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PubMed2002

Leucine aminopeptidase during meiotic development.

We found a leucine aminopeptidase (LAP; EC 3.4.11.1) to be abundant in meiotic prophase tissue of a basidiomycete, Coprinus cinereus. After direct purification of the aminopeptidase component from meiocytes, we cloned the gene by degenerate PCR using partial amino-acid sequences of the purified enzyme and 5' and 3' RACE. It was homologous to the eukaryotic leucine aminopeptidase gene. The recombinant protein possesses the characteristic activities of a Coprinus leucine aminopeptidase (CoLAP) with a molecular mass of 52.4 kDa, and forms a homohexamer. Northern blot and spatial distribution analysis by immunohistochemical staining indicated CoLAP to be abundant in meiotic prophase cells and the supporting cells around meiocytes, but scarce in mycelium cells. Interestingly, from zygotene to pachytene, CoLAP was mostly present in supporting cells around meiocytes, but from diplotene onwards, it was plentiful in meiocytes themselves, suggesting that its expression is required to control some of the biochemical events at meiotic prophase. Moreover, the strong expression of CoLAP mRNA immediately after treatment with methyl methanesulfonate in mycelium implies that CoLAP has a role in somatic DNA repair.

Amino Acid Sequence↗
PubMed1998

Lead content in edible wild mushrooms in northwest Spain as indicator of environmental contamination.

Lead content was determined in wild growing mushrooms collected from two different areas in the Province of Lugo (NW Spain). It has been analyzed by graphite furnace atomic absorption spectrometry in 95 samples of 13 species (7 mycorrhizals and 6 saprophites). In an assessment of lead concentrations, the following factors have been considered: species and ecology, morphological portion, and traffic pollution. The average lead concentration of the samples was 1 ppm dry weight (dw). Saprophite species presented higher levels than mycorrhizal ones (< 1 ppm), Coprinus comatus reaching the maximum mean concentration with 2.06 and 2.79 ppm of dw in the hymenophore and the rest of the fruit body. Morphological portion, statistically, did not show significant difference between the two portions; however, Macrolepiota procera always presented lead high levels in the hymenophore in all samples. The effect due to traffic pollution has been specially observed in Coprinus comatus, presenting the highest concentration with values of 6.51 and 10.43 ppm, respectively, in samples collected in the city center. This species, as other researchers have indicated, could be considered as an indicator by lead contamination. The contribution of mushrooms to the weekly intake of lead was calculated and the possible health risk for the consumer is pointed out. These data are of great importance in view of toxicology and partly environmental protection.

Basidiomycota↗
PubMed1990

Evidence for cross-reactive allergens among basidiomycetes: immunoprint-inhibition studies.

Allergenic cross-reactivity among six basidiomycete species (Calvatia cyathiformis, Coprinus quadrifidus, Psilocybe cubensis, Pleurotus ostreatus, Ganoderma meredithae, and Pisolithus tinctorius) was determined by immunoprint inhibition. Extensive cross-reactivity was demonstrated among Coprinus quadrifidus, Psilocybe cubensis, and Pleurotus ostreatus of the order Agaricales, and Calvatia cyathiformis of the order Lycoperdales. However, G. meredithae (order Aphyllophorales) and Pisolithus tinctorius (order Sclerodermatales) did not demonstrate significant cross-reactivity with the other basidiomycete species. Generally, the two most potent inhibitors were Psilocybe cubensis and Pleurotus ostreatus. Inhibitory dose-response curves of a major allergenic band (isoelectric point, 9.3) were obtained by densitometry. Significant cross-reactivity was demonstrated for the 9.3 band among the species of the order Agaricales and with Calvatia cyathiformis. The most potent inhibitors were again Psilocybe cubensis and Pleurotus ostreatus. Thus, there is substantial allergenic cross-reactivity among the species of the order Agaricales tested and with Calvatia cyathiformis but not between these four species and G. meredithae or Pisolithus tinctorius. These studies support earlier RAST-inhibition observations of shared allergenic epitopes among basidiomycetes, especially epitopes within the Agaricales. The presence of shared epitopes suggests the possibility of devising a panel of skin test reagents representative of a large group of basidiomycetes.

Allergens↗
PubMed1998

Mushroom (Basidiomycete) allergy: diagnosis established by skin test and nasal challenge.

BACKGROUND: Fungal spores are universal components in the air and established as important causes of respiratory allergies. Whereas fungi imperfecti are accepted sources of allergic asthma and rhinitis, the significance of basidiomycetes as respiratory allergens is not established. OBJECTIVES: The aims of the present study were to investigate the rate of sensitization in subjects referred to an allergy clinic to 3 basidiomycetes commonly found in Europe. We demonstrate the clinical relevance of basidiomycete sensitization by active anterior rhinomanometry and identified basidiomycete allergens by immunoblotting. METHODS AND RESULTS: Consecutive outpatient clinic attendees (n = 1207, >12 years of age) were given the skin prick test with a panel of common inhalant allergens and extracts of Boletus, Coprinus, and Pleurotus. To evaluate a cause-and-effect relation between basidiomycete allergens and respiratory allergies, 12 Pleurotus pulmonalis spore-sensitized subjects with respiratory symptoms and 6 control subjects were challenged by anterior rhinomanometry. SDS-PAGE immunoblots were used to identify basidiomycete-specific IgE antibodies in sera from subjects with positive skin prick test results. Of 1207 subjects tested, 48 (4%) reacted to at least 1 of the basidiomycete extracts. Whereas all of the 12 subjects with Pleurotus pulmonalis-positive skin test results showed a significant decrease in nasal air flow (mean 73%), none of the control subjects reacted. Immunoblots revealed several different IgE-binding proteins in Pleurotus and Coprinus extracts. CONCLUSIONS: Our results demonstrate that sensitization to basidiomycetes in subjects with respiratory allergies is frequent. Furthermore, Pleurotus pulmonalis spore extracts can definitely induce respiratory allergy in sensitized subjects. Immunoblot assays disclosed different IgE-reactive bands indicating the existence of basidiomycete allergens.

Adolescent↗
PubMed1998

Characterization of laccases and peroxidases from wood-rotting fungi (family Coprinaceae).

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60 degrees C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.

Amino Acid Sequence↗
PubMed1998

Some characteristics of ammonia fungi 1. In relation to their ligninolytic enzyme activities.

Twenty-six species of ammonia fungi comprising 71 strains were screened for ligninolytic activity using agar plate tests. The tests comprised a wood powder plate test, the Bavendamm reactions, and a Remazol Brilliant blue R (RBBR) decolorization test. The wood powder plate test detected phenol oxidases of Coprinus spp., whereas this method obviously detected no activities from facultative mycorrhizal fungi, such as Hebeloma radicosoides and ectomycorrhiza: H. spoliatum and H. vinosophyllum. With quantitative assays of ligninolytic activity, Coprinus phlyctidosporus, C. echinosporus, Lyophyllum tylicolor, Lepista nuda, L. tarda, Calocybe leucocephala, and Crucispora rhombisperma, which grow on oak-leaf litter, the major phenol-oxidizing enzyme was a laccase. The concentration of urea affected laccase activity; however, urea was not the obligate nitrogen source for the laccase production.

Journal Article↗
PubMed2006

In vitro effects on proliferation, apoptosis and colony inhibition in ER-dependent and ER-independent human breast cancer cells by selected mushroom species.

Breast cancer is the most commonly diagnosed cancer among women in Western countries. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. We have screened 38 species of edible mushroom on human estrogen-receptor positive (MCF-7) and estrogen-receptor negative (MDA-MB-231, BT-20) breast cancer cells to select potential agents with broad-spectrum antitumor activity against breast cancer cells. Water-based extracts of three mushroom species, Coprinellus sp., Coprinus comatus, Flammulina velutipes (CME, CCE and FVE, respectively), were identified as novel anti-breast cancer agents. The anti-tumor activities include: 1) marked growth inhibition of both ER+ and ER- breast cancer cells; 2) induction of rapid apoptosis on both ER+ and ER- cells; 3) significant inhibition of MCF-7 tumor colony formation in vitro. The antiproliferative and cytotoxic activities of the three mushroom extracts were dose-dependent, regardless of the hormone receptor status of the cancer cells. The degree of produced cytotoxicity on ER- breast cancer cells was very high, while the IC50 of mushroom extract CME was found to be as low as 40 microg/ml on MDA-MB-231 cells and the IC50 of mushroom extract FVE was only 30 microg/ml on BT-20 cells. More interestingly, mushroom extracts CME and FVE induced an exceptionally rapid apoptosis on MCF-7 and MDA-MB-231 detected by Annexin V-FITC within 2 h of treatment and DNA fragment end-labeling assay (TUNEL) in 5 h of treatment. Anchorage-independent growth assays indicated that the MCF-7 tumor colony formation rate was reduced by 60% in CCE- and CME-treated cells and nearly completely inhibited (99%) by FVE treatment. These results suggest that mushroom species Coprinus comatus, Coprinellus sp. and Flammulina velutipes contain potent antitumor compounds for breast cancer. Our finding is important due to the lack of chemotherapeutic and chemopreventive agents for ER- human breast cancer.

Agaricales↗
PubMed2005

Annual variation of fungal spores in atmosphere of Porto: 2003.

The seasonal distribution of fungal spore concentration in the city of Porto, Portugal, was continuously studied, from 1 January to 31 December 2003, using a 7-day volumetric Burkard trap. In Portugal, aerobiological studies are scarce, and to our knowledge there are no published data on the atmospheric concentration of fungal spores. The aim of this work therefore is to initiate the aeromycological studies in Porto. The total airborne spore concentration fluctuated between 2 and 2,198 spores/m(3) per day with an daily mean of 403 spores/m(3). The highest airborne spore concentration was found during the summer and the early autumn, while the lowest concentration was registered during the winter. A range of allergenic and phytopathogenic fungal spores was present in the atmosphere of Porto throughout the entire year, although in different concentrations. Among the 22 fungal spore types identified Cladosporium (74.5 %), Ganoderma (11.7 %), Aspergillaceae (2.9 %), Ustilago (2.5 %), Coprinus (1.5 %), Alternaria (1.3 %) and Botrytis (1.3 %) were the most frequent. Seasonally, spore levels of Cladosporium and Alternaria peaked in the autumn and winter, Ganoderma peaked toward autumn, whilst spore levels of the basidiomycete Coprinus fluctuated throughout the year. The total spore concentration was negatively correlated with wind speed and positively correlated with temperature and relative humidity.

Air↗
PubMed1987

[Diagnosis and therapy of mushroom poisoning (II)].

In the second part of this review of mushroom poisonings, the syndromes with intermediate and long lag-times are discussed. They include the coprinus-, phalloides-, gyromitrin- and the orellanus syndrome. The coprinus syndrome occurs whenever alcohol is consumed after a meal containing coprine. The lag-time varies according to the amount and time of alcohol intake. It is very similar to the disulfiram syndrome which is known from the adverse therapy of alcoholism. The lag-time of the phalloides syndrome varies between 7 and 24 hours. It starts with massive gastroenteritis followed by hepatopathia which can lead to hepatic coma and kidney failure. The phalloides syndrome is caused by the amatoxins of the death caps, which inhibit the RNA Polymerase B in the nucleus of the liver cell. The gyromitrin syndrome exhibits also a delayed onset. The hepatotoxicity and the nephrotoxicity are less severe than in the phalloides syndrome. The first metabolite of gyromitrin monomethylhydrazin is responsible for CNS-symptoms such as delirium and convulsions. In contrast to the phalloides syndrome vomiting can be the only leading symptom in gyromitrin poisoning. The orellanus syndrome has the most delayed onset of all mushroom poisonings with 1-3 weeks. It should be thought of in all cases of kidney insufficiency of unknown origin. The orellanines damage the kidney and induce all degrees of kidney insufficiency according to the amount of ingested poison. Terminal kidney failure which requires hemodialysis treatment can occur in severe cases.

Basidiomycota↗
PubMed2005

Laccase-catalysed iodide oxidation in presence of methyl syringate.

The kinetics of potassium triiodide (KI(3)) formation during fungal laccase action was investigated in presence of methyl syringate (MS). The recombinant forms of Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL), and Rhizoctonia solani (rRsL) laccases were used. The triiodide formation rate reached 6.1, 5.5, 6.0, and 2.1 microM/min at saturated rPpL, rCcL, rRsL, and rMtL concentration, respectively, in acetate buffer solution pH 5.5 and in presence of 10 microM of MS and 1 mM of potassium iodide. The triiodide formation rate increased if pH decreased from 6.5 to 4.5. The scheme of laccase-catalysed iodide oxidation includes stadium of MS interaction with oxidized laccase with concomitant production of MS(ox). The reaction of MS(ox) with iodide produced triiodide. The turnover number of MS was 93 and 44 at pH 5.5 for rPpL and rMtL, respectively. The scheme also contained a stadium of reversible reduction of laccase active centre with the mediator explaining the different saturation rate of triiodide production. The fitting kinetic data revealed that the reversibility of the reaction increased for laccases containing lower redox potential of copper type I.

Ascomycota↗
PubMed1992

A fungal mating type protein that regulates sexual and asexual development contains a POU-related domain.

The A mating type factor of the fungus Coprinus cinereus regulates essential steps in sexual development. Here we describe features of one of the four specificity genes of the A42 factor. By transformation we show that the gene regulates not only sexual development but also asexual sporulation. DNA sequence analysis shows that the gene beta 1-1, encodes a protein with a DNA binding motif and is thus likely to be a transcription factor. The DNA binding domain is an unusual homeodomain with D replacing the normally invariant N in the recognition helix and apparent absence of helix II. The homeodomain is linked to a helical region related to the POUs domain, which is part of a bipartite DNA binding domain of certain animal transcription factors. Like POU factors, the beta 1-1 protein has regions rich in serine, threonine and proline which are possible transactivation domains. Putative dimerization domains and sites for post-translational modification are described.

Amino Acid Sequence↗
PubMed1999

First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity.

A protein, with a molecular weight of 20 kDa, and an N-terminal sequence analogous to those of thaumatin-like proteins (TLPs) and thaumatins, was first isolated from the legume of the French bean Phaseolus vulgaris cv Kentucky wonder using a simple procedure involving affinity and ion exchange chromatography. The protein was adsorbed on both CM-Sepharose and Affi-gel Blue Gel. It was the first leguminous TLP-like protein demonstrated to exert antifungal activity against Fusarium oxysporum, Pleurotus ostreatus, and Coprinus comatus but not against Rhizoctonia solani.

Amino Acid Sequence↗
PubMed2001

Isolation of a novel thermolabile heterodimeric ribonuclease with antifungal and antiproliferative activities from roots of the sanchi ginseng Panax notoginseng.

An isolation procedure, consisting of ion exchange chromatography on CM-Sepharose, affinity chromatography on Affi-gel blue gel, and fast protein liquid chromatography on Mono S, was utilized to purify a base-nonspecific, heterodimeric ribonuclease (RNase) with diverse activities from roots of the sanchi ginseng Panax notoginseng. The RNase is unique in that it consists of two different nonglycoprotein subunits with a molecular weight of 27 and 29 kDa, respectively. The latter subunit is characterized by an N-terminal sequence showing remarkable similarity to that of the bitter gourd RNase. The Panax notoginseng RNase demonstrates potent RNase and translation-inhibitory activities. In addition, it exhibits antiproliferative activity toward leukemia L1210 cells and antifungal activity against Physalospora piricola and Coprinus comatus. Its RNase activity is not heat-resistant, unlike most RNases which are thermostable.

Animals↗
PubMed1979

Metabolic products of microorganisms. 181. Chitin synthase from fungi, a test model for substances with insecticidal properties.

Chitin synthase from Coprinus cinereus (Schaeff. ex Fr.) S. F. Gray (= C. lagopus sensu Buller) was used as a model for chitin synthase from insects. The effect of dimilin (difluorobenzuron), captan (trichloromethylsulfonyl fungicide), kitazin P (organophosphorus ester fungicide) and parathion (organophosphorus insecticide) on the fungal enzyme was compared with the effect of nikkomycin (nucleosidepeptide antibiotic).

Agaricales↗
PubMed2000

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines.

Steady-state and single-turnover kinetics for the oxidation of the N-substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4-10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k(cat)/K(m)) varied from 1 x10(7)M(-1)s(-1) to 2.6 x 108 M(-1)s(-1) at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pKa values of 4.9-5.7 and 7.7-9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8x 10(5) M(-1) s(-1) to 2.0 x 10(7) M(-1) s(-1) at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6 x 10(8)M(-1)s(-1) and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0 x 10(7) M(-1) s(-1) and 0.29 eV.

Antiprotozoal Agents↗
PubMed1999

Peroxidase-benzhydroxamic acid complexes: spectroscopic evidence that a Fe-H2O distance of 2.6 A can correspond to hexa-coordinate high-spin heme.

Resonance Raman (RR) spectra have been obtained for single-crystal horseradish peroxidase isozyme C complexed with benzhydroxamic acid (BHA). The data are compared with those obtained in solution by both RR and electronic absorption spectroscopies at room and low (12-80 K) temperatures. Moreover, the analysis has been extended to Coprinus cinereus peroxidase complexed with BHA. The results obtained for the two complexes are very similar and are consistent with the presence of an aqua six-coordinate high-spin heme. Therefore it can be concluded that despite the rather long Fe-H2O distance of 2.6-2.7 A found by X-ray crystallography in both complexes, the distal water molecule can still coordinate to the heme iron.

Coprinus↗
PubMed1990

Luminol chemiluminescence reaction catalyzed by a microbial peroxidase.

A peroxidase produced by microorganisms belonging to the genera Arthromyces and Coprinus was found to be a potent catalyst for the chemiluminescent oxidation of luminol, the luminescence produced per unit of microbial peroxidase protein being well over 100 times as strong as that produced by horseradish peroxidase. No large difference in Km value for H2O2 in the presence of luminol was found between Arthromyces ramosus peroxidase and horseradish peroxidase (7.0 and 15.5 microM, respectively), but Vmax of the Arthromyces peroxidase was 500 times greater than that of the horseradish peroxidase. It was also found that the Arthromyces peroxidase surpasses, beyond expectation, the horseradish peroxidase in the initial velocity of the chemiluminescence reaction with the stopped-flow method. The Arthromyces peroxidase was used for the glucose and cholesterol assays, which were notably more sensitive than the corresponding assays involving the horseradish peroxidase.

Calibration↗
PubMed2006

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity.

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

Animals↗