resistance pattern of biofilm forming Pseudomonas aeruginosa isolated from aquatic environment in South Eastern Nigeria. A total of 272 water samples from Ezillo Water Treatment Plant were examined. Bacteriological analysis of the samples was done using standard microbiological techniques. Biofilm formation was carried out using crystal violet binding assay method while antibiotic studies of was determined using Kirby-Bauer disk diffusion method following Clinical Laboratory Standards Institute (CLSI) guidelines. Plasmid curing analysis was done using acridine orange treatment. The statistical analysis was carried out using statistical analysis software (SAS). A total of 223 isolates of Pseudomonas aeruginosa were confirmed. Data revealed that the level of occurrence was high (85.11%) among the various treatment units in the plant. Biofilm formation among test isolates was high 128 (57.40%). Biofilm forming isolates were resistant to imipenem (39.45%), ceftaxidime (68.79%), ciprofloxacin (69.89%), ofloxacin (70.77%), ertapenem (84.33%) cefepime (85.11%), ticarcillin (89.43%), amikacin (90.44%), gentamicin (91.97%), cefotaxime (93.79%) and were completely resistant to amoxicillin\\clavulanic acid, tobramycin, oxacillin and aztreonam. The multiple antibiotic resistance index (MARI) ranged from 0.64 to 0.93. We report the occurrence of multidrug resistant Pseudomonas aeruginosa in water samples from Ezillo Water Treatment Plant in South Eastern Nigeria, which could be a critical source of infections in the region.

Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role.

Enhancement of Commercial Production of Polymeric PHB Material from Bacterial Strains through Mutagenic Strategies

Poly-β β β β β-Hydroxybutyrate (PHB) is proving itself as an advance polymeric material as well as an alternative for plastic material. The only hinge stopping large scale utilization of PHB at industrial scale and in daily lives is its, economical production. Large scale economical production of PHB depends upon optimization and selection of high PHB producing strains. The present study investigates mutagenic strategies for the improvement of commercial strains for high scale PHB production in industries. Improvement of strains by mutation is very cost effective method and strategically better approach for getting maximum output of PHB. The mutation treatment was performed by Physical and Chemical methods i.e. Ultra violet radiation (Physical) and Acridine Orange treatment (Chemical). Mutation leads to an increase in the yield of PHB as in comparison to original strains which has proved through increase in PHB extraction from the mutated counterparts of Bacillus flexus and Bacillus megaterium. The results revealed that there was an excellent improvement in PHB production by almost 2 folds, when strain treatment was performed with physical mutagen (UV radiation) but no such improvement was seen with chemical mutagen (Acridine Orange). The PHB yield increased marginally from 15.68% to 18.61% with chemical mutagen where as in UV treatment the PHB yield increased exceptionally, from 15.68% to 23.70% in case of Bacillus flexus[Agriculture isolate M1] . In case of Bacillus megaterium UV exposed strain show a phenomenal increase in PHB yield from 11.76% to 25% but no such improvement again was seen in PHB yield through Acridine orange treated colonies i.e. 11.76% to 15.09%. The quality of produced PHB was assessed through FTIR. These results exemplify that physical mutagens can be more effective for improving commercial production of PHB from bacterial strains.

English

This study investigated the loss of resistance of multidrug resistant (MDR) Pseudomonas aeruginosa strains after exposure to dilutions of acridine orange. Five pure axenic strains of P. aeruginosa coded PA1 to PA5 obtained from five infected human sources which included middle ear, urethra, trachea, wound and urine were obtained from the Medical Microbiology Department of the University of Benin Teaching Hospital, Nigeria and stocked on sterile Nutrient agar slants. Slant cultures were sub-cultured aseptically on sterile MacConkey and Blood agar plates and incubated aerobically at 37°C for 24 h to confirm for P. aeruginosa. Gram staining and oxidase test were carried out on resulting colonies. Antibiotic sensitivity test was done by agar disc diffusion method on all confirmed strains on sterile Mueller-Hinton agar plates before and after treatment with acridine orange (AO). P. aeruginosa strains that showed ≤50.0% reduction in resistance markers (RM) after treatment with 0.35, 0.55, 0.75 and 0.95 µg/ml dilutions of AO were noted. Minimum inhibitory concentration (MIC) assay was done using gentamicin on PA5 strain with all four dilutions. All five strains showed 100% resistance against augmentin, nalidixic acid, nitrofurantoin, cotrimoxazole, amoxicillin and tetracycline. Sensitivity was recorded for ofloxacin and gentamicin with 14.6±9.5 and 8.4±4.4 mm zones of inhibition, respectively for all the strains except strain PA3 which was resistant to 8 (100.0%) of antibiotics used. Strains PA1, PA2, PA4 and PA5 were each resistant to 6 (75.0%) of the antibiotics tested. There was loss of RM of 52.1±18.6 and 54.7±37.6% to ofloxacin after treatment with 0.35 and 0.55 µg/ml dilutions, respectively by all MDR P. aeruginosa strains. Loss of RM to gentamicin by strains PA1, PA2, PA4 and PA5 after 0.35 µg/ml acridine orange treatment was recorded as 0.0, 61.5, 58.3 and 60.0%, respectively with a mean±standard error (SE) of 45.0±15.0%. With 0.55 µg/ml dilution, 97.6±28.3% loss of RM was recorded while less than 45.0 and 35.0% loss of RM were recorded for 0.75 and 0.95 µg/ml dilutions, respectively. Acridine orange dilutions of 0.35 and 0.55 µg/ml recorded two-fold (5 µg) and four-fold (2.5 µg) reduction in MIC of gentamicin, respectively. The implications of these findings are discussed. Key words: Resistance loss, Pseudomonas aeruginosa, treatment, dilutions, acridine orange.

A Minimally Invasive Surgery for Bone Metastases Using the Combination of Photodynamic Therapy and Hyperthermia Treatment

Cancer patients with bone metastases in their extremities may require surgical intervention to prevent deterioration in their quality of life due to a pathological fracture or severe bone pain. However, curative surgical interventions sometimes have severe complications due to the status of the original cancers. To avoid the decreased quality of life caused by bone metastasis, minimally invasive surgery that avoids additional surgical morbidity is required. We have established two therapeutic treatments for bone metastasis, a photodynamic acridine orange treatment (AOT) and an electronic magnetic hyperthermia treatment (EMHT). The present study investigated the clinical outcomes of combination therapy with EMHT and AOT for patients with bone metastases in their extremities. Methods: The study included 6 patients with 7 bone cancer metastasis locations. For bone metastases, all patients received intraregional tumor excision supported by AOT, in which photodynamic and radiodynamic therapy kills tumor cells during surgery with minimal damage to normal tissues. After the curettage, bone reconstruction was performed by using magnetic materials with calcium phosphate cement. EMHT was repeatedly performed after surgery. In EMHT, tumor cells are killed with an electric magnetic field generator, and bony union is promoted by electronic stimulation. Results: The mean duration of follow-up was 14 months. During the follow-up period, only one patient experienced a local recurrence, and this recurrence occurred 14 months after surgery. Bony union occurred in 4 of 5 cases (80%), and the pain score was significantly reduced after surgery. Conclusions: In the present study, AOT reduced the invasiveness of surgery. EMHT reduced the tumor growth without major complications and promoted bone formation after surgery. Our clinical results confirmed that EMHT and AOT combination therapy for bone metastasis can preserve limb function without local recurrence or bone absorption.

Antibiogram and plasmid profile of some multi-antibiotics resistant urinopathogens obtained from local communities of southeastern Nigeria

Multi-antibiotic resistant strains of bacteria represent a global medical challenge, having reduced or threatened to completely eliminate the list of reserved, last-resort agents usually deployed in serious and/or life-threatening infections. Even newer and very potent antibiotics are not spared from the emergence of resistant strains of organisms. In this study, the antibiotic resistance profile and the plasmid profile of some multi-antibiotic resistant bacteria strains isolated from the urine samples of fifty volunteers (ages 15-30) from a community in south eastern Nigeria were analyzed. Eight multidrug-resistant bacteria were isolated from the 50 urine samples, of which approximately 60% showed resistance to nitrofurantion, amoxicillin, ciprofloxacin, ampicillin, gentamicin, ampiclox and erythromycin. The MIC of the isolated S. aureus, E. coli, and Klebsiella species to ofloxacin, ciprofloxacin, pefloxacin, and co-trimoxazole were greater than 500 μg/ml. Plasmid profile studies revealed the presence of R-plasmids of size range 11-18 kb. The cultures of resistant isolates irreversibly lost their antibiotic resistance with acridine orange and SDS treatment, which suggests that the resistant genes could be harboured in the plasmids.

Evidence that RDX biodegradation by Rhodococcus strain DN22 is plasmid-borne and involves a cytochrome p-450.

To investigate the biodegradation of the explosive compound RDX in Rhodococcus strain DN22, a bacterium previously isolated for its ability to grow on RDX as sole nitrogen source. Analysis of the rates of RDX degradation and nitrite production indicated that 2 mol nitrite were produced per mole RDX degraded. Cells of strain DN22 had the highest activity against RDX during the exponential phase and low activity in the stationary phase. Nitrite production from RDX was inhibited by metyrapone, menadione, piperonyl butoxide, n-octylamine and carbon monoxide and inducible by pyrrolidine, pyridine and atrazine. Acridine orange treatment yielded RDX-minus derivatives of strain DN22 at a curing rate of 1.5% and all of the cured derivatives had lost a large plasmid. RDX biodegradation in strain DN22 appears to involve a plasmid-encoded cytochrome p-450 enzyme. Plasmid-borne RDX degradation genes could potentially be transferred between bacteria. Our research into RDX metabolism in strain DN22 will facilitate future applications of this bacterium for bioremediation.

The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli.

The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

Induction of Band 3 Aggregation in Erythrocytes Results in Anti-band 3 Autoantibody Binding to the Carbohydrate Epitopes of Band 3

Involvement of band 3 aggregation in the mechanism of anti-band 3 autoantibody binding to the cell surface carbohydrate epitopes of band 3 was investigated. When erythrocytes were treated nonoxidatively with a known protein-aggregating agent acridine orange, protein aggregates of the cell membrane which are insoluble in a nonionic detergent C 12E 8solution were remarkably increased. Analysis of the protein aggregates by SDS–PAGE indicated that they were composed of several species of noncovalently associated membrane proteins including band 3. 125I-labeled anti-band 3 bound to the acridine orange-treated cells, and the binding increased depending on the concentrations of acridine orange used. The binding was inhibited by band 3 and its oligosaccharides but not by the oligosaccharides pretreated with endo-β-galactosidase, an enzyme specifically cleaves poly- N-acetyllactosaminyl saccharide chains of band 3. When erythrocytes were pretreated with endo-β-galactosidase to remove poly- N-acetyllactosaminyl saccharide chains from cell surface prior to acridine orange treatment, the cells did not become susceptible to anti-band 3 binding. The results indicate that induction of band 3 aggregation in erythrocyte membrane leads to anti-band 3 binding to the poly- N-acetyllactosaminyl saccharide chains of band 3. Consistently, membrane proteins including band 3 were found to be aggregated when erythrocytes were oxidized with ADP-chelated Fe 3+under the conditions that induce anti-band 3 binding to the cells. Similar band 3 aggregation was observed on senescent erythrocytes whose carbohydrate epitopes of band 3 had been occupied with anti band 3. These results indicate that anti-band 3 binds to the carbohydrate epitopes of band 3 on erythrocytes when band 3 is aggregated by oxidative and nonoxidative mechanisms.

Loss of penicillin resistance inParacoccus denitrificans induced by mitomycin C and acridine orange

In an attempt to eliminate the penicillin resistance gene of P. dentrificans by curing agents, such as acridine orange (AO) and mitomycin C, it was observed that AO treatment caused temporary phenotypic curing where development of sensitivity was a function of concentration of both the curing agent and benzylpenicillin. However, curing with mitomycin produced sensitive clones at a frequency of 6 X 10(-3) and two permanently cured clones were isolated. Heavy metal resistance and resistance to other drugs, however, remain unchanged in the mitomycin-cured isolate.

Elimination of Xf phage productivity from Xanthomonas campestris pv. oryzae by acridine orange treatment and its effects on the host bacteria.

Elimination of Xf phage productivity from Xanthomonas campestris pv. oryzae by acridine orange treatment and its effects on the host bacteria.

Isolation and characterization of a linear plasmid fromStreptomyces rimosus

A linear plasmid was isolated from a strain of Streptomyces rimosus. This plasmid was separated by agarose gel electrophoresis and its size, about 43 kb, determined both by this method and by electron microscopy. The cleavage pattern of the linear plasmid with 5 restriction endonucleases is given. A protein, which is removed by proteinase K, is probably associated to this plasmid. By ethidium bromides or acridine orange treatment we obtained mutants which had lost their aerial mycelium and their linear plasmid.

Intense, reversible aggregation of intramembrane particles in non-haemolyzed human erythrocytes A freeze-fracture study

Acridine orange, a strongly cationic, membrane-penetrating dye, caused intense aggregation of intramembrane particles in non-haemolyzed human erythrocytes at 5 mM concentration. Simultaneously with the particle aggregation, large, empty, intramembrane particle-free or particle-depleted vesicles were detached from the red cells. Washing the erythrocytes after Acridine orange treatment resulted in complete disaggregation of the intramembrane particles. Less cationic acridine dyes (9-aminoacridine, 5-aminoacridine and Quinacrine) caused much less conspicuous alterations. Rivanol (ethacrine lactate), on the one hand, caused intramembrane particle aggregation in human red cells as well as ribosome aggregation in rabbit reticulocytes when it was dissolved in lactate-buffered sucrose. On the other, Rivanol dissolved in phosphate-buffered saline failed to induce these alterations. Neuraminidase treatment had no effect on the intensity of Acridine orange-induced particle aggregation, but impeded disaggregation. Our results indicate that, in contrast to previous observations, intense and reversible clustering of intramembrane particles is certainly possible in non-haemolyzed erythrocytes. The intramembrane particle aggregation may be due primarily to perturbation of the inner red cell surface by strongly cationic dyes and the presence of sialic acid residues on the outer red cell surface seems to be essential for the reversibility of the process.

アルゴンレーザーによるPhotoradiation Therapyの研究

Effectiveness and mechanism of argon laser photoradiation therapy was studied in cultured cells. Photosensitizing agents used in this study included acridine orange (AO), Adriacin, Cosmegen and Mithramycin. Among these drugs, AO was most effectively photosensitized to cause cell death, whereas other anti-cancer drugs had little additional effects to their own cytocidal effect by laser irradiation. Photoradiation effect was observed only when cells were irradiated after AO treatment. Irradiation of AO or cells separately did not cause cell death. Therefore interaction between AG and cell was considered indispensable in this modality. Until now AO is the most effective photosensitizer in argon laser photoradiation therapy but some disadvantages such as genotoxicity must be overcome before it is widely applied clinically.

Sensitivity of Chloroplast Development in Euglena to Acridine Orange: Bleaching and Recovery from Damage

Treatment of light-grown Euglena cells with acridine orange (AO) at non-lethal concentrations resulted in the permanent loss of their ability to form chloroplasts. However, dark-grown cells were insensitive to AO. Starvation of the cells under light culture or the addition of chloramphenicol (CM) deformed the chloroplasts and made them less AO sensitive. Cells with mature chloroplasts seemed to show the most sensitivity to AO. Illumination with intense visible light of the AO-treated cells, whether they had been light- or dark-grown, made a greater proportion of them liable to be bleached or to be killed. The photosensitization was evident over the range of wavelengths of 450–500 nm, where the absorption maximum of AO is located. When light-grown cells, after AO treatment, were held in darkness in a nongrowth medium for 24 hr, the bleaching effect was reduced. However, if held under a light of 620–670 nm, where cells were not photosensitized but were able to form chloroplasts, the AO-damage was not reversed. The possibility of repair after AO-treatment is discussed.

Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi.

Olive (or oleander) knot is a plant disease incited by Pseudomonas savastanoi. Disease symptoms consist of tumorous outgrowths induced in the plant by bacterial production of indole-3-acetic acid (IAA). Synthesis of IAA occurs by the following reactions: L-tryptophan leads to indoleacetamide leads to indoleacetic acid, catalyzed by tryptophan 2-monooxygenase and indoleacetamide hydrolase, respectively. Whereas the enzymology of IAA synthesis is well characterized, nothing is known about the genetics of the system. We devised a positive selection for the presence of tryptophan 2-monooxygenase based on its capacity to use as a substrate the toxic tryptophan analogue 5-methyltryptophan. Efficient curing of the bacterium of tryptophan 2-monoxygenase, indoleacetamide hydrolase, and IAA production was obtained by acridine orange treatment. Further, loss of capacity to produce IAA by curing was correlated with loss of a plasmid of 34 X 10(6) molecular weight. This plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophan 2-monooxygenase and indoleacetamide hydrolase activities and production of IAA.

Biological Role of Gramicidin S in Spore Functions

Gramicidin-S-negative mutants of Bacillus brevis ATCC9999 have been isolated with a remarkly higher yield after ethidium bromide or acridine orange treatment, than after N-methyl-N'-nitro-N-nitrosoguanidine treatment. Four (MIV, Smr170, R5 and EB 16) of 38 isolated mutants were characterized with respect to the lesion in gramicidin-S-synthesizing activity. The mutants sporulate to the same extent as the parental strain except mutant Smr 170 which sporulates less. However, mutant spores were more heat-sensitive and possessed a reduced level of dipicolinic acid content. No significant difference was observed in the germination time of wild-type and mutant spores. All spores germinated after 80--110 min, but the outgrowth time was different: all gramicidin-S-negative mutants grew out immediately after germination whereas wild-type spores required a lag period of 9--10 h. When the mutants were allowed to sporulate in the presence of gramicidin S, the spores were found to be heat-resistant and their outgrowth postponed to the same period as the parent spores. The addition of gramicidin also eliminated the deficiency of dipicolinic acid. A new class of gramicidin-S-negative mutant, R5, which only activates L-valine and L-leucine, is described. A possible biological function of gramicidin S in the heat-resistance and in the timing of spore outgrowth is discussed.

Comparison of heat shocking and acridine orange treatment in phage typing of nontypable strains of Staphylococcus aureus.

Forty-five percent of 108 nontypable strains of Staphylococcus aureus from clinical and environmental sources were phage typable after heat shock and acridine orange treatment. Although phage patterns were produced by different types of treatment, the same phage groups were present, showing specificity. The newly typable strains appear to be similar in phage group distribution to the primary typable strains typed at the Peter Bent Brigham Hospital during the same time period. Ten of the originally nontypable S. aureus strains that showed lytic patterns after heat shock or acridine orange treatment were subcultured four times from the treated culture and then tested again with bacteriophages. Phage patterns were still present in 8 of the 10 strains without any further treatment. These findings are compatible with the findings of other workers, who have shown that the insensitivity of primary nontypable S. aureus strains to phage is linked to phage-resistant genes in extrachromosomal genetic elements that can be "cured" by both heat shock and acridine treatment.

Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae

The treatment of diploid cultures of yeast with ultraviolet light (UV), γ-rays, nitrous acid (NA) and ethyl methane sulphonate (EMS) results in increases in cell death, mitotic gene conversion and crossing-over. Acridine orange (AO) treatment, in contrast, was effective only in increasing the frequency of gene conversion. The individual mutagens were effective in the order UV > NA > γ-rays > AO > EMS. Prior treatment of yeast cultures in starvation medium produced a significant reduction in the yield of induced gene conversion. The results have been interpreted on the basis of a general model of mitotic gene conversion which involves the post-replication repair of induced lesions involving de novo DNA synthesis without genetic exchange. In contrast mitotic crossing-over appears to involve the action of a repair system independent from excision or post-replication repair which involves genetic exchange between homologous chromosomes.