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Dolichin, a new chitinase-like antifungal protein isolated from field beans (Dolichos lablab).

An antifungal protein, possessing a molecular weight of 28 kDa and an N-terminal sequence resembling chitinases, has been purified from the seeds of the field bean Dolichos lablab. The procedure involved extraction with aqueous buffer, affinity chromatography on Affi-gel blue gel, and ion exchange chromatography on CM-Sepharose. The protein, designated dolichin, exhibited antifungal activity against the fungi Fusarium oxysporum, Rhizoctonia solani, and Coprinus comatus. Dolichin was capable of inhibiting human immunodeficiency virus (HIV) reverse transcriptase and alpha- and beta-glucosidases which are glycohydrolases implicated in HIV infection. It had very low ribonuclease and cell-free translation-inhibitory activities.

Amino Acid Sequence↗

Mungin, a novel cyclophilin-like antifungal protein from the mung bean.

A protein designated mungin, isolated from mung bean (Phaseolus mungo) seeds, possessed activity against the fungi Rhizoctonia solani, Coprinus comatus, Mycosphaerella arachidicola, Botrytis cinerea, and Fusarium oxysporum. The 18-kDa protein also possessed a novel N-terminal sequence with similarity to cyclophilins. It exerts an inhibitory action against alpha- and beta-glucosidases suppresses [(3)H]thymidine in corporation by mouse splenocytes.

Amino Acid Sequence↗

A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum.

From the inner shoots of the chive Allium tuberosum, a single-chained protein with a molecular weight of 36 kDa and an N-terminal sequence manifesting resemblance to chitinases but lacking in cysteine residues characteristic of a cysteine-rich domain present in chitinases of other Allium species, was purified. The isolation procedure entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on DEAE-cellulose and Mono S, and gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It exhibited antifungal activity against Rhizoctonia solani, Fusarium oxysporum, Coprinus comatus, Mycosphaerella arachidicola, and Botrytis cinerea. The IC(50) for its antifungal effect against Botrytis cinerea was 0.2 microM. The antifungal activity was stable after 1 h at pH 1.6 and 12.3, and up to 60 degrees C for 5 min. Incubation of the protein with trypsin or chymotrypsin at an enzyme:substrate ratio of 1:100 and pH 7.6 up to 150 min did not affect its antifungal activity. The protein did not exhibit antibacterial activity. The protein inhibited cell-free translation in a rabbit reticulocyte system with an IC(50) of 0.8 microM, but did not affect the proliferation of mouse splenocytes. It exerted some cytotoxic effect on breast cancer cells and was inhibitory toward HIV-1 reverse transcriptase.

Allium↗

Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein.

A novel single-chained antifungal protein with a molecular weight of 13 kDa displaying an N-terminal sequence with marked similarity to embryo-abundant protein from the white spruce was isolated from the seeds of Ginkgo biloba using ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and then gel filtration on Superdex 75. The protein, designated ginkbilobin, exerted potent antifungal activity against a variety of fungi, including Botrytis cinerea, Mycosphaerella arachidicola, Fusarium oxysporum, Rhizoctonia solani, and Coprinus comatus. Ginkbilobin exhibited a moderate antibacterial action against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. It suppressed the activity of HIV-1 reverse transcriptase and the proliferation of murine splenocytes.

Animals↗

Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum.

The pAN7.1 plasmid containing the E. coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum. Hygromycin-resistant transformants were selected at a frequency of one to five per micrograms of transforming DNA. Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome. The selection system was used to introduce other genes of interest by co-transformation. Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H. cylindrosporum genome with up to 70% efficiency of co-transformation. The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycin-free medium. All transformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.

Basidiomycota↗

Comparative recoveries of airborne fungus spores by viable and non-viable modes of volumetric collection.

The suitability of viable and non-viable volumetric collectors as prevalence indicators for potentially allergenic airborne fungi was studied during 124 paired exposures of the Burkard (Hirst) spore trap and a modified, wind-oriented Andersen sampler. Overall, viable recoveries of several Cladosporium form species varied directly with microscopic spore counts (p less than or equal to 0.0001). However, as spore levels rose, culture plate data progressively underestimated prevailing concentrations (recoveries falling below 5% at levels above 500 spores/M3). Viable collections yielded low estimates of prevalence (20-40%) even at modest Cladosporium levels (less than 100 spores/M3) and substantially understated the abundance and regularity in air of several additional taxa. Spores typical of Penicillium and Aspergillus form species were not sought in spore trap deposits. Careful examination of these failed to reveal typical arthrospores or Fusarium macrospores despite substantial recoveries of corresponding growth in culture. Correlations in the occurrence patterns of arthrospore-forming and non-sporulating colonies with those of Coprinus and 'other basidiospores' (excluding Ganoderma) were noted.

Air Microbiology↗

[Study of the contamination by dermatophytes of a population of small wild mammals in Alsace].

Mature fruit-bodies of Coprinus congregatus grown on modified Bille-Hansen medium were obtained with concentrations of glucose ranging from 5 to 40 g/l and asparagine from 0 to 4 g/l; the carbon/nitrogen ratio varying from 13 to 100. When the ratio C/N is too high (above 100), no visible primordium is formed; when this ratio is under 13, numerous primordia are produced, but they never reach maturity. A positive correlation exists between the C2/N ratio and the time required for producing mature fruit-bodies. The best yield for basidiocarps production is reached when the C/N ratio value is around 30 and glucose concentrations range from 10 to 20 g/l.

Animals↗

Studies on mushroom flavours. 3. Some flavour compounds in fresh, canned and dried edible mushrooms.

The concentrations of 1-octen-3-ol, 5'-GMP and glutamic acid were compared in 8 fresh, 3 canned and 5 dried mushroom species. The highest amounts of 1-octen-3-ol and 5'-GMP were found in the fresh muschrooms. Agaricus bitorquis, Pleurotus ostreatus and Pholiota squarrosa contained 5-7 times as much 1-octen-3-ol as Agaricus bisporus and Calvatia gigantea 58 times as much. Coprinus comatus and Pleurotus ostreatus contained much 5'-GMP. Little 1-octen-3-ol and 5'-GMP were found in most dried and canned mushrooms. Potatoes and tomatoes were analyzed for comparison. Little or no 1-octen-3-ol and 5'-GMP were observed. Glutamic acid was present in most samples in sufficient quantities to have an important influence on the flavour.

Basidiomycota↗

Silver content of wild-grown mushrooms from northern Poland.

Wild-grown and mostly edible species of higher mushrooms collected in 1989-1992 from the districts of Gdańsk, Elblag and Piła (northern part of Poland) have been investigated with regard to their silver content. In total, 527 samples including 25 mushroom species of six families (Agaricaceae, Boletaceae, Cantharellaceae, Hygrophoraceae, Coprinaceae and Russulaceae) were examined. Among the fungi examined only Agaricus campestris and A. augustus were heavy bioaccumulating species and showed the highest concentrations of 35 mg Ag/kg dry weight (1.1-150) and 2.0-6.9 mg/kg, respectively. Silver concentrations exceeding 1.0 mg/kg dry weight were noted in Boletus aestivalis, Lepista nuda, L. personata, and in some specimens of B. edulis, Leccinum scrabum, L. vulpinum, Cantharellus cibarius and Coprinus comatus.

Agaricus↗

A year-round study on functional relationships of airborne fungi with meteorological factors.

Air sampling was conducted in Waterloo, Canada throughout 1992. Functional relationships between aeromycota and meteorological factors were analysed. The meteorological factors were, in descending order of importance: mean temperature, minimum temperature, maximum temperature, mean wind speed, relative humidity (RH), rain, maximum wind speed and snow. The most important airborne fungal propagules in descending order were total fungal spores, unidentified Ascomycetes, Cladosporium, Coprinus, unidentified Basidiomycetes, Alternaria and unidentified fungi. Most airborne fungal taxa and highly significant relationship with temperature, but Aspergillus/Penicillium, hyphal fragments were positively associated with wind speed. In comparison with other airborne fungal taxa, Leptosphaeria and unidentified Ascomycetes were more closely correlated with rain and RH during the growing season.

Air Microbiology↗

[Radioactivity levels in mushrooms collected in the area of Karlsruhe during 1987 and 1988].

Radioactive contamination of wild-growing mushrooms collected in the Northern Black Forest and neighbouring Rhine Valley area has been monitored since the Chernobyl reactor accident. Cs-134, CS-137, Ag-100m and the natural nuclide K-40 were determined by gamma-spectrometry. Pooled data from all species and all locations showed a decrease in the radio-caesium levels from 1986 to 1988, both in mean values and maximum values. Samples of the same species collected at the same location exhibited large differences, although mixed samples rather than individual mushrooms were measured. Accumulation of caesium was observed particularly in Laccaria amethystina, Xerocomus badius, Xerocomus chrysenteron, Cantharellus tubaeformis, Laccaria laccata, and Russula ochroleuca, with maximum values of 3600 (1986), 2000 (1987), and 1200 (1988) Bq/kg of radiocaesium. A large number of species, among them Calocybe gambosa and Coprinus comatus, showed very small accumulations or no caesium. An accumulation of silver was found in Macrolepiota rhacodes and Agaricus arvensis, which contained a maximum of 11 Bq/kg Ag-110m.

Accidents↗

Coprophilous fungi of the horse.

A total of 1267 microfungi, including 35 Myxomycetes, were recorded from the fecal samples of the 60 horses; of these 395 were found on 20 saddle-horse feces, 363 on 20 race-horses and 509 on 20 working horses. Eighty two species representing 53 genera were recorded; of these 7 were Zygomycetes, 18 Ascomycetes, 1 Basidiomycetes and 25 Fungi Imperfecti: 2 Myxomycetes. Common coprophilous fungi are in decreasing order Pilobolus kleinii, Saccobolus depauperatus, Mucor hiemalis, Lasiobolus ciliatus, Podospora curvula, Petriella guttulata, M. circinelloides, Coprinus radiatus, Dictyostelium mucoroides, Sordaria fimicola, C. miser, C. stercorariusm, Acremonium sp., Coprotus granuliformis, Graphium putredinis, Iodophanus carneus, Chaetomium murorum, Podospora communis, P. inaequalis, P. setosa, Saccobolus versicolor and Cladosporium cucumerinum. Species of Myrothecium verrucaria, Actinomucor elegans, Kernia nitida, Spiculostilbella dendritica and Mucor parvispora were found exclusively in working-horses feces. Badhamia sp., Anixiopsis stercoraria, Echinobotryum state of D. stemonitis, Geotrichum candidum and Oidiodendron sp. were found only in saddle-horses feces. Chlamidomyces palmarum, Philocopra sp. were found exclusively in race-horses feces. Notes on infrequent or interesting fungi include Thamnostylum piriforme, Phialocephala dimorphospora, Rhopalomyces elegans and Spiculostilbella dendritica.

Animals↗

The homeodomains of the heterodimeric bE and bW proteins of Ustilago maydis are both critical for function.

In Ustilago maydis, pathogenic development is controlled by the multiallelic b-mating type locus. b encodes two different homeodomain proteins, bE and bW, that dimerize only when they originate from different alleles. The heterodimer is thought to act as a transcriptional regulator of pathogenicity genes. To address the contribution of individual homeodomains to the function of the hetereodimer, amino acids presumed to be critical for the function of homeodomains were altered by introducing deletions and single amino acid substitutions. This analysis demonstrated that both homeodomains are essential for function of the bE/bW complex. All point mutations affecting conserved amino acids in bW resulted in non-functional proteins. For bE the mutational analysis revealed that certain features of the homeodomain, like base-specific contacts and contacts to the sugar phosphate backbone, contribute to function. However, the structural requirements for function of the bE homeodomain appear to be more flexible than those of the bW homeodomain. We compare our data with the related systems in Schizophyllum commune, Coprinus cinereus and Saccharomyces cerevisiae.

Amino Acid Sequence↗

The concentrations and bioconcentration factors of copper and zinc in edible mushrooms.

Copper and zinc contents have been determined in up to 28 species of edible mushrooms from different sites in the province of Lugo (NW Spain). Two hundred thirty-eight collected samples were analyzed by an anodic stripping voltammetric technique using a drop mercury as the working electrode. The results showed that the element concentrations were species-dependent, and the highest metal levels corresponded to the following species: Calvatia utriformis (235.5 mg Cu/kg), Macrolepiota procera (217.8 mg Cu/kg), and Agaricus macrosporus (217.7 mg Cu/kg) and Calvatia utriformis (265.8 mg Zn/kg), Lactarius delicious (231.0 mg Zn/kg), and Agaricus macrosporus (221.3 mg Zn/kg) for Cu and Zn, respectively. All mushroom species bioaccumulated copper and zinc. Nevertheless, some individual samples of the species, such as Hydnum repandum, Cantharellus cibarius, and Coprinus comatus, were bioexclusors (BCF < 1). The hymenophore in mushrooms showed higher mean metal levels than the rest of the fruit bodies, with statistically significant differences. The copper and zinc concentrations were compared to literature data and levels set by legislation. It can be concluded that the consumption of these mushrooms cannot be considered a toxicological risk, and they provide an important nutritional requirement to the diet.

Agaricales↗

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost, the cobia (Rachycentron canadum).

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward D-mannosamine and D(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40 degrees C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60 degrees C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 microM FeCl3. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 microg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 microg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 microM.

Animals↗

Mating-type genes for basidiomycete strain improvement in mushroom farming.

Mushroom production is dependent on the quality of the spawn used to inoculate the cultures. In order to produce high-quality spawn, breeding programs for strains resistant to certain diseases and able to form high-quality fruit bodies under standard growth conditions are necessary. The investigation of the molecular basis for mating provides access to the use of mating-type genes in order to facilitate breeding. For research purposes, two mushroom-forming homobasidiomycetes have been used due to their easy cultivation and sexual propagation on defined minimal media: Schizophyllum commune and Coprinus cinereus. The mating-type genes control formation of the dikaryon from two haploid strains. Only the dikaryon is fertile and able to form mushrooms under the right environmental conditions. These genes are now used in mating-type-assisted breeding programs for economically important mushrooms, especially the white button mushroom, Agaricus bisporus, and the oyster mushroom, Pleurotus ostreatus, aiming at high-yield and high-quality standard mushroom production. Most mushroom species posses two mating-type loci that control their breeding. The genes encoded in the A loci lead to the formation of transcription factors that belong to the class of homeodomain proteins. Active transcription factors are formed by heterodimerization of two proteins of different allelic specificities. In nature, this is only the case if two cells of different mating type have fused to combine the different proteins in one cytoplasm. While fusion in homobasidiomycetes is found irrespectively of mating type, exchange of nuclei between mating mycelia is dependent on the products of the B mating-type loci. The B genes form a pheromone and receptor system that enables the fungi to initiate nuclear migration. The molecular details of the two genetic systems controlling breeding in basidiomycetes are presented in this review.

Basidiomycota↗

Evolution of multispecific mating-type alleles for pheromone perception in the homobasidiomycete fungi.

The evolution of multiple, independent and multispecific mating-type loci is a feature unique to homobasidiomycete fungi. To propose a model of evolution, data assembled for the wood-rotting fungus Schizophyllum commune were analyzed. In one mating-type locus, pheromone receptors and several pheromones are encoded which have been investigated in some detail and can be used to understand the ligand-receptor interactions and activation of signal transduction which are essential to sexual propagation. Previous models for the evolution of new alleles were complicated and involved three subsequent steps (without selectable phenotype) prior to the establishment of a new stable pheromone-receptor pair. This paper presents a model for the evolution of new specificities by recombination and selection that incorporates the multi-state receptor activation recently established for S. commune, explaining differential responses to different pheromones in one receptor molecule. The model takes into account the occurrence of multiple pheromone genes in each locus and unilateral nuclear donor/acceptor strains that may in nature act as steps in the evolution of new specificities. A second homobasidiomycete fungus, Coprinus cinereus, was similarly characterized at the molecular level. Data acquired in this system support the conclusion that the presented model can be generalized.

Basidiomycota↗

Structure and function of a pyrimidine/purine-biased sequence from the 5'-flanking region of the basidiomycete Lentinus edodes gene priA.

The priA gene of the basidiomycete Lentinus edodes possesses a pyrimidine (CT)-rich stretch (26 bp) that includes a short (6-bp) repeat, the elements of which form a mirror repeat at and near the transcriptional initiation sites. A DNA fragment that included this sequence was inserted into pBR322, and the resulting plasmids were introduced into Escherichia coli. Analysis of the susceptibility of these pBR322 derivatives to cleavage by S1 nuclease, following isolation from E. coli, indicated the formation of an open, S1-sensitive structure within and just downstream of the CT/AG-biased sequence. Replacement of two dTMP residues in one of the repeat elements by dGMP resulted in the elimination of the S1-cleavable open structure from the plasmids. To analyze the effect of the CT/AG-biased sequence from priA in the basidiomycete Coprinus cinereus, the integrating vectors pLC2 and pLC2mutCT were used; these contained the wild-type priA promoter and the mutant priA promoter with the aforementioned mutation in the mirror repeat, respectively. The Streptomyces-derived bialaphos resistance gene (bar) was fused downstream of the promoters, and the resulting plasmids, pLC2-bar and pLC2mutCT-bar, were introduced into C. cinereus. Transformants carrying pLC2mutCT-bar grew significantly more slowly on bialaphos-containing agar plates and contained a noticeably lower level of the bar transcript when compared with the transformants obtained with pLC2-bar. These results suggest that an unusual structure induced by the CT/AG-biased sequence is required for efficient gene expression from the priA promoter.

Base Sequence↗