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PubMed2003

Isolation of pisumin, a novel antifungal protein from legumes of the sugar snap pea Pisum sativum var macrocarpon.

An antifungal protein with a novel N-terminal sequence GVGAAYGCFG and a molecular mass of 31 kDa was isolated from the legumes of the sugar snap pea Pisum sativum var. macrocarpon. The protein, designated pisumin, exhibited antifungal activity against Coprinus comatus and Pleurotus ostreatus and much weaker activity against Fusarium oxysporum and Rhizoctonia solani. Pisumin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 6 microM. Pisumin was similar to other leguminous antifungal proteins in that it was adsorbed on Affi-gel blue gel and CM-Sepharose.

Animals↗
PubMed2003

cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis.

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.

Amino Acid Sequence↗
PubMed2003

Differentiation of Agaricus species and other homobasidiomycetes based on volatile production patterns using an electronic nose system.

Comparisons of the qualitative volatile production patterns between seven species of Agaricus, and between two of Volvariella and Pleurotus and one Coprinus species when grown at 25 degrees C on agar media for 14 d were made. There was good reproducibility between the volatile production patterns of the same species using an electronic nose unit with a 14 conducting sensor polymer array. Principle Component Analysis (PCA) showed that it was possible to discriminate between five of the seven Agaricus species, but that some overlap occurred between the others. Cluster analysis showed that there was also overlap between some species with the tropical collection of A. bitorquis separating out from the others. The volatile production profile of the commercial A. bisporus was close to that of a wild species, A. campestris. A. bisporus could be readily differentiated from other non-Agaricus species. This study demonstrates the potential for using electronic nose systems to rapidly differentiate mycelial cultures of homobasidiomycete mushrooms.

Agaricus↗
PubMed2005

Biomechanics of stipe elongation in the basidiomycete Coprinopsis cinerea.

Stipe elongation in fruit bodies of Coprinopsis cinerea (syn. Coprinus cinereus) was examined from a biomechanical perspective. Two strains were studied: the self-compatible Amut Bmut homokaryon that produces normal fruit bodies with relatively short stipes, and mutant B1918 that produces abnormally elongated stipes. Measurements of the pressure exerted by developing mushrooms were made using strain gauges, and these data were compared with measurements of the pressures exerted by vegetative hyphae of the same strains. The experiments demonstrate that AmutBmut hyphae elongating within stipe tissue push with the same pressure (approx. 0.5 atmosphere) as vegetative hyphae growing through their food sources. In purely biomechanical terms, the fruit body may therefore be viewed as a relatively uncomplicated sum of its parts. Analysis of the mutant strain B1918 demonstrated that hyperelongation of the stipe is not associated with any difference in the pressure exerted by the fruit body. The fault in the mechanism of stipe extension in B1918 may be reflected in the increased fluidity of the cell wall of vegetative hyphae of this strain, but further work is necessary to resolve this.

Basidiomycota↗
PubMed2001

Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases.

Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases. However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp. To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site. The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared. The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively). On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)). In contrast, both Fet3p and Cp are rapidly reduced by Fe(II) (k(obs) > 23 s(-)(1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively). Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.

Ceruloplasmin↗
PubMed2001

Isolation of unguilin, a cyclophilin-like protein with anti-mitogenic, antiviral, and antifungal activities, from black-eyed pea.

A protein designated unguilin was isolated from seeds of the black-eyed pea (Vigna unguiculata). It possesses a molecular weight of 18 kDa and an N-terminal sequence resembling that of cyclophilins and the cyclophilin-like antifungal protein from mung beans, and was adsorbed on Affi-gel blue gel and CM-Sepharose. Unguilin exerted an antifungal effect toward fungi including Coprinus comatus, Mycosphaerella arachidicola, and Botrytis cinerea. In addition, unguilin was capable of inhibiting human immunodeficiency virus-1 reverse transcriptase and the glycohydrolases a- and beta-glucosidases which are involved in HIV infection. Unguilin was devoid of lectin and ribonuclease activities. It inhibited methyl-3H-thymidine uptake by mouse splenocytes and it weakly inhibited translation in a rabbit reticulocyte lysate system. Unguilin resembles mungin in some aspects, but differs from it in others.

Amino Acid Sequence↗
PubMed2003

The combined use of electrokinetic remediation and phytoremediation to decontaminate metal-polluted soils: a laboratory-scale feasibility study.

The use of a combination of electrokinetic remediation and phytoremediation to decontaminate two metal-polluted soils has been demonstrated in laboratory-scale reactors. One soil was heavily contaminated with copper, the other with cadmium and arsenic (2500 microg g(-1) Cu; 300-400 microg g(-1) Cd and 230 microg g(-1) As, respectively). Test reactors with two separated chambers, each with a capacity of 5.25 kg soil, were constructed, then the respective chambers were filled with either a mixture of the polluted soil and a control topsoil (75:25) or topsoil alone. Reactors were sown with perennial ryegrass (Lolium perenne cv Elka) and a constant voltage of 30 V was applied continually across the soils in each reactor. Soil sampling took place at the start and the end of the test run, whilst plant foliage was sampled after approximately 3 weeks (both reactors) 6 weeks (Cd soil reactor only) and at the conclusion of each test run (98 days Cu soil, 80 days Cd soil). Soil and plant metal concentrations were measured, together with soil pH. Results showed that in both soils there was a significant re-distribution of metals from anode to cathode in the test reactors, coupled with an enhancement of plant Cu uptake in the cathode region for the Cu soil. Patterns of plant Cd uptake were less clear cut and were not as clearly related to the redistribution of Cd measured in the soil. There was significant acidification of soil at the anode in each test reactor, but soil pH in other parts of the reactor changed little during the course of the experiment. Plant growth was affected at the anode, but was not affected in other parts of the reactor. There was no visual evidence of metal toxicity in the ryegrass in either polluted soil. Some effects on soil fungi were apparent, with a stimulation of Fusarium infection of ryegrass in the cathode region of all reactors and the appearance of sporophores of Coprinus in the same location. It is concluded that the combination of the two techniques represents a very promising approach to the decontamination of metal polluted soils that now requires validation in field conditions.

Arsenic↗
PubMed2005

[Poisoning and drug emergency in practice].

Intoxicated patients make up 5-10% of all patients seen at emergency departments. The management of these patients is not always simple. Many of them are seen after ingestions of relatively non-toxic substances that require minimal medical care, intentional poisoning however often requires the highest standards of medical and nursing care and therefore the admission to an emergency department is mandatory. At admission, the involved substances are often not known since some of the patients are comatose. In such cases, the information from relatives and friends can be very crucial but to get hold of these sometimes essential "hints" is not always easy. Knowledge of the specific toxic agent allows the physician to plan a rational approach to the definitive management of the intoxicated patient after the vital functions have been stabilised. In some cases, very rare intoxications but with typical clinical signs do occur (e.g scromboid fish poisoning, coprinus-syndrome), which needs special diagnostic and therapeutic steps and a great deal of clinical experience. In most cases it is preferable to contact the Poison Control Center for additional advice.

Acute Disease↗
PubMed2002

Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences.

Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe(41) has been substituted by Trp, Val or Ala and the properties of the mutant proteins have been compared with that of the wild-type. The ferric and ferrous states have been studied by resonance Raman, electronic absorption and Fourier-transform infrared spectroscopies, together with their respective fluoride and CO complexes as probes for the integrity of the distal haem-pocket hydrogen-bonding network. The catalytic properties of the mutants, most notably the HRPC-mutant Phe(41)-->Trp (F41W) variant, were also affected. Structural modelling suggests that the bulky indole group of the F41W mutant blocks the distal cavity, inhibiting the binding of fluoride and CO to the haem iron, severely impairing the reaction of the enzyme with H(2)O(2) to form Compound I. Substitution with the smaller side-chain residues Val or Ala resulted in a 2-fold increase in the affinity of the mutants for the aromatic donor benzhydroxamic acid (BHA) compared with the wild-type, whereas the sterically hindered F41W mutant was not able to bind BHA at all. All the mutations studied increased the amount of a ferric six-coordinate aquo-high-spin species. On the other hand, the similarity in the Fe-Im stretching frequencies of the mutants and wild-type protein suggests that the distal haem-pocket mutations do not cause any substantive changes on the proximal side of the haem. Spectra of the HRPC mutant Phe(41)-->Ala-CO and the HRPC mutant Phe(41)-->Val-CO complexes strongly suggested a weakening of the interaction between CO and Arg(38) due to a secondary rearrangement of the haem relative to helix B. The effects observed for these HRP mutants were somewhat different from those noted recently for the analogous Coprinus cinereus peroxidase (CIP) mutants, particularly the Trp mutant. These differences can be reconciled in part as being due to the smaller size of the distal cavity of HRP compared with that of CIP.

Amino Acid Substitution↗
PubMed1972

The microbial biosynthesis of methionine.

1. The enzymes leading to the methylation of homocysteine have been examined in three micro-organisms: a cobalamin-producing bacterium, Bacillus megaterium; a yeast, Candida utilis; and a basidiomycete fungus, Coprinus lagopus. The yeast and the fungus contain negligible endogenous cobalamin. 2. Extracts of each organism catalyse C(1)-transfer from serine to homocysteine with a polyglutamate folate coenzyme. 3. The enzymes generating the methyl group of methionine from C-3 of serine have similar properties in each case, but different mechanisms of homocysteine transmethylation from 5-methyltetrahydrofolates were found. 4. B. megaterium contains an enzyme with properties suggestive of a vitamin B(12)-dependent homocysteine transmethylase, whereas Cand. utilis and Cop. lagopus transfer the methyl group by a reaction characteristic of the cobalamin-independent mechanism established for Escherichia coli. 5. The specificity of each transmethylase for a 5-methyltetrahydropteroylpolyglutamate is consistent with the results of analyses of endogenous folates in these organisms, which showed only conjugated forms. 6. None of the extracts catalysed methionine production from S-adenosylmethionine and homocysteine. 7. These results are compared with results now available for methionine synthesis in other organisms, which show a considerable diversity of mechanisms.

Bacillus megaterium↗
PubMed2001

Hypogin, a novel antifungal peptide from peanuts with sequence similarity to peanut allergen.

A protein designated hypogin, with a prominent suppressive action on the growth of the fungi Mycosphaerella arachidicola, Fusarium oxysporum and Coprinus comatus, was isolated from seeds of the peanut Arachis hypogaea. The protein inhibited human immunodeficiency virus (HIV) reverse transcriptase and enzymes associated with HIV infection including alpha-glucosidase and beta-glucosidase. The proliferative response of mouse splenocytes was attenuated in the presence of the protein. The protein exhibited a molecular mass of 7.2 kDa in tricine gel electrophoresis and gel filtration on Superdex 75 and an N-terminal sequence resembling peanut allergen Ara H1. The isolation procedure involved affinity chromatography on Affi-gel blue gel and ion-exchange chromatography on CM-Sepharose. The protein was adsorbed in both chromatographic media.

Allergens↗
PubMed1995

Seven newly discovered intron positions in the triose-phosphate isomerase gene: evidence for the introns-late theory.

The gene encoding the glycolytic enzyme triose-phosphate isomerase (TPI; EC 5.3.1.1) has been central to the long-standing controversy on the origin and evolutionary significance of spliceosomal introns by virtue of its pivotal support for the introns-early view, or exon theory of genes. Putative correlations between intron positions and TPI protein structure have led to the conjecture that the gene was assembled by exon shuffling, and five TPI intron positions are old by the criterion of being conserved between animals and plants. We have sequenced TPI genes from three diverse eukaryotes--the basidiomycete Coprinus cinereus, the nematode Caenorhabditis elegans, and the insect Heliothis virescens--and have found introns at seven novel positions that disrupt previously recognized gene/protein structure correlations. The set of 21 TPI introns now known is consistent with a random model of intron insertion. Twelve of the 21 TPI introns appear to be of recent origin since each is present in but a single examined species. These results, together with their implication that as more TPI genes are sequenced more intron positions will be found, render TPI untenable as a paradigm for the introns-early theory and, instead, support the introns-late view that spliceosomal introns have been inserted into preexisting genes during eukaryotic evolution.

Amino Acid Sequence↗
PubMed1998

A role for heterodimerization in nuclear localization of a homeodomain protein.

The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (beta-glucuronidase) gene and showing that beta-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.

Journal Article↗
PubMed1983

5S rRNA sequences from eight basidiomycetes and fungi imperfecti.

The 5S rRNA sequences from the basidiomycetes or fungi imperfecti Rhizoctonia crocorum, Rhizoctonia hiemalis, Exobasidium vaccinii, Trichosporon oryzae, Tilletia controversa, Tilletiaria anomala, Dacrymyces deliquescens and Coprinus radiatus were determined. With the exception of Exobasidium, these sequences conform to the association previously found between septal pore type and sequence. The sequence from the supposed ascomycete anamorph Rhizotonia hiemalis clearly is allied with basidiomycete sequences.

Base Sequence↗
PubMed2000

Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences.

Phylogenetic relationships of mushrooms and their relatives within the order Agaricales were addressed by using nuclear large subunit ribosomal DNA sequences. Approximately 900 bases of the 5' end of the nucleus-encoded large subunit RNA gene were sequenced for 154 selected taxa representing most families within the Agaricales. Several phylogenetic methods were used, including weighted and equally weighted parsimony (MP), maximum likelihood (ML), and distance methods (NJ). The starting tree for branch swapping in the ML analyses was the tree with the highest ML score among previously produced MP and NJ trees. A high degree of consensus was observed between phylogenetic estimates obtained through MP and ML. NJ trees differed according to the distance model that was used; however, all NJ trees still supported most of the same terminal groupings as the MP and ML trees did. NJ trees were always significantly suboptimal when evaluated against the best MP and ML trees, by both parsimony and likelihood tests. Our analyses suggest that weighted MP and ML provide the best estimates of Agaricales phylogeny. Similar support was observed between bootstrapping and jackknifing methods for evaluation of tree robustness. Phylogenetic analyses revealed many groups of agaricoid fungi that are supported by moderate to high bootstrap or jackknife values or are consistent with morphology-based classification schemes. Analyses also support separate placement of the boletes and russules, which are basal to the main core group of gilled mushrooms (the Agaricineae of Singer). Examples of monophyletic groups include the families Amanitaceae, Coprinaceae (excluding Coprinus comatus and subfamily Panaeolideae), Agaricaceae (excluding the Cystodermateae), and Strophariaceae pro parte (Stropharia, Pholiota, and Hypholoma); the mycorrhizal species of Tricholoma (including Leucopaxillus, also mycorrhizal); Mycena and Resinomycena; Termitomyces, Podabrella, and Lyophyllum; and Pleurotus with Hohenbuehelia. Several groups revealed by these data to be nonmonophyletic include the families Tricholomataceae, Cortinariaceae, and Hygrophoraceae and the genera Clitocybe, Omphalina, and Marasmius. This study provides a framework for future systematics studies in the Agaricales and suggestions for analyzing large molecular data sets.

Agaricales↗
PubMed1977

The time and duration of meiosis.

Ever since meiosis was recognized as a process there has been a continuing interest in its temporal aspects. Two main types of meiotic timing experiments have been conducted: first, experiments to estimate the duration of meiosis (and sometimes its stages); second, experiments to locate the sensitive stage(s) when exposure of meiocytes to various treatments can affect meiotic chromosome behaviour (e.g. pairing or recombination). Such experiments have played an important role in increasing our understanding of the meiotic process. The duration of meiosis has been estimated in about 70 organisms, including two prokaryotes (yeast and Chlamydomonas) and the following eukaryotes: 1 Basidiomycete (Coprinus lagopus), 2 Gymnosperms (Larix decidua and Thuja plicata gracilis). at least 39 angiosperms, and at least 26 animal species. The duration of female meiosis has been estimated in far fewer species than male meiosis. However, estimates of the duration of female meiosis are available for 6 angiosperms. Drosophila melanogaster, Xenopus laevis, and several mammals. Comparison of these data shows that the duration of meiosis is one of the most variable aspects of the meiotic process, ranging from less than 6 h in yeast to more than 40 years in the human female. Developmental holds at different stages of meiosis are common in plants and animals, and inevitably prolong the meiotic division. However, even among species without developmental holds, the duration of meiosis is very variable. For instance, in animals it ranges from about 1-2 days in male Drosophila melanogaster to more than 24 days in male Homo sapiens and several Orthopterans. Despite the large variation in the duration of meiosis three generalizations can be made: (i) first prophase is always very long compared with the remaining meiotic stages, (ii) the rate of meiotic development is very slow compared with the rate of development in dividing somatic meristem cells of the same organisms under the same conditions, (iii) the duration of meiosis is characteristic of the genotype and species. Four main factors have been recognized which effect or determine the duration of meiosis, namely (1) environmental factors (e.g. temperature); (2) nuclear DNA content; (3) ploidy level of the organism; and, (4) the genotype. Because nuclear DNA content plays a major role in determining the duration of meiosis, it has been suggested that DNA influences the rate of meiotic development in two ways: first through its informational content (the genotype), and second indirectly by the physical and mechanical effects of its mass independently of its informational content (i.e. the nucleotype). Thus, the observed duration of meiosis is the result of a complex genotype-nucleotype-environment interaction. With the obvious exception of variation caused by developmental holds, changes in the duration of meiosis usually involve proportional changes in the durations of all its stages...

Animals↗
PubMed1994

Gene arrangement and sequence of the 5S rRNA in Filobasidiella neoformans (Cryptococcus neoformans) as a phylogenetic indicator.

We cloned the 5S rRNA gene and determined its organization in the four genes encoding rRNAs in a ribosomal DNA repeat unit of Filobasidiella neoformans, the teleomorph of Cryptococcus neoformans. The 5S rRNA gene contained 118 nucleotides and was located 1 kb upstream from the 18S rRNA gene within the 8.6-kb fragment of the ribosomal DNA repeat unit. The sequence of the 5S rRNA gene from F. neoformans was more similar to the sequence of the 5S rRNA gene from Tremella mesenterica than to the sequences of the 5S rRNA genes from Filobasidium species. The arrangement of the rRNA genes in F. neoformans closely resembles the arrangement of the rRNA genes in mushrooms such as Schizophyllum commune, Agaricus bisporus, and Coprinus cinereus in that the 5S rRNA-coding region not only is located within the repeat unit that encodes the other rRNAs but also is transcribed in the same direction as the other rRNA genes. This is the first description of the arrangement of rRNA genes in a species belonging to the Heterobasidiomycetes.

Base Sequence↗
PubMed1981

Solubility of (1 leads to 3)-beta-D/(1 leads to 6)-beta-D-glucan in fungal walls: importance of presumed linkage between glucan and chitin.

In Saccharomyces cerevisiae, Neurospora crassa, Aspergillus nidulans and Coprinus cinereus most of the alkali-insoluble (1 leads to 3)-beta-D/(1 leads to 6)-beta-D-glucan of the wall can be extracted with dimethyl sulphoxide. The same fraction, and in Saccharomyces cerevisiae a small additional fraction, can be extracted by a destructive procedure involving 40% NaOH at 100 degrees C. The small fraction of the glucan which resists this treatment becomes soluble after a subsequent treatment with HNO2 indicating that it is covalently linked to chitin in the wall. In contrast, in Schizophyllum commune and Agaricus bisporus, nearly all the (1 leads to 3)-beta-D/(1 leads to 6)-beta-D-glucan appears to be held insoluble by linkage to chitin.

Agaricales↗