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Wild edible mushrooms are very popular for both their flavors and nutritional values. However, some mushroom species can be harmful to human health as they accumulate some elements in excessive amounts. The aim of this study was to determine the concentrations of Al, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, P, Se, and Zn of some wild edible (Agaricus arvensis, A. bitorquis, A. sylvaticus, Amanita vaginata, Armillaria mellea, Clavariadelphus pistillaris, Clitocybe nebularis, Clitopilus prunulus, Hygrophorus marzuolus, H. russula, Lactarius volemus, Lycoperdon molle, and Macrolepiota mastoidea) and non-edible mushroom species (A. citrina, Auricularia mesenterica, Chanterellus melanoxeros, Chondrostereum purpureum, Clathrus ruber, L. controversus, L. helvus, and L. zonarius) collected from Belgrad forest (Istanbul, Turkey). Daily intakes of element (DIE) and health risk index (HRI) values of the edible mushroom species were also calculated. The concentrations of the elements in question were determined to be in the ranges of 9.7-556.8, 2.5-2226.7, 0.06-2.52, 0.03-13.17, 3.74-100.19, 13.3-507.4, 2635.0-28614.0, 493.0-2412.0, 6.97-3150.73, 0.29-13.26, 0.38-3.67, and 9.1-293.8 mg/kg, respectively. The Cd concentration of H. russula (DIE: 1.08, HRI: 1.08), Cr concentration of C. nebularis (DIE: 5.64, HRI: 1.88), and the Cu concentration of M. mastoidea (DIE: 42.94, HRI: 1.07) were above the reference values. The results showed that the long-term consumption of H. russula, C. nebularis, and M. mastoidea collected from Belgrad forest can have a negative impact on human health. Therefore, it was concluded that the element concentrations of edible wild mushrooms in this region should be examined periodically.

We studied neuroprotective, antioxidative, antimicrobial and cytotoxic activity of acetone extracts of Clitocybe geotropa and Clitocybe nebularis. The ability of extracts to inhibit acetylcholinesterase was tested in order to examine neuroprotective potential of selected mushroom species. DPPH, superoxide anion and reducing power assays were used in assessing the antioxidative potential of studied mushrooms. Total phenolics content was estimated as pyrocatechol equivalent. Antimicrobial activity was evaluated through microdilution method. MTT assay was utilized in determining the cytotoxic activity of extracts. The results demonstrated that studied extracts express relatively strong neuroprotective, antioxidative, antimicrobial and moderate cytotoxic activity. The results of acetylcholinesterase inhibition for Clitocybe geotropa were in range 46.95–14.82% and for Clitocybe nebularis 36.73–12.49%. DPPH assay’s IC50 for Clitocybe geotropa were 246.58 μg/mL and for Clitocybe nebularis 46.49 μg/mL. IC50 in superoxide anion assay was 151.36 μg/mL for Clitocybe geotropa and 35.69 μg/mL for Clitocybe nebularis. In reducing power assay the absorbance for Clitocybe geotropa was in range 0.0708–0.0132 and for Clitocybe nebularis 0.0324–0.0141. Total phenolics content of Clitocybe geotropa was 95.71 µg PE/mg and of Clitocybe nebularis 93.94 µg PE/mg. MIC values for Clitocybe geotropa were in range 0.78–25 mg/mL and for Clitocybe nebularis 3.12–25 mg/mL. Cytotoxic activity of extracts was moderate, IC50 values for Clitocybe geotropa ranged from 124.2 to 155.3 μg/mL and for Clitocybe nebularis from 134.03 to 199.03 μg/mL. Both mushroom species are edible and can be referred to as functional food.

Aims We aimed to expand the microbial biocatalyst platform to generate essential oxyfunctionalized standards for pharmaceutical, toxicological and environmental research. In particular, we examined the production of oxyfunctionalized nonsteroidal anti-inflammatory drugs (NSAIDs) by filamentous-fungi. Methods and results Four NSAIDs; diclofenac, ibuprofen, naproxen and mefenamic acid were used as substrates for oxyfunctionalization in a biocatalytic process involving three filamentous-fungi strains; Beauveria bassiana, Clitocybe nebularis and Mucor hiemalis. Oxyfunctionalized metabolites that are major degradation intermediates formed by Cytochrome P450 monooxygenases in human metabolism were produced in isolated yields of up to 99% using 1 g l-1 of substrate. In addition, a novel compound, 3',4'-dihydroxydiclofenac, was produced by B. bassiana. Proteomic analysis identified CYP548A5 that might be responsible for diclofenac oxyfunctionalization in B. bassiana. Conclusions Efficient fungi catalysed oxyfunctionalization was achieved when using NSAIDs as substrates. High purities and isolated yields of the produced metabolites were achieved. Significance and impact of the study The lack of current efficient synthetic strategies for oxyfunctionalization of NSAIDs is a bottleneck to perform pharmacokinetic, pharmacodynamic and toxicological analysis for the pharmaceutical industry. Additionally, oxyfunctionalized derivatives are needed for tracking the fate and impact of such metabolites in the environment. Herein, we described a fungi catalysed process that surpasses previously reported strategies in terms of efficiency, to synthesize oxyfunctionalized NSAIDs.

summary The extension rates of Clitocybe nebularis (Batsch ex Fr.) Kummer strains on 2% malt agar were only 30–40%, of those, up to 3.4 mm d−1, observed in woodland at equivalent exponential mean temperatures. Extension of mature field systems was accomplished by mycelial annuli or arcs 30-40 cm wide, differentiated into a leading edge of mycelial cords followed by a zone of dense, diffuse mycelium which bleached litter components, and a trailing edge of greyish, Used mycelium. Disruption of mature annuli by natural obstacles or experimental re-orientation within the mycelial band resulted in regression of the affected segment of mycelium. Localized lysis following encounter with an obstacle by immature patches of mycelium with a diameter of 30–50 cm, led to polarized development of the residual mycelium. Strains from different fruit bodies were somatically compatible when paired on 2% malt agar if sampled from the same ring, but incompatible if from different rings, resulting in mutual antagonism and formation of a persistent demarcation zone. By contrast, collision between adjacent systems in woodland culminated in mutual obliteration of the interaction fronts. C nebularis was non-combative when paired against other decomposer basidiomycetes on 2% malt agar, being either replaced or deadlocked but not replacing mycelia of these fungi. The implications of these observations are discussed in terms of emerging concepts of ecological strategies, foraging theory and polarity in mycelial collectives.

Five new peptaibols, longibramides A-E (1-5) with 11 amino acid residues, were isolated from a fungus Trichoderma longibrachiatum Rifai DMG-3-1-1, which was isolated from a mushroom Clitocybe nebularis (Batsch) P. Kumm collected from coniferous forest in the subboreal area of northeast China. The structures of longibramides A-E were determined by their spectroscopic data (NMR and MS-MS spectra), their absolute configurations were determined by X-ray diffractions and Marfey's analyses. The X-ray diffractions of longibramides A, B and the similar CD spectra of A-E showed that they all had α-helix conformations. Longibramides B and E showed moderate cytotoxicities against BV2 and MCF-7 cells, and also showed some inhibitory effects against methicillin-resistant Staphylococcus aureus MRSA T144. L-trans-Hyp was not commonly found in natural peptaibols, which was the 6th or 10th amino acid residue in longibramides C-E . The X-ray diffractions of longibramides A and B afforded the accuracy conformations of their secondary structures, which maybe help to interpret the structure-activity relationships of the family of peptaibols in the future.

Lectins have been increasingly utilized as carriers for targeted drug delivery based on their specific binding to glycans located on mammalian cells. This study employed two lectins, B subunit of bacterial Shiga holotoxin (Stx1B) and fungal Clitocybe nebularis lectin (CNL), for surface display on the lactic acid bacterium Lactococcus lactis. The specific adhesion of these engineered, lectin-displaying L. lactis to cancer cells was evaluated. The expression and surface display of both lectins on L. lactis were demonstrated by western blotting and flow cytometry, respectively. MTS assays revealed that recombinant Stx1B had no effect on Caco-2 cell viability at concentrations of &le, 25 &micro, g/mL, whereas CNL was non-toxic even at relatively high concentrations of &le, 250 &micro, g/mL. Stx1B bound to Caco-2, HT-29 and HeLa cells after 1 h of incubation. CNL bound to Caco-2 cells and recognized several glycoproteins in HT-29 and Caco-2 cell homogenates of which a 70 kDa protein predominated. Confocal microscopy revealed adhesion of Stx1B-displaying L. lactis to HeLa, Caco-2, and, to a lesser extent, HT-29 cells, CNL-displaying L. lactis showed a relatively similar level of adherence to HT-29 and Caco-2 cells. Thus, lectin-displaying L. lactis might serve as a carrier in targeted drug delivery when coupled to a therapeutic moiety.

Clitocybe mushrooms have long been recognized for their various therapeutic potential and medicinal properties. A few members of the genus are considered edible and many others are poisonous. This study investigated the ethanolic extracts obtained from C. nebularis (CN) and I. geotropa (IG) mushrooms for phenolic content and antioxidant, antiproliferative, antimicrobial, and antibiofilm activities. The data from ultra-performance liquid chromatography and Fourier transform infrared spectroscopy analysis of the mushrooms were presented for the first time. According to the results, both ethanolic extracts contain high levels of phenolic (catechin, myricetin, quercetin, rutin, gallic acid, vanillic acid) compounds. Fourier transform infrared spectroscopy results may suggest the presence of clitopycin in CN extract. The ethanol extract of CN scavenged about 79% and the IG 78% of the free 2,2-diphenyl-1-picrylhydrazyl radicals. Additionally, the CN and IG extracts inhibited glutathione-S-transferase by 10%-18% at all concentrations. The CN extract effectively inhibited aldose reductase by 30%-80% at all concentrations. Besides, the CN extract showed promising antiproliferative activity on HT-29 and MCF-7 cell lines. On the other hand, CN and IG extracts displayed inhibitory effects on some multidrug-resistant Gram-positive bacteria and effectively inhibited biofilm production. The obtained results showed that C. nebularis and I. geotropa extracts presented inhibition of biofilm production. Therefore, C. nebularis was demonstrated to be a potential source of natural medicine.

Agaricus bisporus mannose-binding protein (Abmb) was discovered as part of the mushroom tyrosinase (PPO3) complex, but its function in the mushroom has remained obscure. The protein has a β-trefoil structure that is common for Ricin-B-like lectins. Indeed, its closest structural homologs are the hemagglutinin components of botulinum toxin (HA-33) and the Ricin-B-like lectin from Clitocybe nebularis (CNL), both of which bind galactose, and actinohivin, a recently discovered mannose-binding lectin from actinomycetes. Here we show that Abmb is evolutionarily related to them, which are lectins with a β-trefoil fold. We also show for the first time that Abmb can exhibit typical lectin agglutination activity but only when in the complex with mushroom tyrosinase. This is unexpected and unique because the two proteins are not evolutionarily related and have different activities. Lectin and tyrosinase major role in defense mechanism as well as Abmb and PPO3 gene regulation during the early stages of the development of mushroom fruiting bodies suggested that Abmb has likely a function in defense against bacterial infection and/or insect-induced damage.

U ovom radu utvrđivana je kontaminacija, bio koncentracija i distribucija žive u samoniklim gljivama roda Clitocybe, vrstama Clitocybe nebularis i Clitocybe inversa, uz procjenu i rizik unosa žive u ljudski organizam putem prehrambenih proizvoda. Uzorci gljiva i gornji horizont tla prikupljeni su na osam nezagađenih i geografski udaljenih područja kako bi se spriječila lokalna ili regionalna emisija žive. Koncentracija žive u klobuku gljiva varirala je od 0,183 ± 0,025 do 0,621 ± 0,745 mg kg-1 suhe tvari, u stručku od 0,071 ± 0,012 do 0,312 ± 0,208 mg kg-1 suhe tvari. Raspon koncentracija žive u gornjem horizontu tla manji je u odnosu na anatomske dijelove gljive, a vrijednosti su se kretale od 0,012 ± 0,005 do 0,025 ± 0,006 mg kg-1 suhe tvari. Istraživanjem je utvrđena niska koncentracija žive u gljivama vrste Clitocybe, kao i u gornjim horizontima tla prikupljenih iz nezagađenih područja. Klobuk u odnosu na stručak u obje analizirane vrste gljiva akumulira višu koncentraciju žive. Vrsta Clitocybe nebularis pokazala se vrlo dobrim bio akumulatorom žive, sa vrijednostima bio koncentracijskog faktora od 10,42 do 24,26. Istraživanjem je utvrđeno da vrste Clitocybe nebularis i Clitocybe inversa ne predstavljaju opasnost unosa žive u ljudski organizam prilikom konzumacije istih. The contamination, bio-concentration and distribution of mercury (Hg) in wild mushrooms of the genus Clitocybe such as Clitocybe inversa, Clitocybe nebularis were studied, and the possible dietary intake and risk for human consumers in Croatia was estimated. Mushrooms, together with the associated forest topsoils were collected from 8 unpolluted and geographically distant areas, far from local or regional emission sources. The Hg contents were in the range 0.183 ± 0.025 to 0.621 ± 0.745 mg kg−1 dry matter in caps and 0.071 ± 0.012 to 0.312 ± 0.208 mg kg−1 in stems. The corresponding topsoil concentrations varied over a relatively narrow range between sites, from 0.012 ± 0.005 to 0.025 ± 0.006 mg kg−1 dry matter. Overall, the study results showed low levels of mercury both, in edible Clitocybe mushrooms and forest topsoils from background (unpolluted) forested areas in Croatia. The morphological distribution showed considerably greater concentrations of mercury in the caps relative to the stems. C. nebularis showed good ability to bioconcentrate Hg, with bioconcentration factors (BCF) values in the range 10.42 to 24.26. The data suggests that Clitocybe mushrooms from unpolluted areas in Croatia can be considered as a low risk food from the point of view of the tolerable Hg intake.

Clitocybe nebularis lectin (CNL) is present in fruiting bodies of clouded agaric along with several similar isolectins that are all small and stable proteins. It is a beta-trefoil type lectin forming homodimers that are essential for its functionality. It binds specifically N,N’-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacDiNac) and human blood group A determinant-containing glycan epitopes. Its most probable function is to defend fruiting bodies against predators and parasites. In addition, an endogenous regulatory function is possible for CNL, as indicated by its interaction with fungal protease inhibitors sharing the beta-trefoil fold. CNL is toxic to insects, nematodes and amoebae, as well as to leukemic T-cell lines. Bivalent carbohydrate binding is essential for the toxicity of CNL, against both invertebrates and cancer-derived cell lines. In addition, CNL exhibits potent immunostimulation of human dendritic cells, resulting in a strong T helper cell type 1 response. Based on its unique characteristics, CNL is a promising candidate for applications in human and veterinary medicine as well as in agriculture, for plant protection.

The light subunit of mushroom Agaricus bisporus tyrosinase (LSMT) is a protein of unknown function that was discovered serendipitously during the elucidation of the crystal structure of the enzyme. The protein is non-immunogenic and can penetrate the intestinal epithelial cell barrier, and thus, similar to its structural homologue HA-33 from Clostridium botulinum, may be potentially absorbable by the intestine. LSMT also shares high structural homology with the ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which has been shown to display biological activity against leukemic cancer cells and dendritic cells. Therefore, we evaluated the biological activity of LSMT. An in vitro assay suggested that LSMT presentation to most of the cancer cell lines studied has a negligible effect on their proliferation. However, inhibition of cell growth and a slight stimulation of cell proliferation were observed with breast cancer and macrophage cells, respectively. LSMT appeared to be relatively resistant against proteolysis by trypsin and papain, but not bromelain. Challenges with gastric and intestinal juice suggested that the protein is resistant to gastrointestinal tract conditions. This is the first report on the biological characteristics and implication of LSMT. (C) 2017 Elsevier B.V. All rights reserved.

The synthesis of valuable metabolites and degradation intermediates of drugs, like non-steroidal anti-inflammatory drugs (NSAIDs), are substantially for toxicological and environmental studies, but efficient synthesis strategies and the metabolite availability are still challenging aspects. To overcome these bottlenecks filamentous fungi as microbial biocatalysts were applied. Different NSAIDs like diclofenac, ibuprofen, naproxen and mefenamic acid could be oxyfunctionalized to produce human metabolites in isolated yields of up to 99% using 1 g L−1 of substrate. Thereby the biotransformations using Beauveria bassiana, Clitocybe nebularis or Mucor hiemalis surpass previous reported chemical, microbial and P450-based routes in terms of efficiency. In addition to different hydroxylated compounds of diclofenac, a novel metabolite, 3’,4’-dihydroxydiclofenac, has been catalyzed by B. bassiana and the responsible P450s were identified by proteome analysis. The applied filamentous fungi present an interesting alternative, microbial biocatalysts platform for the production of valuable oxyfunctionalized drug metabolites.ImportanceThe occurrence of pharmaceutically active compounds, such as diclofenac and its metabolites, in the environment, in particular in aquatic systems, is of increasing concern because of the increased application of drugs. Standards of putative metabolites are therefore necessary for environmental studies. Moreover, pharmaceutical research and development requires assessment of the bioavailability, toxicity and metabolic fate of potential new drugs to ensure its safety for users and the environment. Since most of the reactions in the early pharmacokinetics of drugs are oxyfunctionalizations catalysed by P450s, oxyfunctionalized metabolites are of major interest. However, to assess these metabolites chemical synthesis often suffer from multistep reactions, toxic substances, polluting conditions and achieve only low regioselectivity. Biocatalysis can contribute to this by using microbial cell factories. The significance of our research is to complement or even exceed synthetic methods for the production of oxyfunctionalized drug metabolites.

BACKGROUND Data of mushroom nitrate content from scientific studies is limited. There have been two such recent investigations (mainly regarding certain cultivated species). To obtain comparable analytical data, we analyzed 134 samples of 54 taxa gathered and prepared by our department. RESULTS The mushroom species were evaluated according to their nutritional types: saprotrophic, mycorrhizal and wood-decaying groups. Low and relatively invariable contents were found in the mycorrhizal (216.5 mg kg−1 dry matter (DM) and wood-decaying groups (228.6 mg kg−1 DM), but in the saprotrophic group we observed a wide variability (151.4–12 715 mg kg−1 DM). CONCLUSION A considerable nitrate content was found in samples of seven ‘accumulator’ species (Clitocybe nebularis, C. odora, Lepista nuda, L. personata, L. irina, Macrolepiota rachodes and M. procera). The toxicological relevance of daily uptake of acceptable nitrate content via mushrooms only is not presumable, but the ‘accumulator’ saprotrophic species can be ‘contributors’ to our nitrate intake in foods. © 2015 Society of Chemical Industry

The recently identified fungal protease inhibitors cnispin, from Clitocybe nebularis, and cospin, from Coprinopsis cinerea, are both β-trefoil proteins highly specific for trypsin. The reactive site residue of cospin, Arg27, is located on the β2-β3 loop. We show here, that the reactive site residue in cnispin is Lys127, located on the β11-β12 loop. Cnispin is a substrate-like inhibitor and the β11-β12 loop is yet another β-trefoil fold loop recruited for serine protease inhibition. By site-directed mutagenesis of the P1 residues in the β2-β3 and β11-β12 loops in cospin and cnispin, protease inhibitors with different specificities for trypsin and chymotrypsin inhibition have been engineered. Double headed inhibitors of trypsin or trypsin and chymotrypsin were prepared by introducing a second specific site residue into the β2-β3 loop in cnispin and into the β11-β12 loop in cospin. These results show that β-trefoil protease inhibitors from mushrooms exhibit broad plasticity of loop utilization in protease inhibition.

Current knowledge of the medicinal properties of Basidiomycetes mushroom species of the genus Clitocybe and of the biological activity of C. nebularis fruiting bodies is reviewed. The main focus is the therapeutic potential of lectins from C. nebularis. Species of the genus Clitocybe, including C. nebularis, have not been traditionally considered as medicinal mushrooms; however, recent studies have demonstrated their antitumor, immunomodulatory, and antioxidative properties, their antimicrobial (antiviral, antibacterial, and antifungal) activities against various bacteria and fungi, as well as their potential use in therapy for alcoholism and as psychotropic agents. These activities have been shown to be due to various compounds, either isolated or in extracts, mainly polysaccharides but also phenols, ribonucleosides, and proteins. These include laccase, protease inhibitors, and lectins. C. nebularis has been shown to be rich in a variety of lectins and isolectins with distinct carbohydrate-binding specificities, showing versatile biological activities. They exhibit immunostimulatory and adhesion-/phagocytosis-promoting properties, as well as toxicity in various invertebrates. Mushroom species of the genus Clitocybe, including C. nebularis, thus constitute a valuable source of compounds showing diverse biological activities with a broad potential for applications in biomedicine or biotechnology. On the basis of such evidence reviewed here, we propose that C. nebularis and other Clitocybe species can be considered to be medicinal mushrooms.

The goal of this research was a comprehensive analysis of four wild edible mushroom species, Cantharellus cinereus, Clavariadelphus pistillaris, Clitocybe nebularis and Hygrocybe punicea, which have not been analyzed so far. Extracts of different polarities have been prepared and evaluated for their antioxidant activities by DPPH, ABTS, FRAP, TRP and CUPRAC methods. For all extracts, total phenolic content was determined. Based on the analysis, it was shown that solvent type had a significant effect on the antioxidant capacities of mushroom extracts, so water extracts showed the highest activity. Furthermore, the analysis includes determination of mineral composition, fatty acid profiles and antimicrobial activity. Unsaturated fatty acids, which are very important for human health, are dominant in the studied mushroom species. Linoleic and oleic acid consist of over 50 % of the total fatty acid composition. Seventeen biologically important and toxic elements have been analyzed by ICP-OES and ICP-MS and results showed that the element concentrations were species-dependent. Also, it has been found that analyzed mushrooms did not show any antimicrobial activity. Chemometric analysis was used to understand the connection between the extracts of different polarities. This is the peer-reviewed version of the following article: Dimitrijević, M. V.; Mitić, V. D.; Nikolic, J. S.; Djordjevic, A. S.; Mutić, J.; Stankov-Jovanović, V. P.; Stojanovic, G. S. First Report about Mineral Content, Fatty Acids Composition and Biological Activities of Four Wild Edible Mushrooms. Chemistry and Biodiversity 2019, 16 (2), 2–11. [https://doi.org/10.1002/cbdv.201800492]

In vitro Kirby-Bauer disc-diffusion assays coupled with bio-imaging software techniques were used to assess native forest dwelling “fairy ring” forming fungi (Clitocybe nebularis) and co-habitant forest tree-root colonising non pathogenic, antibiotic producing bacteria (Bacillus licheniformis, Bacillus pumilis) for their antagonism towards Japanese larch dieback oomycetes phytopathogens which also affects ornamental alternative hosts. The aqueous extracts of C. nebularis exhibited the highest clearance (inhibitory) zone of 21.4 × 105 pixels = 573%) against Phytophthora ramorum than growth/clear zone Cartesian integrates recorded in untreated (control) disc (3.7 × 105 pixels = 100%) over 3-day incubation. The fairy ring fungal extracts also exhibited substantive antagonism against P. kernoviae (147%), P. lateralis (347%) and a solanaceous crop infecting P. infestans (86%). Quite encouragingly, the soil oomycete phytopathogen P. ramorum was inhibited strongly (mean ~ 177%) by both forest bacilli. Aqueous extracts of non-forest antifungal herbaceous plants (garlic and elderberry) expressed similar inhibitory effects (mean ~ 70%). A seaweed fungal elicitor component fucoidan showed moderate levels (mean ~ 85%) of antagonism against P. ramorum, P. kernoviae, P. lateralis and P. infestans. The results in this in vitro study highlight the intensity and vigor of antagonistic forest microflora and non-forest herbaceous antifungal agents such as garlic and other plant extracts as serious candidates for suppression of the oomycete Phytophthora pathogenic fungi in forest soils. This study calls for urgent scoping and impact assessment studies in pot experiments and mini-plot forest trials to gauge the fitness of these natural resources for field level potential biotechnological applications to combat the devastating dieback disease in the native woodlands and horticulture.

A lectin like protein designated as LSMT is recently discovered in Agaricus bisporus. The protein adopts very similar structure to Ricin-B like lectin from Clitocybe nebularis (CNL) and HA-33 from Clostridium botulinum (HA-33), which both recognize sugar molecules that decorate the surface of the epithelial cells of the intestine. A preliminary study in silico pointed out potential capability of LSMT to perform such biological activity. Following that hypothesis, we demonstrated that LSMT is indeed capable of penetrating out from a dialysis tube of the mice intestine origin. Furthermore, the protein appeared not to evoke the immune response upon introduction into mice, unlike its structural homologs. This is the first report on the biological implication of LSMT that might lead to its application. (C) 2016 Elsevier Inc. All rights reserved.

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.

Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a β-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the β2 and β3 strands, distinguishing cospin from other β-trefoil-fold serine protease inhibitors in which β4-β5 or β5-β6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.

A novel lectin, isolated from the basidiomycete mushroom Clitocybe nebularis and termed C. nebularis lectin (CNL), exhibits an immunostimulatory effect on the most potent antigen-presenting cells, the dendritic cells (DCs). Treatment of human monocyte-derived DCs with CNL in doses from 1 to 10 μg/ml resulted in a dose-dependent induction of overall DC maturation characteristics. Exposure of DCs to CNL for 48 hr resulted in extensive up-regulation of co-stimulatory molecules CD80 and CD86, as well as of the maturation marker CD83 and HLA-DR molecules. Such CNL-matured DCs (CNL-DCs) were capable of inducing a T helper type 1-polarized response in naive CD4+ CD45RA+ T cells in 5-day allogeneic co-cultures. The allostimulatory potential of CNL-DCs was significantly increased relative to untreated controls, as was their capacity to produce several pro-inflammatory cytokines such as interleukin-6, interleukin-8 and tumour necrosis factor-α. By using a specific Toll-like receptor 4 (TLR4) signalling inhibitor, CLI-095, as well as Myd88 inhibitory peptide, we have shown that DC activation by CNL is completely dependent on the TLR4 activation pathway. Furthermore, activation of TLR4 by CNL was confirmed via TLR4 reporter assay. Measurement of p65 nuclear factor-κB and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CNL stimulation of DCs revealed primarily an increase in nuclear factor-κB activity, with less effect on the induction of p38 MAPK signalling than of lipopolysaccharide-matured DCs. The CNL had the ability to activate human DCs in such a way as to subsequently direct T helper type 1 T-cell responses. Our results encourage the use of mushroom-derived lectins for use in therapeutic strategies with aims such as to strengthen anti-tumour immune responses.

Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators.

Research of the hyphosphere of ten species of basidial macromycetes permitted them to be divided into two groups by the character of impact on bacteria. In the first group of basidiomycetes (Albatrellus ovinus, Gomphidius glutinosus, Clitocybe nebularis, Gymnopus confluens, Coltricia perennis, Ramaria stricta), inhibition of bacteria by the macromycetes was observed; in the second group (Geastrum fornicatum, Calocera viscosa, Clavariadelphus ligula, Lycoperdon perlatum), there was a stimulation that manifested itself in the change in the indicators of the total number of bacteria, share of cells with a damaged external membrane, and suppression of development of several genera. The obtained results speak for the diversity of the relations between the basidiomycetes and bacteria in this soil locus.

Basidiomycete mushrooms are a rich source of unique substances, including lectins, that could potentially be useful in biotechnology or biomedical applications. Lectins are a group of carbohydrate-binding proteins with diverse biological activities and functions. Here, we demonstrate the presence of a number of lectins in the basidiomycete mushroom Clitocybe nebularis. Glucose-, galactose-, sucrose-, lactose-, and Sepharose-binding lectins were isolated from fruiting bodies using affinity chromatography on Sepharose-immobilized sugars or on Sepharose. The lectins were characterized biochemically and their binding specificities examined by agglutination and agglutination inhibition assays. In addition, insecticidal and anti-nutritional properties of the lectins were studied against a model organism, fruit fly (Drosophila melanogaster), and Colorado potato beetle (Leptinotarsa decemlineata). Of the several basidiomycete mushrooms screened, C. nebularis extract showed the most potent insecticidal activity. Sucrose-binding lectin showed the strongest activity against D. melanogaster, followed by lactose- and galactose-binding lectins. Feeding bioassays with Colorado potato beetle revealed that C. nebularis extract exhibited high anti-nutritional activity against the insect; and of those tested, only lactose-binding lectin, named CNL showed the effect. Mushroom C. nebularis is shown to be rich in a variety of lectins with versatile biological activities, including insecticidal and anti-nutritional effects. C. nebularis lectins could thus have potential for use as natural insecticides.

Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue. The binding is associated with a peptide-bond flip of glycine that occurs before or concurrently with the inhibitor docking. Mycocypins possess a beta-trefoil fold, the hallmark of Kunitz-type inhibitors. It is a tree-like structure with two loops in the root region, a stem comprising a six-stranded beta-barrel, and two layers of loops (6 + 3) in the crown region. The two loops that bind to cysteine cathepsins belong to the lower layer of the crown loops, whereas a single loop from the crown region can inhibit trypsin or asparaginyl endopeptidase, as demonstrated by site-directed mutagenesis. These loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.

We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K i in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family.

A new family of cysteine protease inhibitors from the basidiomycete Macrolepiota procera has been identified and the family members have been termed macrocypins. These macrocypins are encoded by a family of genes that is divided into five groups with more than 90% within-group sequence identity and 75-86% between-group sequence identity. Several differences in the promoter and noncoding sequences suggest regulation of macrocypin expression at different levels. High yields of three different recombinant macrocypins were produced by bacterial expression. The sequence diversity was shown to affect the inhibitory activity of macrocypins, the heterologously expressed macrocypins belonging to different groups showing differences in their inhibitory profiles. Macrocypins are effective inhibitors of papain and cysteine cathepsin endopeptidases, and also inhibit cathepsins B and H, which exhibit both exopeptidase and endopeptidase activities. The cysteine protease legumain is inhibited by macrocypins with the exception of one representative that exhibits, instead, a weak inhibition of serine protease trypsin. Macrocypins exhibit similar basic biochemical characteristics, stability against high temperature and extremes of pH, and inhibitory profiles similar to those of clitocypin from Clitocybe nebularis, the sole representative of the I48 protease inhibitor family in the merops database. This suggests that they belong to the same merops family of cysteine protease inhibitors, the mycocypins, and substantiates the establishment of the I48 protease inhibitor family.

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcalpha1-3(Fucalpha1-2)Galbeta-containing carbohydrates, and GalNAcbeta1-4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.

The current state in heavy metals contamination and mineral nutrition of beech forests near the aluminium plant Žiar nad Hronom (Central Slovakia) was evaluated based on soil analysis and on element analysis of potential fungal and plant indicator species. The research was conducted in two monitoring plots with high (PMP Žiar nad Hronom) and low (BEES Kremnicke vrchy Mts.) immission loads, respectively. Supply of macronutrients (Ca 2+ , Mg 2+ , K + ) was found sufficient to sustain forest growth at both sites. Differences in trace metal concentrations between these two localities were confirmed to be persistent. In the more polluted site, trace metal contents were found to be higher in the A horizon compared to the B horizon, indicating an essentially anthropogenic origin of the current metal levels and a strong binding of certain trace metals to soil organic matter. Different distribution patterns were found for calcium, magnesium, sodium, copper, cadmium, lead in plant and fungal biomass. In the herbal plant species Carex pilosa and Dentaria bulbifera , increased concentrations of potassium in aboveground biomass and a tendency to retain copper in the root biomass were observed. Tolerable cadmium concentrations were exceeded in plant samples from PMP Žiar nad Hronom. Both cadmium and lead concentrations were above the recommended limits in fruit bodies of many fungal species, which may hence constitute a health hazard if consumed. The highest concentrations of heavy metals were found in Agrocybe praecox for Pb (48.77 mg kg −1 ), in Gymnopus aquosus for Cd (4.75 mg kg −1 ), in Polyporus varius for Zn (364.97 mg kg −1 ) and in Clitocybe nebularis for Cu (34.10 mg kg −1 ).

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-1) associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.

Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.

Ergosterol (ergosta-5 ,7,22-trien-3β-ol ) is a compound found only in fungi. Ergosterol has medical interest, as it is easily converted into Vitamin D2. In the previous work we have studied 22 species of true fungi. The highest quantity of ergosterol was found in basidiomes of Clouded Agaric (Clitocybe nebularis (Fr.) Kumm). Meanwhile, there is no data on the purification of ergosterol from this fungus in the literature.The aim of research.The aim of research is to elaborate a rational method of obtaining ergosterol from basidiomes of Clouded Agaric and assess the perspectives of the raw material for its purification.Materials and methods.In order to choose the best method of ergosterol purification, it was obtained in two ways: 1 ) by alkaline hydrolisis that was used in the preparation of ergosterol from baker yeast and 2) by the extraction of raw matherial by methanol with following chromatography on silica gel.A substance with high ergosterol level was purified from marc basidiomes of Clouded Agaric by methanol extraction with following of freezing the methanol extract and the following chromatography on silica gel. This substance was dissolved in hexane and purified by chromatography on silica gel by washing the column sequentially with hexane, hexane - ethyl acetate (6:1 ), hexane -ethyl acetate -methanol 4:2:1. The obtained fractions were dried and weighed.The analysis of the obtained compounds was carried out by gas-liquid сhromatography - mass spectrometry , Lieberman-Burchard reaction and TLC on silica gel. Chromatograms were spraied with saturated sodium antimony in chloroform.Results and discussionPure ergosterol was obtained from dried baker's yeast and basidiomes marc of Clouded Agaric by alkaline hydrolysis followed by methylene chloride extraction of raw material and crystallization from ethanol and mixtures of benzene with ethanol (yield 0.71% and 0.52% ).Methanol extraction of dried basidiomes marc of Clouded Agaric, concentration and freezing, helped to obtain the substance, which according to the GLC -MS contained up to 91.1% of ergosterol. Chromatography of the substance obtained on silica gel allowed to obtain pure ergosterol with minor losses and higher purity, than by the crystallization.ConclusionsWe found a high content of ergosterol in Clouded Agaric basidiomes using gas-liquid chromatography-mass spectrometry. Almost pure ergosterol was sedimented by freezing of methanol extract.Authentic sample of ergosterol obtained from baker's yeast by TLC confirmed the identification of Clitocybe nebularis ergosterol.It was shown that chromatographic purification on a column of silica gel had advantages over alkaline hydrolysis of raw matherial and allowed to obtain pure ergosterol in a single stage with a higher yield (2.54 % of pure ergosterol). This method of purification is also an opportunity to obtain other substances which are in the methanol extract., Источником получения витамина D2 в промышленности является эргостерин, поэтому поиск его дешевых и богатых источников является актуальным. Среди исследованных нами 22 видов настоящих грибов его содержание оказалось самым высоким в говорушке серой (Clitocybe nebularis (Fr.) Kumm.). Поэтому с целью разработки рациональной методики его получения осуществлена очистка эргостерина из сушеных выжимок базидиом 2 методами: щелочного гидролиза и экстракции сырья метанолом со следующей хроматографией на силикагеле. Установлено, что хроматографическая очистка на колонке силикагеля имеет преимущества над щелочным гидролизом сырья, позволяя получить чистый эргостерин в одну стадию с высоким выходом (2,54%) и открывает возможность получения других веществ, которые содержатся в метанольной вытяжке., Джерелом отримання вітаміну D2 у промисловості є ергостерин, тому пошук його дешевих і багатих джерел є актуальним. Серед 22 видів справжніх грибів, які ми дослідили, його вміст виявився найвищим у грузлика димчатого (Clitocybe nebularis (Fr.) Kumm.). З метою розробки раціональної методики його отримання здійснили очистку ергостерину з сушених вичавок базидіом двома методами: лужного гідролізу та екстракції сировини метанолом із наступною хроматографією на силікагелі. Встановили, що хроматографічна очистка на колонці силікагелю має переваги над лужним гідролізом сировини, даючи змогу отримати чистий ергостерин в одну стадію з високим виходом (2,54%) і відкриває можливість виділення інших речовин, які містяться в метанольній витяжці.

Fungi are members of a large group of eukaryotic organisms that include yeasts and molds, as well as the most familiar member, mushrooms. Fungal lectins with unique specificity and structures have been discovered. In general, fungal lectins are classified into specific families based on their amino acid sequences and three-dimensional structures. In this chapter, we provide an overview of the approximately 80 types of mushroom and fungal lectins that have been isolated and studied to date. In particular, we have focused on ten fungal lectins (Agaricus bisporus, Agrocybe cylindracea, Aleuria aurantia, Aspergillus oryzae, Clitocybe nebularis, Marasmius oreades, Psathyrella velutina, Rhizopus stolonifer, Pholiota squarrosa, Polyporus squamosus), many of which are commercially available and their properties, sugar-binding specificities, structural grouping into families, and applications for biological research being described. The sialic acid-specific lectins (Agrocybe cylindracea and Polyporus squamosus) and fucose-specific lectins (Aleuria aurantia, Aspergillus oryzae, Rhizopus stolonifer, and Pholiota squarrosa) each showed potential for use in identifying sialic acid glycoconjugates and fucose glycoconjugates. Although not much is currently known about fungal lectins compared to animal and plant lectins, the knowledge accumulated thus far shows great promise for several applications in the fields of taxonomy, biomedicine, and molecular and cellular biology.

The molecular mass of clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, has been determined by analytical ultracentrifugation and gel exclusion chromatography. The result is in agreement with the formula mass of 16.8 kDa, demonstrating that the inhibitor is a monomer in aqueous solution. This enables the kinetics of unfolding and refolding to be interpreted in terms of folding in a kinetically two state, highly cooperative transition from the thermally unfolded state.