Photodynamic inactivation and its repair in mycoplasmas

Photodynamic inactivation is the loss in viability observed when organic dye-treated cells are exposed to visible light and molecular oxygen. The photodynamic inactivation of mycoplasmas, the smallest free living cells, has been studied. Depending on the extent of inactivation in Acholeplasma laidlawii, photodynamic induced damage can be repaired if the irradiated cells are incubated in the dark in buffer. Analysis of the DNA of these cells shows that photodynamic inactivation induces single strand breaks which can be repaired during liquid holding. To examine possible damage to the cell membrane, glucose uptake was studied as a permeability measure. Neither acriflavine nor photodynamic inactivation had any measurable effect on membrane permeability.     

The enhancement of the mutagenic effect of ultraviolet radiation inEscherichia coli by caffeine and acriflavine

The mutational synergism of caffeine and acriflavine was studied in five types ofEscherichia coli mutants induced by u. v.-radiation. The following types of mutations were compared: streptomycinrresistance (strain B/r), streptomycin-independence (strain Sd-4), and reversions to prototrophy (strains WP-14 pro⁻, WP-2 try⁻, and WP-2 try⁻ hcr⁻). In all hcr⁺ strains tested the presence of caffeine or acriflavine in a post-irradiation plate medium slightly decreases the survival of u.v.-irradiated cells and increases considerably the frequency of induced mutations. The mutational synergism of caffeine and acriflavine in the str-r and str-i mutants is observed only within the range of low doses. The abovementioned dose-dependence of the synergistic effect is discussed from the point of view of qualitative difference between the premutational damage caused by low and high doses. The post-irradiation treatment by caffeine slightly increases the frequency of induced prototrophs also in the WP-2 hcr⁻ strain. This finding is explained by the inhibition of the residual HCR-activity of the strain. The post-irradiation mutational synergism of acriflavine was not found in the WP-2 hcr⁻ strain.     

Protection of Normal, Lysogenic, and Pyocinogenic Strains Against Ultraviolet Radiation by Bound Acriflavine

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.     

Effect of acriflavine on ultraviolet inactivation of Acholeplasma laidlawii

An increased sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawiiAn increase sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawii cells were plated on medium containing either acriflavine or chloramphenicol. Chloramphenicol reduced liquid holding recovery (dark repair) to about 10 percent of that in untreated irradiated cells. In acriflavine treated cells no dark repair could be observed and there was a progressive degradation of cell DNA during holding. While the primary effect of acriflavine may be to inhibit excision repair, since ultraviolet-irradiated Mycoplasma gallisepticum (cells which lack an excision repair mechanism) show a slight increase in inactivation when plated on medium containing acriflavine, the dye must also have some other effects on ultraviolet repair processes. Acriflavine treatment of A. laidlawii cells before ultraviolet irradiation has a protective effect, as seen by an increased cell survival.     

Fusarium oxysporum Forms Heterokaryons with Fusarium redolens

Vegetative compatibility among three isolates of Fusarium oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens from diseased lupins was investigated. Pairings between five mutants originated from each isolate revealed two compatibility groups. The first VCG comprised race 1 of F. oxysporum f. sp. lupini and one isolate of F. oxysporum var. redolens; the second VCG comprised race 2 of F. oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens. Heterokaryon formation was observed in many pairings involving mutants of both taxa. These findings provide evidence of the conspecificity of these two taxa and they support Gordon 's classification (1952) according to which F. redolens is actually F. oxysporum.     

Evidence for repair of ultraviolet-induced damage in Cystobacter species (Myxobacterales).

Cystobacter species strain CK 1 does not grow with more than 0.2 microgram/ml acriflavine. Spontaneous two-step mutants growing with 2 microgram acriflavine per ml have been selected. One mutant (strain CK3) was used to investigate the effect of repair inhibitors. Both strains exhibit pronounced shoulders in their UV dose curves of inactivation. Acriflavine (AF), coumarin (CU), and caffeine (CA) when incorporated in the post-irradiation plating medium decreased survival of irradiated cells. Post-treatment with 2 microgram acriflavine/ml abolished the shoulder of the curve. Caffeine (1600 microgram/ml) and coumarin (350 microgram/ml) reduced it only to about 40%. It is concluded that probably two repair mechanisms are present. Pre-treatment of the cells with 2 microgram acriflavine/ml for two hours before UV-irradiation resulted in a constant dose enhancement factor of 1.9. The protective effect is increased with the time of treatment with acriflavine. This may indicate that pyrimidine dimers are responsible for UV-inactivation.     

The action of acriflavine on yeast protoplasts

Summary The effect of acriflavine on protoplasts of a strain ofSaccharomyces carlsbergensis has been compared with the effect on intact cells of the same strain. The strain was typical of the species in that little or no mutation occurred when young cells were exposed to acriflavine at concentrations up to 50 μg/ml for 4 hours. However, after exposure of protoplasts, prepared by the action of snail crop juice, to acriflavine at concentrations up to 10 μg/ml for the same period, the rate of oxidation of acetate was reduced by 45% whereas the rate of fermentation of glucose remained unchanged. It is concluded that the susceptibility of cells to acriflavine may be dependent on the resistance of cell wall material to penetration by the dye, thus controlling the rate of entry into the cytoplasm.     

Instability in the ctMR2 strain of Drosophila melanogaster: role of P element functions and structure of revertants

Summary Simultaneous multiple transpositions and longterm genetic instability have been described in the ct ^MR2 strain of Drosophila melanogaster and its derivatives. This strain originated from a cross that was dysgenic in the P-M system. While spontaneous instability declined over 2 years, instability has been reactivated by backcross to the progenitor P element bearing strain MRh12/Cy. We show here using germline transformation that active P factor alone cannot mimic the effect of this cross, suggesting that MRh12/Cy contains some other activator. In addition, we have observed that ct ⁺ exceptional progeny arise in the F1 s well as the F2 generations. Molecular analysis of X chromosomes from some ct ⁺ progeny indicates that phenotypic reversion of the ct mutation can arise through two unrelated mechanisms.     

Modeling of <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial growth kinetics and optimal fed-batch fermentation for beauvericin production

Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20–50 g l⁻¹ glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l⁻¹) than in the batch culture (194 mg l⁻¹). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.     

Chlorate and nitrate reduction in the phototrophic bacteriaRhodobacter capsulatus andRhodobacter sphaeroides

Chlorate or trimethylamine-N-oxide (TMAO) added to phototrophic cultures ofRhodobacter sphaeroides DSM 158 increased both the growth rate and the growth yield although this stimulation was not observed in the presence of tungstate. This strain, exhibited basal activities of nitrate, chlorate, and TMAO reductases independently of the presence of these substrates in the culture medium, and nitrate reductase (NR) activity was competitively inhibited by chlorate. Phototrophic growth ofRhodobacter capsulatus B10, a strain devoid of NR activity, was inhibited only by 100 mM chlorate. However, growth of the nitrate-assimilatingR. capsulatus strains E1F1 and AD2 was sensitive to 10mm chlorate, and their NR activities were not inhibited by chlorate. Both NR and chlorate reductase (CR) activities of strain E1F1 were induced in the presence of nitrate or chlorate respectively, whereas strain AD2 showed basal levels of these activities in the absence of the substrates. A basal TMAO reductase (TR) activity was also observed when these strains ofR. capsulatus were cultured in the absence of this electron acceptor. These results suggest that chlorate and TMAO can be used as ancillary oxidants byRhodobacter strains and that a single enzyme could be responsible for nitrate and chlorate reduction inR. sphaeroides DSM 158, whereas these reactions are catalyzed by two different enzymes inR. capsulatus E1F1 and AD2.     

Identification of Fusarium species causing basal rot of onion in East Azarbaijan province,Iran and evaluation of their virulence on onion bulbs and seedlings

One of the economically important diseases of onion is the basal rot caused by various Fusarium species. Identification of the pathogenic species prevalent in a region is indispensable for designing management strategies, especially to develop resistant cultivars. Eighty Fusarium isolates are obtained from red onion bulbs on infected fields of East Azarbaijan province. Inoculating the onion bulbs with 38 selective isolates indicated that 17 isolates were pathogenic on onion. According to the morphological and molecular characteristics, these isolates were identified as F. oxysporum, F. solani, F. proliferatum and F. redolens. This is the first report of F. redolens on onion in Iran. On the other hand, the virulence of each pathogenic isolate was evaluated on onion bulbs and seedlings. F. oxysporum which causes severe rot and damping-off was considered as a highly virulent species in both conditions. While, F. proliferatum was considered as the most destructive on onion bulbs. Rot ability of F. solani was not considerable, and only the 4S isolate caused pre- and post-emergence damping-off more than 50%. Finally, F. redolens with less pathogenicity on onion bulbs was identified as the most virulent isolate on onion seedlings, which was explanatory of its importance on farm.     

Use of Rep‐PCR for Genetic Diversity Analyses in Fusarium culmorum

Fusarium culmorum is a pathogen of economically important grain crops. In this work, Rep‐PCR was used to identify genetic diversity in F. culmorum isolates which have been collected from wheat fields in Turkey. Reproducible genomic fingerprints were amplified in each strain by PCRs of prokaryotic repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC) and BOX sequences. Totally 104 molecular markers were evaluated and similarity comparisons were shown as a dendrogram. The average genetic diversity was 52.3% ranging from 15.8% to 88.7% according to the Rep‐PCR data. Cluster analysis showed agreement with the distance of sampling locations. The highest genetic similarity (84.2%) was determined between two F. culmorum isolates (F1 and F2) originated from the same agro‐ecological region. Our results showed that Rep‐PCR is convenient and rapid for genetic diversity analyses and strain differentiation in F. culmorum.     

Lack of surface receptors not restriction-modification system determines F4 phage resistance inStreptococcus bovis II/1

The resistance ofStreptococcus bovis strain II/1, the producer ofSbvI restriction endonuclease, to F4 phage infection was demonstrated by the double-agar-layer method. Despite the presence of restriction endonucleaseSbvI which can cleave F4 phage, DNA to numerous fragmentsin vitro, the evidence that adsorptio inhibition is the most important defence mechanism in phage resistance ofS. bovis II/1 strain was obtained by adhesion experimentsin vivo. Electron microscopy of phage-host mixtures showed many phage particles on the bacterial surface of phage-sensitiveS. bovis 47/3 control strain in comparision with no phage particles seen onS. bovis II/1 (phage-resistant) strain surface.     

Photodynamic effects of deuteroporphyrin on Gram-positive bacteria

The photodynamic effects of deuteroporphyrin (DT) on the growth and viability ofStaphylococcus aureus, Streptococcus faecalis, andBacillus cereus are described. By exposure to light and DT, the growth ofSta. aureus andStr. Faecalis strains was markedly inhibited, whereasB. cereus was undergoing lysis. Counting the viable bacteria during the DT treatment revealed that more than 99% of the initial bacterial population was killed within the first 30 min of treatment. The pH dependence of the photodynamic inhibitory effect shows that it is best obtained at pH 6.5. No temperature dependence in the range of 37°–44°C could be detected. The best photodynamic effect was achieved when the culture was in the mid-log phase. DT-treated bacteria, when grown in the dark or in the presence of horse serum albumin, were unaffected by the porphyrin action. Electron microscopic examinations ofSta. aureus andStr. faecalis showed the appearance of well-developed mesosomes within the cell cytoplasm.B. cereus preparations have not shown any mesosome formation but showed some lysed cells as well as some spores. None of the mentioned effects by DT and light could be observed on Gramnegative bacteria such asEscherchia coli orPseudomonas aeruginosa.     

Expression of pigmentation genes following electroporation of albino<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth. The growth rates of all eight color variants were significantly greater than the recipient and donor strains under some culture conditions. Spectrophotometric analysis of the pigments extracted from the color variants revealed the pigments had absorbance spectra different from the DNA donor strain. These color variants may have resulted from transformation with wild-type DNA, mutation reversion, or activation of alternative pathway(s)—i.e., new mutations—that resulted in pigment production.     

Enhanced UV Sensitivity of Thiobacillus ferrooxidans Resulting from Caffeine and Acriflavine Treatment of Irradiated Cells

This study was aimed at identifying the roles of caffeine and acriflavine, two repair inhibitors, on UV sensitivity of iron-oxidizing Thiobacillus ferrooxidans ATCC 13728. The UV-dose response survival curve was inflected in nature, suggesting the population heterogeneity of the isolate. Caffeine and acriflavine potentiated the UV-induced killing of the organism. With the increase in concentrations of these compounds, the extent of survival decreased. Similarly, the inhibitory effects of caffeine and acriflavine increased with the increase in dose of UV-irradiation. The cells irradiated with 10 s (equivalent to 5.6 × 10⁻⁵ J/m²/s) of UV-exposure tended to become resistant to the inhibitory effects of caffeine and acriflavine, as evidenced by the time course study of recovery. The cells appear to stage a dramatic recovery from UV damage in the presence of caffeine (3.0 mg/ml) and acriflavine (20 μg/ml) over a period of 25–30h and 35–40h respectively, when grown in the presence of energy sources. Received: 4 December 2000/Accepted: 10 January 2001     

E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells

The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.     

The effect of ultrasound and its combination with radiation on the genetic material ofSaccharomyces cerevisiae

Ultrasonication at 20 kHz, intensity 35 W/cm² and amplitude 15–25 μm of a diploid strain ofSaccharomyces cerevisiae was found to act as a weak mutagen with maximum efficiency at the 20 % survival of the cells. Under these conditions, the frequency of reversion of the suppressible allele ilv1-92 increased ten times, the frequency of mitotic gene conversion four times. Doses leading to survivals lower than 20 % led to a slight increase in the frequency of cytoplasmic respiration-deficient mutants. Submutagenic doses applied immediately after γradiation or UV light did not substantially increase the effect of these physical agents on the genetic material of the yeast strain investigated. Application of ultrasound prior to UV radiation did not considerably influence the effect of the radiation either.     

Using C60 fullerenes for photodynamic inactivation of mosquito iridescent viruses

This article describes the photodynamic inactivation of mosquito iridescent virus (MIV) Aedes flavescens in the presence of water-soluble C₆₀ fullerenes. It has been observed that the photodynamic inactivation of MIV for about 1?h reduces the infectious titre of the virus in large wax-moth larvae Galleria mellonella to 4.5 lg ID₅₀/mL. The influence of the C₆₀ concentration on its anti-viral activity was tested in the concentration range from 1 to 0.001?mg/mL. It has been found that C₆₀ is able to inactivate the iridovirus even in low concentrations. Consequently, the findings of this work suggest that photoexcited C₆₀ fullerenes can be successfully used for the inactivation of iridoviruses in biological systems.     

Photobiological activity of exogenous and endogenous porphyrin derivatives in Escherichia coli and Enterococcus hirae cells

Photodynamic treatment, the combined application of a photosensitiser and visible light, represents a new and promising approach for the inactivation of microorganisms. The photosensitising potentials of exogenous zinc-phthalocyanine-tetrasulphonate (ZnPsTS), tetraphenylporphyrins (TPPs) and endogenous porphyrin derivatives were tested and compared on Gram-negative and Gram-positive bacteria, Escherichia coli B. and Enterococcus hirae, respectively. The synthesis of endogenous porphyrins was induced by 5-aminolevulinic acid (δ-ALA). The porphyrin- or δ-ALA-treated cells were irradiated with white light. The photosensitising efficiency of endogenous derivatives on both types of bacteria is ZnPcTS < TP(4-OGluOH)₃P < TP(4-OGluOH)₄P. However, neither exogenous derivatives exhibit appreciable photosensitising activity for disinfection application. ALA-induced photodynamic treatment showed good potential for the inactivation of Escherichia coli cells, but not towards Enterococcus hirae cells. The failure of photosensitisation of the Enterococcus hirae strain selected indicates that apart from the Gram-positive character, other structural elements of the membrane can influence the result of photodynamic treatments. Received: 13 October 1999 / Accepted: 1 January 2001