Presence Of Acriflavine Research Articles

Some observations on the lethal effects of near-ultraviolet light on Escherichia coli, compared with the lethal effects of far-ultraviolet light.

Abstract— Near‐ultraviolet light (365.5 nm) reduces the ability of Escherichia coli B/r and B8‐1, to form colonies on nutrient agar after irradiation. This lethal effect is distinct from that obtained after far‐u.v. irradiation (253.7 nm) because the far‐u.v. sensitive and resistant strains are equally susceptible to near‐u.v. Variation in susceptibility to ultraviolet light during growth is more marked for near‐u.v. than for far‐u.v. The number of survivors after near‐u.v. irradiation of log phase cells is affected by several post‐irradiation treatments; more cells survive if growth immediately after irradiation occurs at higher temperatures (unlike far‐u.v.). Also, the presence of acriflavine and caffeine in the nutrient agar decreases the number of survivors (in common with far‐u.v.).

“Excision repair” and dose-modification: Questions raised by radiobiological experiments with acriflavine

If bacteria are in contact with acriflavine before exposure to UV irradiation, they are strongly protected. With Escherichia coli B and 6 resistant and sensitive mutants, 30-min contact with acriflavine at 5 μg/ml confers protection which is strictly dose-modifying, and is the same for all variants, viz. dose-reduction factors of 10 and 15 for aerobic and anoxic conditions respectively. There is good chemical evidence for interpreting the protection as stemming from intercalation of the dye molecules between bases which are consequently inhibited from photochemical interaction between neighbours. Strict dose-modification is seen also when effects of UV are modified by enzymatic photoreactivation, which is known to monomerize pyrimidine dimers. Dose-modification is to be expected if an agent or procedure changes the probability that a given photochemical event will occur, or that it will remain in lethal form. It should also be expected, therefore, if a photochemical product is “excised” from DNA. However, “excision-repair,” which has been widely postulated to account for differences in sensitivity between variants of a strain, or for modification of UV damage, fails to act as a dose-modifying agent. Some of this failure has been attributed to the inactivation of the postulated “repair enzymes” by the radiation, but evidence from our experiments shows that this explanation is unlikely. Further experiments in which the lethal effects of UV were enhanced by the presence of acriflavine in the growth medium after irradiation throw doubt on the assumption that in this role acriflavine acts by inhibiting “excision repair”.

Effect of Quinacrine Hydrochloride and Acriflavine Hydrochloride on Development and Stability of Resistance to Dimetridazole by Tritrichomonas foetus

Quinacrine hydrochloride had no effect on the course of resistance development to dimetridazole by Tritrichomonas foetus in experimentally infected hamsters. Also in hamsters, exposure of a dimetridazole-resistant strain of T. foetus to acriflavine hydrochloride did not effect a reversion to the dimetridazole-sensitive state. Although streptomycin-resistant strains of bacteria survive and multiply when cultured in media containing quinacrine hydrochloride, or certain polyamines, and streptomycin, streptomycin-sensitive strains cultured under the same conditions do not develop a tolerance for the antibiotic. The literature on this interesting facet of drug resistance has been summarized by De Courcy and Sevag (1967) and by Warren et al. (1967). Moreover, McLoughlin and Gardiner (1968) found that quinacrine hydrochloride did not prevent Eimeria tenella from developing resistance to glycarbylamide. Mukherjea and Ray (1956) found cytochemical aberrations, including alteration in nuclear DNA, in Tritrichomonas foetus that had been cultured in the presence of acriflavine. Exposure to acriflavine, in vivo or in vitro, also induces dyskinetoplasy in several genera of the order Kinetoplastida. Trager and Rudzinska (1964) and Stuart and Hanson (1967) have summarized the literature on this phenomenon. Moreover, Mitsuhashi et al. (1961) and Watanabe and Fukasawa (1961) reported that transmissible drug resistance could be eliminated in vitro by culturing multiple resistant strains of Shigella flexneri or Escherichia coli in medium containing acriflavine. The cells became sensitive to all drugs to which they previously had been resistant. McLoughlin and Chute (1968) described a similar reversion by an amprolium-resistant strain of Eimeria tenella that was serially propagated in groups of chickens fed mash containing acriflavine. McLoughlin (1967) found that T. foetus developed resistance to dimetridazole and that Received for publication 29 October 1968. the degree of resistance persisted virtually unchanged for long periods. The present report describes the influence of quinacrine hydrochloride on the development of dimetridazole resistance by T. foetus, and the effects of acriflavine on the stability of this resistance. MATERIALS AND METHODS Quinacrine hydrochloride Hamsters, intravaginally infected with a dimetridazole-sensitive strain of Tritrichomonas foetus, were visually grouped by size into 3 lots of 10 and one of 5. Each lot was weighed and the various dosages were calculated on the basis of mean group weight. Hamsters of the first 3 groups were given an initial course of medication with quinacrine hydrochloride, low level dimetridazole, or a combination of the two. All four groups were then treated with dimetridazole at a level of 100 mg/kg. In the initial course of medication, the animals were treated for 5 consecutive days, rested for 2 days, and then treated for an additional 5 days. They were given a second 2-day rest period prior to initiation of the 5-day test treatment. Aqueous solutions of the medicaments were used and were adjusted so that 1 ml contained the appropriate dosage per hamster. With dimetridazole at the 100-mg level, it was necessary to slightly acidify the medium to increase solubility. The solutions were administered per os with a modified hypodermic needle and syringe. Vaginal secretions from principal and control hamsters were examined for trichomonads microscopically and by cultural techniques utilizing Diamond's medium (Diamond, 1957) after each treatment period. The protocol used for each group is given in Table I. Acriflavine hydrochloride The methods used for grouping, treating, and examining the hamsters were the same as in the studies with quinacrine hydrochloride. The principals were infected with dimetridazole-resistant T. foetus, Strain 4711 (McLoughlin, 1967). The protocol used for these trials is given in Table II.

Fine Structure of Bacteria Grown in the Presence of Acriflavine

The effect of acridine derivatives on bacterial growth has been shown to be dependent upon the concentration used, pH, temperature, and the position of the amino substituents on the acridine molecule. These factors, in turn, affect the amount of acridine bound to cell constituents. Many bacterial species, when grown in media containing acridities, become filamentous, or pleomorphic, or growth may be entirely prevented. The fine-structure of two species of bacteria treated with acriflavine was investigated. Both were Gram-positive bacilli, one was a Corynebacterium, and the other an aerobic spore-bearing Bacillus.Organisms were incubated at 21°c in the presence of concentrations of acriflavine ranging from 0.25 ug/ml to 12 ug/ml in phosphate buffered peptone water yeast extract medium at pH 7.5. Viable counts were carried out and the amount of acriflavine bound, either reversibly or irreversibly, was estimated at 450 mu, using a DB spectrophotometer. Cultures were observed by light microscopy and, after four days growth, were processed for electron microscopy by fixation in veronal-acetate pH 6.1 buffered 0.8% Os O4 for one hour and embedding in Epon 812 resin.

Oxidative phosphorylation in yeast. IV. Combination of a nuclear mutation affecting oxidative phosphorylation with cytoplasmic mutation to respiratory deficiency

1. 1. An attempt was made to superimpose a cytoplasmic mutation for respiratory deficiency ρ − over a nuclear mutation for oxidative phosphorylation deficiency by treatment of an oxidative phosphorylation-deficient yeast mutant op 1 with acriflavine. Unlike wild-type yeast, cells of the mutant op 1 grown in the presence of acriflavine did not form visible colonies after plating on solid medium with glucose as carbon source. 2. 2. Growth of the op 1 mutant in acriflavine-supplemented medium was limited. After about 10 generations the cells of the mutant ceased to multiply. They were then unable to respire, lacked cytochrome b and aa 3 but seemed not to be dead since they fermented and assimilated glucose and did not stain with methylene blue. 3. 3. In the culture of the op 1 cells growing in the presence of acriflavine a considerable fraction of cytoplasmic respiration-deficient mutants was detected by crossing the cells with a neutral cytoplasmic mutant of opposite mating type. 4. 4. Revertants from the op 1 mutant which had grown in the presence of acriflavine gave rise to normal, viable respiration-deficient colonies after plating on solid medium. 5. 5. It has been concluded that specific combination of the cytoplasmic ρ − and nuclear op 1 mutations in a cell resulted in loss of multiplication ability. Implications of such a cumulative effect for the nature of op 1 and ρ − mutations are discussed.

Drug Resistance in Eimeria tenella. VII. Acriflavine-Mediated Loss of Resistance to Amprolium

An amprolium-resistant strain of Eimeria tenella was propagated serially in chickens fed mash containing 0.05% acriflavine hydrochloride. Increased sensitivity to amprolium was evident after the first passage. After six passages in the presence of acriflavine, the response of the experimental strain was approximately the same as that of an amprolium-sensitive control strain. Exposure to acriflavine, in vitro or in vivo, induces dyskinetoplasy in a number of genera of the order Kinetoplastida. The literature on this phenomenon has been summarized by Trager and Rudzinska (1964) and by Stuart and Hanson (1967). Mukherjea and Ray (1956) found cytochemical aberrations including alteration in nuclear DNA in Tritrichomonas foetus that had been cultured in the presence of acriflavine. Moreover, Mitsuhashi, Harada, and Kameda (1961) reported that transmissible (F factor) drug resistance could be eliminated in vitro by culturing multiple resistant strains of Shigella flexneri and Escherichia coli in medium containing acriflavine. The cells became sensitive to all drugs to which they previously had been resistant. The present report describes the response of an amprolium-resistant strain of Eimeria tenella to propagation in chickens fed mash containing acriflavine. MATERIALS AND METHODS Three-week-old chicks were used. They were grouped by weight as suggested by Gardiner and Wehr (1950) into lots of 10, and each lot was started on the appropriate mash 24 hr before the infected groups were inoculated with approximately 100,000 sporulated Eimeria tenella oocysts. In the initial passage, groups of birds fed unmedicated mash, mash containing 0.0125% amprolium, or mash containing 0.05% acriflavine hydrochloride were given oocysts from an amproliumsensitive strain. In subsequent passages of this strain, oocysts recovered from the unmedicated group were used to inoculate the three groups of the succeeding passage. Similar groups of birds initially were given oocysts of a strain that had been serially passed through 64 groups of birds fed mash containing amprolium. In subsequent passages, oocysts recovered from the unmedicated and from the acriflavine-medicated birds were used to inoculate the respective groups of the next Received for publication 22 March 1968. passage. This protocol is presented as a flow sheet in Figure 1. An uninfected, unmedicated group was carried at each passage to allow comparison of weight gains. On the 8th day after inoculation, the surviving birds were weighed and necropsied. The ceca were removed and cecal lesions evaluated. Oocyst cultures were made from the ceca and cecal contents of the respective infected groups, and the number of oocysts produced per surviving bird was estimated for each group. Weight gains, oocyst production, mortality rates, and cecal lesion scores obtained at each passage were used to calculate the Anticoccidial Indices for the respective groups as suggested in the Merck Bulletin on Amprolium (1960). These Indices form the basis for the comparisons made.

Changes in Sensitivity to Acriflavine of Escherichia coli Grown in Media of Different Glucose Contents

SUMMARY: Organisms of Escherichia coli K-12 strains sensitive and resistant to acriflavine were plated on media with and without acriflavine after growth in media containing different concentrations of glucose. Proportionally more organisms produced colonies, in the presence of acriflavine, after growth in media containing a high concentration of glucose than in media with lower glucose contents. The final pH value of the growth medium was low with the high glucose media. With the resistant strain, the number of bacteria which survived acriflavine increased as the final pH value of the medium from which bacteria were harvested was decreased, but the initial glucose concentration rather than the final pH value of the culture was more influential in increasing survival with the sensitive strain. The acriflavine-binding capacity of the bacteria was affected by the initial glucose concentration of culture medium probably indirectly through a change of pH. Acriflavine sensitivity of the bacteria varied with the amount of acriflavine bound. The acriflavine-binding capacity of bacteria modified by the pH value of culture medium was stabilized in the course of several doublings of bacteria in that medium. The glucose concentration of the medium affected the acriflavine sensitivity of the sensitive strain through some mechanism other than the change of pH.

The loss of kinetoplastic DNA in two species of Trypanosomatidae treated with acriflavine.

The effects of acriflavine on two species of Trypanosomatidae, Crithidia luciliae and Trypanosoma mega, have been investigated. It has been observed that kinetoplastic (i.e. mitochondrial) DNA is lost in a high percentage of acriflavine-treated cells. Resting flagellates, from stationary-phase or hemin-deficient cultures, are considerably more resistant to the acridine than are flagellates from a log-phase culture. When the kinetoplast has retained some DNA and still remains visible in stained smears, it appears reduced in size, and its ultrastructure is extremely abnormal: the DNA fibrils, clearly visible in normal kinetoplasts, are condensed; they appear as an electron-opaque, apparently homogeneous mass, separated from the membranes by a space of low electron-opacity. Analyses of DNA extracts, with high speed centrifugation in CsCl density gradients, revealed that the satellite band, presumably kinetoplastic DNA, is lost by trypanosomes grown for 5 days in the presence of acriflavine. Radioautography was used to study the effects of acriflavine on thymidine-(3)H incorporation in C. luciliae. At the concentration which affects the kinetoplast specifically, the dye produces an 87% inhibition of thymidine incorporation in this organelle. The kinetics of this inhibition suggest a direct effect on replication. No decrease in incorporation occurs in the nucleus. These results lead to the conclusion that loss of kinetoplastic DNA is due to continued growth and cell division in the absence of kinetoplastic DNA replication. Several hypotheses are discussed concerning the specificity of the dye's action upon the replication of extrachromosomal DNA.

On the relationship between respiratory activity and lipid composition of the yeast cell

1. 1. Phospholipids, total fatty acids, sterols and hydrocarbons were determined in respiratory-competent and respiratory-deficient cells of Saccharomyces cerevisiae grown aerobically and anaerobically. 2. 2. In a respiratory-competent strain, the previously-reported reduction in the content of phospholipids, fatty acids, and sterols of cells grown anaerobically on glucose in medium supplemented with ergosterol and Tween 80, relative to aerobically-grown cells, was confirmed. Anaerobically-grown cells from medium lacking ergosterol contained more phospholipids and fatty acids, and from medium lacking both ergosterol and Tween 80 contained more phospholipids, than cells from the supplemented medium. 3. 3. Changes in the lipid fractions during respiratory adaptation of anaerobically-grown respiratory-competent yeast were followed. The level of phospholipids rose during the first 2 h of adaptation, the level of other fractions rising during several hours approximately parallel with the development of respiratory activity. 4. 4. When anaerobically-grown respiratory-competent yeast was aerated in the presence of acriflavin or chloramphenicol, no respiratory adaptation took place. However, the content of phospholipids and of total fatty acids increased in a manner similar to that in cells during respiratory adaptation, whereas the content of sterols increased less. The cells aerated in the presence of cycloheximide had no respiratory ability, the content of phospholipids was similar to that in the cells aerated with acriflavin or chloramphenicol, the content of fatty acids was intermediate between aerobically- and anaerobically-grown cells, and the content of sterols was markedly reduced and approximated that of non-aerated anaerobically-grown cells. 5. 5. The respiratory-deficient mutant, derived from the competent strain, showed the same pattern of lipid composition as the original strain. The amount of lipids was reduced in anaerobically-grown cells. The lipid composition of aerobically-grown mutant cells was the same as that of aerobically-grown wild yeast, except for the phospholipid content which was lower in the mutant.

Differential effects of acriflavine and caffeine on various ultraviolet-irradiated Escherichia coli strains and T1 phage.

Abstract Caffeine and acriflavine are known to inhibit repair of UV lesions in DNA of host-cell-reactivating [HCR(+)] Escherichia coli strains. Large differences were observed between various strains: B, B/r(λ) and K12(λ), carrying extra UV sensitivity relative to B/r and K12S, respectively, show much greater repair inhibition than the latter strains. This seems to be related to the (normally) rather incomplete repair in B, B/r(λ) and K12(λ), which was inferred from results on liquid-holding recovery 6 . Both incompleteness of repair and its great susceptibility to inhibition by caffeine or acriflavine could be explained by the assumption that UV lesions related to the extra sensitivity require the repair early . This assumption seems plausible at least for the extra sensitivity resulting from lysogenicity. Decreased UV survival in the presence of acriflavine or caffeine was also found in HCR(−) strains. B s−1 and B syn − , differing considerably in their UV sensitivities, show equal survival (at levels between 1 and 10 −4 ) if incubated with 7.5 ωg/ml acriflavine. The possible interpretation that all HCR(−) strains tested exhibit residual repair activity in the absence of acriflavine or caffeine, is supported by the finding that HCR(−) strains also show a slight residual HCR activity, inhibitable by acriflavine or caffeine, toward UV-irradiated phage Tr. However, part of the decreased cell survival might be due to synergistic effects between UV and acriflavine. At the highest applicable concentration, acriflavine is a stronger repair inhibitor than caffeine. However, neither substance decreases UV survival of HCR(+) cells or phage infecting HCR(+) cells to the level to which survival of HCR(−) cells or phage infecting HCR(−) cells is decreased by acriflavine or caffeine.

EFFECTS ON IRRADIATED MICRO-ORGANISMS OF GROWTH IN THE PRESENCE OF ACRIFLAVINE.

Investigations were made on the effects of acriflavine on lethal damage induced in microorganisms by ionizing as well as by ultraviolet radiation. Growth curves for E. coli B and E. coli B/r on solid medium incorporating 5 gamma /ml acriflavine showed that the initial lag phase was prolonged, but the generation times were unaffected. The most striking characteristic of acriflavine is that it affects all strains in the same sense, the incorporation of acriflavine in the medium bringing additional radiation damage to light. In every case the effect was greater after ultraviolet than after ionizing radiation, and, with the latter, the treatment was more effective if suspensions were irradiated in anoxic than in aerobic conditions. (P.C.H.)

Transmission of colicinogeny between strains of Salmonella typhimurium grown together.

SUMMARY: Ability to produce colicines I, E1, E2, K or B was transferred to Salmonella typhimurium strain LT2 by growth in broth with suitable colicinogenic strains of Escherichia coli or Shigella sonnei. When LT2 (colI), i.e. carrying the colicine I factor, or LT2 (colB) were grown overnight in broth with LT2 col - (non-colicinogenic), c. 50 % of the latter became colicinogenic; LT2 (colE2) and LT2 (colK) did not transmit; LT2 (colE1) transmitted to only c. 0.1 % of the acceptor population. But LT2 carrying either colI or colB in addition to colE2, colK or colE1, transmitted both factors. When overnight broth cultures of LT2 (colI) and LT2 col - were mixed and incubated c. 40 % of the latter acquired colI by 20 hr. (when the viable count had doubled); but only c. 0.02 % acquired colI in 3 hr. The low initial transfer results from the fact that in a stock culture of LT2 (colI) only c. 1/5000 bacteria are ‘competent donors’, able to transmit colI. The later large increase in the proportion of colicinogenic bacteria probably results from ‘epidemic spread’ of the colI factor amongst the acceptor population, initiated by the few acceptor bacteria which originally receive it. It is supposed that most bacteria which have just acquired colI become competent donors. In a doubly colicinogenic strain most competent donors transmit both colicine factors. Aeration by shaking during incubation interfered with transmission of colicinogeny, probably by abolishing the prolonged phase of slow growth of unaerated cultures. Growth in the presence of acriflavine did not ‘cure’ LT2 (colI) or LT2 (colI) (colE2) of colicinogeny, nor of ability to transmit. LT2 (colE1) and LT2 (colE2) supported the epidemic spread of colI or colB about as well as did LT2 col -; but in LT2 (colK) the spread of colI was greatly reduced and that of colB somewhat reduced. The prior presence in an acceptor strain of one of the readily transmissible factors, colI or colB, did not interfere with the epidemic spread of the other. But LT2 (colI) did not become a competent donor on accepting colE2 and, by inference, colI from LT2 (colI) (colE2).

Inactivation by ultraviolet light of an acriflavine-sensitive gene function in phage T4D.

Mutants of phage T4D can be isolated which multiply in the presence of concentrations of acriflavine inhibitory to the growth of wild-type phage. Bacteria mixedly infected with resistant and sensitive phage are unproductive in the presence of acriflavine. Irradiation of the sensitive phage with ultraviolet light results in inactivation of the function of the gene for acriflavine sensitivity, permitting the mixedly infected complexes to yield phage. This function is inactivated in an exponential manner and has a cross section comparable to cross sections of other gene functions in T4D.