Presence Of Acridine Orange Research Articles

Different pH requirements for entry of the two picornaviruses, human rhinovirus 2 and murine encephalomyocarditis virus

The entry into cells of human rhinovirus 2 (HRV 2) and murine encephalomyocarditis (EMC) virus was studied by the use of light-sensitive virus grown in the presence of acridine orange (HRV 2) and neutral red (EMC). HeLa cells were protected against infection with HRV 2 by NH 4Cl, monensin, and other compounds known to increase the pH of intracellular vesicles. Preincubation of the cells with the same compounds reduced the ability of the cells to bind [ 35S]methionine-labeled HRV 2, apparently due to inhibition of recycling of endocytosed receptors back to the cell surface. The cells were also protected against infection when HRV 2 was bound to cells on ice and the cells were then incubated at 37° with the different compounds. This indicates that low pH is also necessary for some event in the entry process taking place after the virus is bound to the cells. In contrast, compounds which increase the pH in acidic intracellular compartments did not protect mouse L-cells against infection with EMC-virus, and the entry of the virus was inhibited by low pH in the medium. This inhibition was partly overcome by the presence of the ionophore monensin, which elevates the pH in endosomes and lysosomes. Possibly, EMC virus enters the cytosol from vesicles with neutral or slightly alkaline pH.

The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae. A cyclic-AMP-dependent activation of phosphofructokinase 2.

When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate. These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature. Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate. Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange. When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase. Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase. This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km. Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate. From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S. cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme. In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.

Deletions in Klebsiella pneumoniae R plasmids induced by growth in the presence of acridine orange at high temperature.

The generation in vivo of plasmids deleted at specific sites in strains of Klebsiella pneumoniae containing R plasmids, by treatment with high concentrations of acridine orange (1.2 mg/ml) at 42 degrees C are reported. These deletions seem to be site specific because loss of specific restriction fragments after digestion with restriction enzymes was demonstrated.

Possible Involvement of a Megaplasmid in Nodulation of Soybeans by Fast-Growing Rhizobia from China

Several isolates from a newly described group of fast-growing acid-producing soybean rhizobia, Rhizobium japonicum, were analyzed for plasmid content. All contained from one to four plasmids with molecular weights of 100 x 10 or larger. Although most of the isolates shared plasmids of similar size, the restriction endonuclease (BamHI, EcoRI, and HindIII) patterns of the plasmids from three of the isolates were vastly different. Growth in the presence of acridine orange was effective in producing mutants cured of the largest plasmid in one of the strains. These mutants also lost the ability to form nodules on soybeans. High-temperature curing of a smaller plasmid in another strain did not lead to loss of nodulating ability or alteration of symbiotic effectiveness on soybean cultivars. The identities of all of the isolates and mutants were ascertained by immunofluoresence and immunodiffusion. The new fast-growing strains of R. japonicum may provide a better genetic system for the study of the soybean symbiosis than the slow-growing R. japonicum, not all of which can be shown to contain plasmids.

Electron microscopical studies of the effects of aromatic hydrocarbons on sea urchin embryos

The effects of mono-, di-, and tricycfic aromatic hydrocarbons on the early development of Paracentrotus lividus were studied. In the light microscope effects were registered as inhibition of differentiation and formation of abnormal larvae with excess mesenchyme. With benzene, acridine, and aminoacridine cell separation and rapid disintegration of the embryos were observed, whereas with acridine orange the embryos developed into active larvae with a characteristic abnormal appearance. In the electron microscope weak effects were studied such as more distinct yolk granules than in the control. An increase in the number of lipid droplets was observed after treatment in phenanthrene and low concentrations of acridine orange. In the presence of acridine orange and Azur A the yolk granules appeared as more or less empty vesicles.

Photodynamic effects of dyes on bacteria: V. Mutagenensis by acridine orange and 460-nm or 500-nm light instranis of Escherichia coli that differ in repair capability

With acridine orange (AO) and monochromatic 460-nm light, the mutation rate of the wild-type strain of Escherichia coli (SP2) 3-fold higher than that of the endonuclease-deficinet strain WP2s ( uvrA). In addition, the mutation rates of the recombiination-deficient strains WP10 ( recA) and B s−1 ( uvrB lex A) were also about 3-fold less than that of teh wild-type straian. This observation is in striking contrast to the earlier results with AO and 500-nm light in which strains WP10 and B s−1 yielded mutation rates that were 12-fold and5-fold greater, respectively, that the wild-type response. The relatively large decrease in mutation reate when the uvrA endonuclease was absent together with structural condiseration in the binding of AO to DNA lead us to propose that the major lesions leading to mutations produced by 460-nm light in the presence of AO may be true DNA single-strand breaks that occur before DNA replication.

Photodynamic effects of dyes on bacteria: IV. Lethal effects of acridine orange and 460- or 500-nm monochromatic light in strain of Escherichia coli that differ in repair capability

• Lethal effects of acridine orange (AO) and monochromatic 460- or 500-nm light were measured in strains of Escherichia coli that differ in repair capability. Strains WP10 ( recA) and B s-1 ( lexA uvrB) were more sensitive by factors of 17 to 281 (based on F 37 values) than the strian which tested the most resistant in the presence of AO at both wavelengths, WP2s ( uvrA). The difference in sensitivity between the reA and WP2s strains was greater with AO and 500-nm light (281-fold) than with 460-nm light (115-fold) suggesting that although DNA lesions produced by either 460-nm or 500-nm light in the presence of AO are rendered non-lethal by rec + lex + -dependent repair processes, such repair is more efficient for the lesions produced by 500-nm light. • Survival curves with AO and 460-nm light were either of the simple exponential type (WP2 and B s−1) or were exponential with a small (WP10) or moderate (WP2s) shoulder. In contrast, the survival curves with AO and 500-nm light were of the 2-component typ for the 4 strains tested. The resistant component made up 7–75% of the population. The distinctly different survival-curve shapes obtained at the 2 wavelenghts indicate that difference DNA lesions are produced by AO and 460- and 500-nm light. The data obtained for lethality with AO and 500-nm and light are consistent with result previously reported, which indicate that the very high mutation rates obtained with recA strains are the consequence of the absence of rec + and lex + repair of DNA lesions.

PHOTODYNAMIC AND SUNLIGHT INACTIVATION OF ESCHERICHIA COLI STRAINS DIFFERING IN NEAR‐UV SENSITIVITY AND RECOMBINATION PROFICIENCY

Abstract— Stationary cells of four Escherichia coli strains exhibiting all four possible combinations of genes controlling near‐UV sensitivity (nur vs nur+) and recombination proficiency (far‐UV sensitivity; recA1 us recA+) have been inactivated by visible light in the presence of acridine orange (AO, 10µg/ml) and sunlight. The results demonstrate that strains sensitive to near‐UV inactivation are also sensitive to inactivation by visible light in the presence of AO and sunlight irrespective of the recA allele carried by the strain. These results may be interpreted to mean that major mechanisms of inactivation of stationary E. coli cells by near‐UV, visible light in the presence of AO and sunlight are similar and not closely related to the mechanism of inactivation by far‐UV.

Photodynamic effects of dyes on bacteria III. Mutagenesis by acridine orange and 500-nm monochromatic light in strains of Escherichia coli that differ in repair capability

In the presence of acridine orange (AO) and monochromatic 500-nm light, the recombination-deficient strain of Escherichia coli, WP10 ( recA), showed a 15-fold increase in mutation rate over the wild-type (WP2) strain. Under the same conditions, strain B s−1 ( uvrB lesA lon) showed a 5-fold increase in mutation rate over strain WP2. In contrast, the endonuclease-deficient strain, WP2s ( uvrA), showed a lower AO-500 nm mutation rate than wild-type. The extremely high mutation rate of the recA strain cannot be due to error-prone inducible SOS repair since the inducible recA + function is absent. Repair of the AO-500 nm-induced lesions is likely due to a recA +-dependent, error-free, recombination process. It is concluded that the high mutation rates with AO-500 nm light obtained in chemostat cultures of recA ad lexA strains occur as a consequence of errors during semi-conservative DNA replication in the presence of unrepaired DNA lesions.

Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis subsp. diacetylactis : Evidence for Plasmid-Linked Citrate Utilization

The use of Streptococcus diacetylactis as a flavor producer in dairy fermentations is dependent upon its ability to produce diacetyl from citrate. Treatment of S. diacetylactis strains 18-16 and DRC1 with acridine orange resulted in the conversion of approximately 2% of the DRC1 population and 20% of the 18-16 population to citrate negative, which is indicative of the involvement of plasmid deoxyribonucleic acid (DNA). Growth in the presence of acridine orange also resulted in the appearance of 2% lactose-negative derivatives in S. diacetylactis 18-16 and 99% lactose-defective, proteinase-negative derivatives in S. diacetylactis DRC1. Cesium chloride-ethidium bromide equilibrium density gradients of cleared lysate material from each strain revealed the presence of covalently closed circular DNA. Samples of this covalently closed circular DNA were subjected to agarose gel electrophoresis to determine the plasmid composition of each strain. S. diacetylactis 18-16 was found to possess six plasmids, of approximately 41, 28, 6.4, 5.5, 3.4, and 3.0 megadaltons (Mdal). S. diacetylactis DRC1 contained six plasmids, of approximately 41, 31, 18, 5.5, 4.5, and 3.7 Mdal. Variants of S. diacetylactis 18-16 which failed to produce acetoin plus diacetyl from citrate (citrate negative) were missing a 5.5-Mdal plasmid. Lactose-negative mutants of the same strain were devoid of a 41-Mdal plasmid. Lactose-defective, proteinase-negative mutants of S. diacetylactis DRC1 were missing a 31-Mdal plasmid. The citrate-negative mutants of S. diacetylactis DRC1 isolated in this study did not possess a 5.5-Mdal plasmid. Thus, we have evidence that there is a correlation between the ability to utilize citrate and the presence of a 5.5-Mdal plasmid. A relationship was also noted between lactose fermentation and proteinase activity and plasmid DNA in S. diacetylactis.

Physical properties of poly (3,N4-ethenocytidylic acid).

Homopolymers of etheno CMP have been prepared by the action of polyribonucleotide phosphorylase upon etheno CDP. At alkaline pH the optical properties are consistent with a structure consisting of partially helical single-stranded chains whose helical regions are stabilized by base stacking. At acid pH the degree of helicity increases markedly. The degree of cooperativity displayed by the helix leads to coil transition induced by pH or temperature is less than for the case of polyribocytidylic acid. In the presence of acridine orange the alkaline form develops a strong extrinsic CD spectrum.

On the Mechanism of the Acridine Orange Sensitized Photodynamic Inactivation of Lysozyme II. Kinetics in Presence of N-Acetylglucosamine

The photodynamic deactivation of lysozyme in presence of acridine orange is caused by a reaction between singlet oxygen formed via the dye triplet state and the protein. In order to identify the region where the singlet oxygen reacts with the protein we have investigated the kinetics of the deactivation in presence ofthe inhibitor of the enzymatic reaction N-acetylglucosamine (GlcNAc). The overall experimental rate constant becomes slower with increasing saccharide concentrations. As we can exclude experimentally that this kinetical effect is caused in presence of the saccharide by a physical quenching of singlet oxygen or of the dye triplet state it has to be assumed that GlcNAc protects the surrounding of its bindings place at subsite C of the enzymatic center sterically against an attack of singlet oxygen. In this region three tryptophan residues are located, which could be sensitive against singlet oxygen. Surprisingly, however, it has been found that only those species are protected, in which a second saccharide molecule is bound to the protein, probably at subsite E at the enzymatic center, where no sensitive amino acid side chains are located.

Conductivity, Emf, and Spectrophotometric Study on the Participation of Na+ and Cl− into Acridine Orange-Poly(α,l-Glutamic Acid) Complex

Abstract Conductivity, emf and spectroscopic investigations were made for the dimerization process of Acridine Orange (AO) and for the complex formation of AO with poly(α,l-glutamic acid) (PLG) in water. The increase of the dimer species of AO by adding the neutral salt was quantitatively explained, taking the ionic strength into account. From the emf measurements, the inclusions of Cl− into PLG–AO complex were concluded to be negligible, whereas Na+ was included into this complex to a fairly appreciable extent. The association of Na+ with carboxylate groups in the PLG–AO complex was described on the basis of the ordinary law of mass actions. The association constant Ki was unaffected by the presence of AO even in the helical region of PLG. These results strongly support Schwarz’s theory on cooperative binding.

On the mechanism of the acridine orange sensitized photodynamic inactivation of lysozyme. I. Basic kinetics.

The kinetics of the photodynamic desactivation of lysozyme in presence of acridine orange as the sensitizer have been investigated in detail varying oxygen, protein, dye concentration, ionic strength and pH value. The kinetics can be approximately described as an over all pseudo-first-order rate process. Changing the solvent from water to D2O or by quenching experiments in presence of azide ions it could be shown that the desactivation of lysozyme is caused exclusively by singlet oxygen. The excited oxygen occurs via the triplet state of the dye with a rate constant considerably lower than that to be expected for a diffusionally controlled reaction. Singlet oxygen react chemically (desactivation, k=2.9 X 10(7) m(-1) sec(-1)) and physically (quenching process, k=4.1 X 10(8) m(-1) sec(-1)) with the enzyme. The kinetical analysis shows that additional chemical reaction between singlet oxygen and lysozyme would have only little influence on the kinetics of the desactivation as long as their products would be enzymatically active and their kinetical constants would be less than about 1 X 10(8) m(-1) sec(-1)).

Isolation of a hybrid F' factor-carrying Escherichia coli lactose region and Salmonella typhimurium histidine region, F42-400 (F' ts114 lac+, his+): its partial characterization and behavior in Salmonella typhimurium.

Episome F' ts114 lac+ (F42-114) was transferred into Salmonella typhimurium carrying an F'his+ (FS400) episome, and fused episome F' ts114 lac+, his+ (F42-400) was obtained. Episome F42-400 could be transferred to S. typhimurium, Escherichia coli and Klebsiella pneumoniae. Identification of the episome was based on: (i) temperature sensitivity of the Lac+ and His+ phenotypes; (ii) the fact that F- segregants, obtained after temperature curing or acridine orange curing, were simultaneously Lac- and His-; and (iii) linkage of lac+ with his+ in episomal transfers to E. coli and S. typhimurium. The frequency of episome transfer was influenced by the genotype of the donor. Plasmid LT2, prevalent in S. typhimurium LT2 strains, was suggested to be responsible for the low fertility of S. typhimurium donors. Episome F42-400 was capable of chromosome mobilization, and the extent of chromosome mobilization was not influenced by the presence or absence of the histidine region on the donor chromosome. Growth in a defined medium with acridine orange was able to cure F42-400. The frequency of curing was increased (the frequency of His+ cells was 0.0001%) if the cells were grown at 40 C in the presence of acridine orange. Selection for temperature-resistant Lac+, His+ derivatives in a strain without histidine deletion yielded Hfr strains. However, similar and stronger selections in strains without the chromosomal histidine region failed to yield Hfr strains. Our inability to obtain Hfr's in strains without the chromosomal histidine region was explained by assuming that the episome F42-400 has lost the F sites involved in integration into the S. typhimurium chromosome.

Behavior of a hybrid F' ts114 lac+, his+ factor (F42-400) in Klebsiella pneumoniae M5a1.

Episome F' ts114 lac+, his+ (F42-400) was transferred from Salmonella typhimurium to Klebsiella pneumoniae. From the progeny, a strain of K. pneumoniae able to retransfer the episome was obtained. The His+ phenotype in this strain is temperature sensitive. Escherichia coli female-specific phages phiII, W31, and T3 were shown to plate on K. pneumoniae. From phiII we obtained two derivatives; phiIIK, which plates only on K. pneumoniae, and phiIIE, which plates only on E. coli. Growth of phages T3 and phiIIK was inhibited by F42-400 in K. pneumoniae. Growth in presence of acridine orange in a defined medium at 40 C resulted in a high level of curing. The frequency of His+ cells after growth in acridine orange at 40 C was 0.001%. An extensive search to detect chromosome mobilization by F42-400 in K. pneumoniae, under different experimental conditions, was negative. We cannot exclude the possibility that the low transfer efficiencies prevented our detection of chromosome mobilization. A search among temperature-resistant, acridine orange-curing-resistant, or galactose-resistant derivatives of the K. pneumoniae donor strain failed to reveal any chromosome transfer. Our failure to detect Hfr's may be a result of: (i) the peculiarity of episome F42-400, (ii) the peculiarity of K. pneumoniae chromosome, or (iii) low transfer efficiency. K. pneumoniae-modified F42-400 and phage 424 were restricted by E. Coli K-12. E. coli K-12-modified episome F42-400 and phage 424 were restricted by K. pneumoniae. E. coli C failed to restrict F42-400 modified with K. pneumoniae specificity. The ability of K. pneumoniae to accept F42-400 is less, by about a factor of 50, than that of E. coli C. As an explanation for the differences in the behavior of E. coli C and K. pneumoniae in ability to receive F42-400 it was suggested that recipient bacteria have specific sites for interaction with the F-pilus tip; these are present in E. Coli C, leading to high transfer efficiency, whereas they may not be present (or if present, are not accessible) in K. pneumoniae, leading to low transfer efficiency.

Irradiation of Escherichia coli in the visible spectrum with a tunable organic-dye laser energy source.

Pulsed laser energy was shown to be effective in inhibiting the growth of Escherichia coli. The irradiation source was derived from a tunable organic-dye laser utilizing rhodamine 6G (590 plus or minus 5 nm) solutions as lasing media. The organisms, suspended in nutrient broth, were irradiated both with and without an exogenous photosensitizer. One photosensitizer (toluidine blue) did not appreciably alter the inhibitory effect observed. In the presence of acridine orange, however, some additional growth occurred.

Irradiation of Escherichia coli in the Visible Spectrum with a Tunable Organic-Dye Laser Energy Source

Pulsed laser energy was shown to be effective in inhibiting the growth of Escherichia coli. The irradiation source was derived from a tunable organic-dye laser utilizing rhodamine 6G (590 plus or minus 5 nm) solutions as lasing media. The organisms, suspended in nutrient broth, were irradiated both with and without an exogenous photosensitizer. One photosensitizer (toluidine blue) did not appreciably alter the inhibitory effect observed. In the presence of acridine orange, however, some additional growth occurred.

The intermediate in the photodegradation of DNA in the presence of acridine orange

The intermediate in the photodegradation of DNA in the presence of acridine orange

Liquid holding recovery of photodynamic damage in E. coli.

Escherichia coli cells lose viability when irradiated with visible light in the presence of acridine dyes. There has been some controversy about whether such photodynamic damage can be repaired. Several investigations on different E. coli strains failed to reveal any significant difference in the sensitivity to photodynamic damage between radiation‐resistant E. coli B/r and radiation‐sensitive E. coli Bs‐1 strains (Uretz, 1964; Janovska et al., 1970) suggesting the absence of repair. On the contrary, other investigators (Rupp, 1966; Harm, 1968) strongly predicted the existence of a dark‐repair mechanism for such damage. No systematic study of liquid‐holding recovery of photodynamic damage has yet been reported and knowledge of repair capacity for photodynamic damage is still incomplete. In the present communication, liquid‐holding‐repair capability of E. coli B/r and Bs‐1 for photodynamic inactivation in the presence of acridine orange or acriflavine has been investigated. These studies show that B/r can repair photodynamic damage while Bs‐1 is deficient in this ability.