Effect of itraconazole on Staphylococcus aureus biofilm and extracellular vesicles formation.

Staphylococcus aureus is a leading cause of a wide range of clinical chronic infections mainly due to the establishment of a biofilm. Biofilm, a population of bacteria within a self-produced matrix of extracellular polymeric substance, decreases the susceptibility to antibiotics, immune defenses and contributes to antimicrobial resistance. To date antibiotic combination has been considered a strategy to combat S. aureus infection, but this approach does not solves the main pharmacokinetic problem caused by biofilms, consisting in insufficient drug penetration within the structure. Therefore, new antimicrobial agents that could overcome this resistance need to be discovered. Fighting staphylococcal resistance and biofilm formation is an important goal of the pharmaceutical research. Some fungicide has been observed to have antibacterial effect. anyway their use as antibiotics on S.aureus has been poorly studied. The aim of this work was to investigate the effect of the fungicide itraconazole (IT) on S. aureus biofilm formation and explore by SEM the morphological alteration after treatment. A strong biofilm disaggregation and morphologically different extracellular vesicles (EV) production were observed starting from sublethal IT doses. This suggests that IT resistance phenomena on the part of S. aureus are more difficult to establish respect other antibiotics. The adjuvant properties of IT could be used to combat bacterial biofilm and/or to improve antibiotic treatment. Moreover, because the production of EV represents a secretory pathway involved in intercellular communication shared to mammalian cells, fungi, and bacteria, our study is important to increase information that can be generalized to higher organisms.

Expression and characterization of two new alkane-inducible cytochrome P450s from Trichoderma harzianum.

n-Dodecane and fatty acids were good inducers of cytochrome P450 (CYP) and the ω-hydroxylase of lauric acid, which is a marker for ω-hydroxylation of n-alkanes, in Trichoderma harzianum. A cDNA, containing an ORF of 1520 bp, encoding a CYP52 of 520 amino acids, was isolated by RACE. Another n-alkane-inducible CYP was identified by LLC-MS/MS analysis of a microsomal protein band induced by n-dodecane in a library of T. harzianum. This suggests that T. harzianum has a CYP-dependent conversion of alkanes to fatty acids allowing their incorporation into lipids.

Spontaneous chromosome loss and colcemid resistance in lymphocytes from patients with myotonic dystrophy type 1.

Myotonic Dystrophy type 1 (DM1) is one of the many inherited human diseases whose molecular defect is the expansion of a trinucleotide DNA sequence. DM1 shares with fragile X syndrome (FMR1), another "unstable triplet syndrome", several molecular features not present in the remaining triplet diseases. As FMR1 is also characterised by chromosome instability at the site of the expanded triplet, lymphocytes from DM1 patients and healthy donors were cultured for micronucleus (MN) analysis, in order to verify if DM1 is also prone to chromosome instability. A FISH analysis was also carried out to detect the presence of centromeric sequences in the observed MN. The data indicate that DM1 patients present a percentage of centromere-positive MN significantly higher than controls, suggesting that chromosome loss is the main mechanism underlying the origin of the increased spontaneous instability. To further assess the proneness to instability of cells of DM1 patients, cultures from patients and controls were treated in vitro with growing concentrations of two different mutagens: colcemid, a "pure" aneugen compound whose target is tubulin, and mytomicin C, a strong clastogen. The results show that the patient group is significantly less sensitive to colcemid. These data, together with FISH analysis, suggest the presence, in DM1 patients, of an already damaged tubulin, which becomes no more sensitive to the effect of colcemid and which could be the main defect underlying the aneugenic effects in DM1.

Metabolism of 5-methoxypsoralen by Saccharomyces cerevisiae.

Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects. The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450. Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism. As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture. The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite. As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation. Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.

Conditions that influence the genetic activity of potassium dichromate and chromium chloride in Saccharomyces cerevisiae.

Potassium dichromate and chromium chloride were analyzed for their ability to induce mitotic gene conversion and point reverse mutation in D7 diploid strain of S. cerevisiae. We used cells from the stationary phase of growth with and without metabolic activation (S9 hepatic fraction) and cells from the logarithmic phase, that contain a high level of cytochrome P-450 and have a greater permeability. In the present work we confirmed the genetic activity of K2Cr2O7 in cells from the stationary phase, with and without S9 fraction and in cells from the logarithmic growth phase. A slight increase in genetic activity was observed in experiments with CrCl3 using phosphate buffer, but no genetic effects were noted in Tris-HCl buffer. Our studies suggest that phosphate ion may be the carrier responsible of the entrance of trivalent chromium in the cells. The higher cellular permeability may account for the different results obtained with both compounds in cells from the stationary and logarithmic phases of growth.

Inhibition of yeast cytochrome P-450 by ammonium metavanadate.

Incubation of diploid D7 strain cells of Saccharomyces cerevisiae (grown in 20% glucose) in the presence of ammonium metavanadate (AMV) led to a decrease in the cytochrome P-450-dependent monooxygenase system (cytochrome P-450 level and 7-ethoxycoumarin O-deethylase). The electrophoretic analysis of microsomal fractions of yeast cells treated with metavanadate revealed a decrease in the intensity of the bands corresponding to a M(r) in the range of 51,000-58,000 Da compared with those observed in controls, i.e., cells grown in 20% glucose. Analysis of the cytochrome P-450 transcript showed that AMV treatment reduced the mRNA level. Our results suggest that AMV inhibits the yeast cytochrome P-450 system by acting at both the pre- and post-transcriptional levels.

Genetic and biochemical investigation on chloral hydrate in vitro and in vivo.

Chloral hydrate (CH), a metabolite of trichloroethylene (TCE), was studied in vitro using the D7 diploid strain of Saccharomyces cerevisiae, with and without a mammalian microsomal activation system (S9 fraction), and in vivo by intrasanguineous host-mediated assay (HMA). The in vivo effects on the hepatic microsomal monooxygenase induced by CH in mice pretreated with beta-naphthoflavone (beta-NF) and Naphenobarbital (PB) were also investigated. Chloral hydrate induced a significant increase of mitotic gene conversion in D7 strain both in vivo and in vitro. The enzymatic determinations in mice showed a decrease in aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities (about 37% and 29% respectively) after one acute dose of CH. Moreover, stability experiments, carried out in the conditions of the liver microsomal assay (LMA), showed an increase of residual activity, after 1 h of preincubation with respect to the control (about 22% and 9% for APD and p-NAD respectively).

Genetic and biochemical studies on perchloroethylene 'in vitro' and 'in vivo'.

Perchloroethylene (PCE) was tested in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in suspension tests with and without a mammalian microsomal activation system (S9) and 'in vivo' by the intrasanguineous host-mediated assay. In addition, enzyme alteration studies were performed in mice non-pretreated or pretreated with phenobarbital + beta-naphthoflavone. PCE did not induce any genetic effect either 'in vitro' or 'in vivo'. In the suspension test, PCE was more toxic without metabolic activation and less toxic with mammalian microsomal activation. The enzymatic determinations showed an increase of the aminopyrine demethylase activity and of the level of cytochrome P-450.

Cytochrome P-450 factors determining synthesis in strain D7 Saccharomyces cerevisiae. An alternative system to microsomal assay.

Certain aspects of cytochrome P-450 induction were studied in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in order to obtain cells containing a high level of metabolizing enzymes. The highest level of cytochrome P-450 was reached during the logarithmic growth phase in a 20%-glucose liquid medium. Yeast cells harvested in these conditions were used in the mutagenesis test with dimethyl nitrosamine (DMNA) as a positive control and with styrene (Sty). Both substances gave positive results, whereas Sty never showed any mutagenic activity in the conventional test with stationary growth phase cells and external metabolic activation. The test with cells from the logarithmic growth phase is proposed as a possible alternative to the liver-microsome assay, and its reliability is discussed.

Genetic activity of vinylidene chloride in yeast.

Vinylidene chloride (VDC) was tested for its ability to induce both point mutation and mitotic gene conversion in a diploid strain (D7) of the yeast Saccharomyces cerevisiae in a suspension test with and without a mammalian microsomal activation system, and in the intrasanguineous host-mediated assay in mice. In suspension tests with D7, VCD was toxic but not genetically active without microsomal activation. When a mouse liver 10 000 X g supernatant was included in the suspension tests, dose-related increases in both point mutation and mitotic gene conversion were seen at survival levels greater than 50%, at doses of VCD above 20 mM. In the host-mediated assay, VDC induced both point mutation and mitotic gene conversion when recovered from the liver and kidneys after both acute and sub-acute dosing. Yeasts recovered from the lungs showed little, if any, increase in either point mutation or mitotic gene conversion.

Stability of microsomal mono-oxygenase during incubations for the liver microsomal assay with S9 fractions of mouse liver under various inductions.

Aminopyrine-N-demethylase and p-nitroanisole-O-demethylase activities were determined in incubation mixtures for the liver microsomal assay at time zero and after 1 h of incubation in the conditions for the mutagenic assay. The experiments were performed with the S9 liver fraction of mice in the basal state and induced with sodium phenobarbital, beta-naphthoflavone or both. Lipid peroxidation was also determined. The experiments were repeated with female mice and also in the presence of styrene, as an example of a xenobiotic substance. The activity of pNAD was much more stable than that of APD in all the conditions tested. The pattern of stability, however, was similar for the two activities: more stable than controls with S9 fractions from beta-NF-induced mice, less stable than controls in PB-induced mice, intermediate between controls and PB-induced mice in those with combined induction by PB + beta NF. Styrene 50 mM in the incubation mixtures led to a marked inactivation of enzymic activity, similar in all cases and reaching about 90% in 1 h. S9 fractions from female mice gave enzymes slightly more stable in almost all cases. Lipid peroxidation was appreciably more elevated in basal than in induced animals. It was concluded that, for a mutagenesis test on an unknown xenobiotic, S9 fractions from mice following PB and beta-NF induction are to be preferred from the point of view of activation.

Study of the optimal temperature for the liver microsomal assay with mice S9 fractions.

The effect of temperature on enzymatic activity and stability was studied with respect to the monooxygenase activities of aminopyrine-N-demethylase (APD) and p-nitroanisole O-demethylase (pNAD) under incubation conditions for the liver microsomal assay. The activities of S9 liver fractions of mice induced with sodium phenobarbital and beta-naphthoflavone were determined during a period of preincubation in a range of temperatures from 30 to 44 degrees C. The greatest value of the mean specific activity was found at 40-42 degrees C for both APD and pNAD. The rapid increase of lipid peroxidation after 1 h of incubation at temperatures higher than 42 degrees C can provide an explanation of the enhancement of the rate of inactivation. In order to determine whether biological response is affected by the modifications induced by temperature in the metabolic activating system, tester strain D7 of Saccharomyces cerevisiae was used to assay the genetic activity of the well known premutagenic agent cyclophosphamide by incubating the mixtures both at the traditional temperature of 37 degrees C and at 42 degrees C. We suggest that the use of more favourable conditions for LMA with respect to enzymatic activity, than the traditional ones could improve the reliability and the sensitivity of such tests.

Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH.

The genetic effects of variation in pH in culture media and in suspension tests were examined in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. Deviation from the normal pH of 6.24 in the liquid culture medium, has a significant effect on cellular growth and on mitotic gene conversion at the trp5 locus. Frequencies of reversion at the ilv I-92 locus and of mitotic crossing-over at the ade2 locus are not significantly influenced. Suspension tests, performed using phosphate buffer (pH 5.8), strongly confirm the original results. Our data suggest that the increase in mitotic gene conversion under various conditions of pH is due to a specific effect of pH itself on the cells of S. cerevisiae. In fact, increases were obtained using the same pH in both cellular growth and non-growth conditions. The maximum effect detected with both procedures was obtained at pH 5.8; in the growth test, at this pH, gene conversion frequency appeared to be most pronounced, being about 10 times higher than that of the control. These results suggest that pH exerts its specific action both on growing and non-growing yeast cells, and the difference in induction of genetic effect between these two conditions is probably due to a time factor.

Study of spermine and spermidine effects on Saccharomyces cerevisiae. Polyamine production in different growth conditions and in the presence of interleukin-2.

The role of polyamines during cell growth is still uncertain. Yeast cells possess an amine pathway similar to that described for animal cells. We studied the relationship between growth and polyamine production in yeast cells of Saccharomyces cerevisiae grown under various conditions. Polyamines were determined in homogenates of yeast cells by a rapid enzymatic assay, confirmed by thin-layer chromatography. Polyamine production is dependent on the growth conditions. The effects of spermine, spermidine, and interleukin-2 on yeast cells were investigated. Exogenous polyamines affected the endogenous pathway, and interleukin-2 treatment increase yeast growth and polyamine production.

Induction of cytochrome P-450 by 5-methoxypsoralen in the yeast Saccharomyces cerevisiae.

Yeast cells (D7 strain) incubated in the presence of 5-methoxypsoralen (5-MOP) increase the activity of the monooxygenase system cytochrome P-450 dependent (cytochrome P-450 level and 7-ethoxycoumarin-O-diethylase activity). Northern analysis of cytochrome P-450 specific RNA shows that 5-MOP treatments induce an increase in mRNA. The induction of cytochrome P-450 appears to occur at the transcriptional level. The capacity of 5-MOP to induce the cytochrome P-450 system in eukaryotic cells, in which it is known to be involved in the metabolism of the psoralen, may decrease the availability of the compound for photo-induced genotoxic reactions, which may explain the good tolerance in patients.

Expression profiling of two stress-inducible genes encoding for miraculin-like proteins in citrus plants under insect infestation or salinity stress.

The expression of two genes, namely Mir1 and Mir3 and the abundance of their encoded proteins, the putative miraculin-like proteins, MLP1 and MLP3, showing similarity to the Kunitz family of protease inhibitors, were monitored in the leaves of the citrus variety, 'Clementine' after Tetranychus urticae infestation and elicitor treatments, or in the leaves of three other diploid citrus: 'Willow leaf', 'Cleopatra' mandarins and 'Trifoliate' orange, as well as their respective doubled diploids and the allotetraploid somatic hybrid 'FLHORAG1' under salt stress. RT-PCR and 2-DE indicated that Mir1 and Mir3 and their products were present at low-basal expression in all citrus genotypes. Both genes and products were induced in the 'Clementine' leaves infested by T. urticae, but a contrasting profile was observed under elicitor treatments. Under salt stress, the two genes showed an expression pattern contrasting each other and depending on the genotypes. 'Cleopatra' mandarin, 'Trifoliate' orange and 'FLHORAG1' presented overexpression of Mir3 and MLP3 and decreased levels of Mir1 and MPL1. The opposite behaviour was found in 'Willow leaf' mandarin. The positive correlation of the expression profile of the two genes with that of a gene encoding a putative apoplastic cysteine protease (CysP) might suggest a possible interaction of the respective encoded proteins during the response to biotic stress. Under salt stress, CysP and Mir 1 showed a similar expression pattern but only at transcript level. The possible occurrence of post-translational CysP regulation is discussed.

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

Cloning, tissue expression, and inducibility of CYP 3A79 from sea bass (Dicentrarchus labrax).

Multiple members of the CYP3A subfamily have been identified and intensively studied in mammals as they represent prominent CYP enzymes involved in drug metabolism. Also in fish, some CYP3A genes have been identified by cDNA cloning and immunological techniques, but relatively little is known about their function, distribution, and inducibility. In this study, a novel CYP3A, designated as CYP3A79 was isolated from adult male sea bass, an economically valuable species in fisheries. The sea bass CYP3A79 that was cloned contained an open-reading frame of 1512 bp that encoded a 504 amino acid protein and shared a high-sequence identity with medaka, killifish, and trout CYP3As. Interestingly, CYP3A79 also shares five of six substrate recognition sites (SRS) with the SRS of other fish CYP3As, suggesting an evolutionary conservation of the function of these enzymes. In this fish, we also investigated the expression of CYP3A79 and its susceptibility to induction by various compounds including clotrimazole and dehydroepiandrosterone, two strong ligands of zebrafish PXR. The expression of CYP3A79 mRNA was detected by RT-PCR only in the intestine and liver. The immunoblot analysis by antitrout CYP3A27 confirmed the presence of a CYP3A-like protein in the microsomes of these tissues, but, in addition, a immunoreactive protein with this antibody was also observed in the heart microsomes, suggesting the presence of other CYP3A isoforms in this fish. Accordingly, the southern blot analysis of genomic DNA indicated that multiple CYP 3As may be present in sea bass. All attempts to induce 6beta-testosterone hydroxylase, as a marker of CYP3A79, by dexametasone, 17beta-estradiol, pregnenolone 16alpha-carbonitrile, corticosterone, clotrimazole, and dehydroepiandrosterone failed. On the contrary, the administration of 17beta-estradiol, pregnenolone 16alpha-carbonitrile, and corticosterone strongly inhibited this activity and, in parallel, reduced the expression of CYP3A79 transcript. Thus, the sea bass CYP3A79 appears to be resistant to induction, suggesting that this enzyme and likely other CYP3As are regulated differently compared to those of mammals.

Multiple inducible cytochromes P-450 in yeast.

A form of cytochrome P-450 is produced in Saccharomyces cerevisiae during the logarithmic growth phase. This form is inhibited by metyrapone, tetrahydrofuran, and carbon oxide which are inhibitors of ethanol-inducible cytochromes P-450 of mammals. Addition of Na-phenobarbital to the 0.5% glucose liquid medium induced a form of cytochrome P-450 inhibited only by metyrapone. Thus, yeast possess multiple forms of cytochrome P-450 that are individually inducible.