Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite.

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Grape variety related trans-resveratrol induction affects Aspergillus carbonarius growth and ochratoxin A biosynthesis.

The paper reports the results of a study performed to investigate the influence of the grape variety on the growth of Aspergillus carbonarius on grape berries and the correlation between the amount of ochratoxin A (OTA) and the content of trans-resveratrol produced after fungal contamination. Variations in the amount of OTA produced by the fungus are observed depending on both grape variety and on the induction of trans-resveratrol determined during the infection. The obtained data suggest that if an increase in trans-resveratrol production in grape berries occurs early after the fungal infection, the berry exploits this compound to control OTA synthesis. If the increase in trans-resveratrol concentration is delayed after fungal infection (40 h), a control of OTA accumulation can not be achieved. The possibility of exerting significant control of OTA biosynthesis by this phytoalexin seems to rely in the promptness of its production, as occurs also in other fungus plant interactions and, in turn, seems to be dependent also on grape cultivar. In this fungus-plant system, trans-resveratrol appears to represent a defence-related compound toward A. carbonarius and OTA contamination.

Early detection of toxigenic fungi on maize by hyperspectral imaging analysis.

Fungi can grow on many food commodities. Some fungal species, such as Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Fusarium spp., can produce, under suitable conditions, mycotoxins, secondary metabolites which are toxic for humans and animals. Toxigenic fungi are a real issue, especially for the cereal industry. The aim of this work is to carry out a non destructive, hyperspectral imaging-based method to detect toxigenic fungi on maize kernels, and to discriminate between healthy and diseased kernels. A desktop spectral scanner equipped with an imaging based spectrometer ImSpector- Specim V10, working in the visible-near infrared spectral range (400-1000 nm) was used. The results show that the hyperspectral imaging is able to rapidly discriminate commercial maize kernels infected with toxigenic fungi from uninfected controls when traditional methods are not yet effective: i.e. from 48 h after inoculation with A. niger or A. flavus.

Lentinula edodes enhances the biocontrol activity of Cryptococcus laurentii against Penicillium expansum contamination and patulin production in apple fruits.

Penicillium expansum is a post-harvest pathogen of apples which can produce the hazardous mycotoxin patulin. The yeast Cryptococcus laurentii (LS28) is a biocontrol agent able to colonize highly oxidative environments such as wounds in apples. In this study culture filtrates of the basidiomycete Lentinula edodes (LF23) were used to enhance the biocontrol activity of LS28. In vitro L. edodes culture filtrates improved the growth of C. laurentii and the activity of its catalase, superoxide dismutase and glutathione peroxidase, which play a key role in oxidant scavenging. In addition, LF23 also delayed P. expansum conidia germination. The biocontrol effect of LS28 used together with LF23 in wounded apples improved the inhibition of P. expansum growth and patulin production in comparison with LS28 alone, under both experimental and semi-commercial conditions. The biocontrol effect was confirmed by a semi-quantitative PCR analysis set up for monitoring the growth of P. expansum.

Apyap1 affects aflatoxin biosynthesis during Aspergillus parasiticus growth in maize seeds.

It is demonstrated that, in fungal cells grown in synthetic media, the Apyap1 gene is implicated in the modulation of aflatoxin biosynthesis following the perturbation of redox balance. This study suggests that an association between oxidative stress and aflatoxin biosynthesis also occurs in maize seeds. We used DeltaApyap1, a strain in which the gene Apyap1 was disrupted, to verify whether this oxidative stress-related transcription factor, by affecting cell redox balance, can have a role in the modulation of aflatoxin synthesis. The amount of hydroperoxides (ROOH) produced by wild type (WT) and DeltaApyap1, both grown in potato dextrose broth, was assayed in the filtrate. In maize seeds (30 g), inoculated with WT and DeltaApyap1conidia and incubated at 30 degrees C for 15 days, lipoxygenase activity (LOX), lipoperoxides (LOOH) production, fungal growth and aflatoxin biosynthesis was analysed. It was observed that DeltaApyap1 released more hydroperoxides in the culture media and more aflatoxins in seeds, possibly through stronger stimulation of LOX, which, in turn led to greater LOOH production in the seeds. On the basis of the results, a hypothesis regarding strategies to control aflatoxin synthesis is formulated.

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the Pleurotus eryngii complex.

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man's selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods-amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes-to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.

Oxidant/antioxidant balance inAspergillus parasiticus affects aflatoxin biosynthesis.

A close correlation between lipoperoxide formation in cells ofAspergillus parasiticus and aflatoxin biosynthesis has been established in rich and poor media in which oxidative stress was induced by addition of cumene hydroperoxide, a lipoperoxidation inducer. The presence of hydroperoxides of linoleic acid inA. parasiticus mycelia was analysed by liquid chromatography-mass spectrometry (LC-MS). This relation appears to be driven by activation of certain oxidative stress related transcription factors, such asyap1-like,skn7-like andhsf2-like. Activation of these factors then leads to the promotion of transcription of genes encoding antioxidant-related enzymes, such as superoxide dismutase, catalase and glutathione peroxidase.The incomplete seavenging of intracellular oxidation inA. parasiticus cells can lead to aflatoxin biosynthesis. The relationship between oxidative stress and aflatoxin biosynthesis is indicated by the high correlation among increased activity of lipoperoxidation and the antioxidant defence system with formation of aflatoxins.With regard to the relationship of oxidative stress and aflatoxin biosynthesis, the mechanism of action of butylated hydroxyl anisole (BHA), an antioxidant compound, in the control of aflatoxin biosynthesis was also investigated. Results indicate this compound can act,per se, by inhibiting lipoperoxidation and by inducing antioxidative defence responses of the fungal cell.

Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production.

Biosynthesis of aflatoxins, toxic metabolites produced by Aspergillus parasiticus, is correlated to the fungal oxidative stress and cell ageing. In this paper, the mechanism underlying the aflatoxin-inhibiting effect of the Lentinula edodes culture filtrates was studied by analysing their anti-oxidant activity and beta-glucan content. Mushroom beta-glucans are pharmacologically active compounds stimulating anti-oxidant responses in animal cells. L. edodes lyophilised filtrates stimulate A. parasiticus anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and aflatoxin inhibition was better correlated with beta-glucan content than with anti-oxidant activity of the filtrates. RT-PCR analyses on treated mycelia showed a delay in the activation of aflR, and norA, genes of aflatoxin cluster and a synchronous activation of hsf2-like, a homologue of a yeast transcription factor involved in oxidative stress responses. The first evidence of hsf2-like in A. parasiticus and its activation during aflatoxin biosynthesis is reported. L. edodes filtrates could play a role as external stimulus affecting the anti-oxidant status in the fungal cell that, in turn, leads to aflatoxin inhibition. In the fungal cell, beta-glucans present in the filtrates could stimulate the activation of transcription factors related to anti-oxidant response and anti-oxidant enzyme activity with a contemporaneous delay of aflatoxin genes transcription, which led to a marked reduction of aflatoxin production. This research suggests new perspectives to set suitable strategies against aflatoxins and L. edodes could be considered a promising tool.

Genetic diversity and population structure of the Italian fungi belonging to the taxa Pleurotus eryngii (DC.:Fr.) Quèl and P. ferulae (DC.:Fr.) Quèl.

A study using allozymes and PCR fingerprinting was conducted to estimate the genetic diversity of Italian populations of two economically important cultivated fungal taxa, Pleurotus eryngii and P. ferulae. Very little is known about the genetic diversity distribution pattern of these taxa. Heterozygote deficiency was observed at few loci; in fact the inbreeding coefficients were not high, which demonstrates that mechanisms restrain the inbreeding act at the local level. Estimates of genetic differentiation indicated a pattern of greater variation within, rather than between, populations. These results were supported by AMOVA analysis, which attributed a low proportion of the total genetic variation to large geographical scale divergence, and indicated that most of the genetic diversity was because of differences within populations. This distribution pattern of genetic variation of P. eryngii and P. ferulae populations seems to be the result of high gene flow, by efficient basidiospore dispersal, and outcrossing mechanisms, which restrain inbreeding within populations.

Oxidative stress, growth factor production and budding in potato tubers during cold storage.

In order to verify the role played by oxidation in the budding of potato tubers (Solanum tuberosum L. cv. Kennebec), the physiological events occurring below bud at 4 degrees C have been studied for a period of 6 months. The low temperature storage induced an increase in the degree of unsaturation and a decrease in the ratio of saturated/unsaturated fatty acids of membrane polar lipids with a subsequent increase of lipid hydroperoxides (LOOH). Cold stress increased both enzymatic antioxidative activities (superoxide dismutase, SOD, E.C.1.15.1.1; catalase, CAT, E.C.1.11.1.6), and alpha-tocopherol levels thus protecting membrane's polyunsaturated lipids. Between 0 and 15 days of storage SOD/CAT ratio, alpha-tocopherol, LOOH levels and the degree of lipid unsaturation showed strong variations. After 30 to 120/150 days the antioxidative system seemed to reach a homeostasis different from that of time 0, accompanied by a constant increase of indole-3-acetic acid (IAA) after 60 days. The antioxidative system, after 150 days, lost its efficiency while LOOH levels were maintained higher than time 0 and IAA concentration was sufficient to allow sprouting.

Early physiological and cytological events induced by wounding in potato tuber.

The response of potato tuber (Solanum tuberosum L. cv. Kennebec) to mechanical wounding was investigated at different times. Changes in the levels of indole-3-acetic acid (IAA), polyunsaturated fatty acids (PUFAs) and lipid hydroperoxides (LOOHs) were monitored up to 120 min after wounding and related to the cytological events occurring up to 24 h. Twenty minutes after injury, an increase in IAA and LOOH levels and a decrease in the levels of PUFAs was observed. Wounding induced mitoses in differentiated (parenchyma) cells starting at 120 min, and promoted an increase of mitotic activity in the meristematic cells (procambium and bud dome), after 360 min. The inhibition of the increase in LOOHs and IAA by lipoxygenase (LOX) inhibitors, as well as the ability of in vitro peroxidated linoleic acid to enhance IAA production, suggest a close relationship among lipoperoxidation, IAA and mitotic activity in the response of potato tuber cells to injury, resulting in a specific growth response, i.e. bud growth and periderm formation.

Ergosterol oxidation may be considered a signal for fungal growth and aflatoxin production in Aspergillus parasiticus.

The addition of compounds able to peroxidize cell lipids (carbon tetrachloride (CCl4), cumene hydroperoxide (CUH), or linoleic acid hydroperoxide (LAH)) to 5-day-old Czapek-Dox Medium cultures of Aspergillus parasiticus induces a significant reduction of the tri-unsaturated ergosterol (ERG) levels in fungal microsomes and mitochondria, whereas the concentrations of the di-unsaturated linoleic acid (LA; C18:2 n-6) are unaffected. Aflatoxin (AFT) output follows ERG reduction and is associated with both a renewal of fungal growth and a slow increase of ERG concentration in subcellular membranes. We suggest that, by analogy with the regulatory role played on cell proliferation and metabolism by polyunsaturated fatty acid by-products (eicosanoids) in mammalian membranes, by-products of ERG oxidation may be considered triggers sufficient to induce both further fungal growth and AFT biosynthesis.

Effect of different inhibitors of sterol biosynthesis on both fungal growth and aflatoxin production.

Azole antifungals are reported to interfere with fungal growth by selective impairment of the P-450 dependent 14 alpha-demethylase system key to biosynthesis of ergosterol (ERG), thus leading to the depletion of this sterol in fungal membranes and to the accumulation of methylated precursors. We have investigated whether azole antifungals ketoconazole, miconazole, econazole, or itraconazole were able to modify the sterol and fatty acid patterns of a toxigenic strain of Aspergillus parasiticus, inducing the growth of mycelium depleted of ergosterol (ERG) and rich in less oxidizable sterols. It had been demonstrated that oxidation of ERG is correlated to aflatoxin biosynthesis. However, in this study no alteration of sterol or fatty acid patterns was observed after 7, 14, and 21 days of incubation of A. parasiticus in the presence of sublethal doses of azole antifungals. Specific production of aflatoxin was unaffected. Among the four antifungals tested, itraconazole was the strongest inhibitor of fungal growth and aflatoxin production while ketoconazole was the least effective.

Cytological events induced by arachidonic acid in potato tubers.

Necrosis of potato tuber cells is enhanced, after 24 h incubation, by arachidonic acid (AA), an elicitor of the hypersensitive response in Solarium tuberosum L. and also by Fenton's reagents (a hydroxyl radical-generating system) either alone or in association with AA. The phenomenon is independent of tuber age. In aged tubers, phenylthiourea (PTU) causes a significant reduction of AA-induced cell necrosis. Necrosis observed in the presence of PTU alone is lower than in other treatments, and neither different from the controls nor affected by-tuber age. Cell size is not affected by treatments or ageing. Nuclear hypertrophy occurs independently of tuber ape, with the highest values after treatment with AA and Fenton's reagents. Nucleolar extrusion is observed in all treatments but earliest in the presence of AA. AA also enhances the number of lignified parenchymal cells and, to a lesser extent, the number of traeheary elements.

Effect of some food preservatives on aflatoxin production.

The effect of some food preservatives, such as sorbic (SA) and propionic (PA) acids, on aflatoxin production in synthetic media or in moistened (20%) wheat seeds, was investigated. The preservatives tested, added to synthetic media at sublethal concentrations both at the inoculum and after 5 days of incubation, stimulated aflatoxin production by Aspergillus parasiticus. Sorbic and propionic acids are metabolized by the fungus in vivo and in vitro. Lower concentrations of PA and SA (0.05 to 0.1% w/w) in wheat seeds are ineffective against both fungal growth and aflatoxin production, whilst the combined use of butylated hydroxy toluene (BHT), and PA or SA was more effective in controlling aflatoxin production than their use as single components.

Novel study on the elicitation of hypersensitive response by polyunsaturated fatty acids in potato tuber.

A GC-MS procedure was carried out for the simultaneous and unequivocal quantitation of both potato phytoalexin (rishitin and lubimin) accumulation and the rate of disappearance of polyunsaturated fatty acids (PUFA) and some of their esters tested as possible elicitors. Potato 5-lipoxygenase and lipolytic acyl hydrolase play a key role in hypersensitive response (HR) induction. As expected, arachidonic acid, its hydrolysable esters, and eicosapentaenoic acid elicited much higher HR than the other PUFA tested, although the latter were equally affected by potato 5-lipoxygenase. Hydroxyl radicals appear to be actively involved in the browning process. The polyaminoacid poly-L-lysine did not show any eliciting activity.

Relationship between lipids and aflatoxin biosynthesis.

This paper describes the key role of lipids on fungal growth and of lipoperoxidation on the output of aflatoxin biosynthesis both 'in vitro' and 'in vivo'. 'In vitro' BHA, BHT and cysteamine, depending their concentration, are capable of reducing or blocking aflatoxin output induced by lipoperoxides or halomethanes in cultures of Aspergillus flavus or A. parasiticus without affecting fungal growth. 'In vivo' BHA and BHT significantly reduced aflatoxin production on wheat, maize and sunflower inoculated with aflatoxigenic Aspergilli essentially by preventing fungal growth. 'In vivo' the seeds surface lipids represent a very important carbon source for fungal growth.

Effect of different antioxidants and free radical scavengers on aflatoxin production.

Different antioxidants and free radical scavengers on aflatoxin production are analysed. The different compounds at different concentration were used: buthylated hydroxyanisole (BHA), buthylated hydroxytoluene (BHT), α-tocopherol (vitamin E), ascorbic acid (vitamin C), reduced glutathione, cysteine, cysteamine. The above compounds were tested in culture ofAspergillus parasiticus supplemented with carbon tetrachloride, a potent stimulating agent of aflatoxin biosynthesis.Cysteamine and BHA highly inhibited the aflatoxin production induced by carbon tetrachloride, the inhibition decreased by lowering the concentration.On the contrary, vitamin E, vitamin C, reduced glutathione and cysteine further enhanced the carbon tetrachloride stimulating effect. The addition of the above compounds did not significantly affect the growth of the fungal mycelia.

Stimulation of aflatoxin biosynthesis by lipophilic epoxides.

Epoxy fatty acids added to the culture media either with the inoculum or at the end of exponential growth phase stimulated aflatoxin production by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. This effect did not appear when the unsaturated fatty acids used for the synthesis of the epoxides and the polyhydroxyacids (which can be considered to be derived from the opening of the oxirane ring) replaced the epoxides in the culture media. No significant differences were detected in the lipid fractions (diglycerides, sterols, triglycerides, free fatty acids, sterol esters) extracted from the mycelia grown in the presence of any of the fatty acid derivates.