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PubMed1995

Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies.

A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL. ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn(2+)-depleted ProB can regain the activity with Zn2+, Co2+, or Mn2+. Specific cleavage of Pro29-Lys30 in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the alpha-amino group of lysyl residues. ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn2+ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.

Amino Acid Sequence↗
PubMed2002

Alpha-glucosidase inhibitor from the seeds of balsam pear (Momordica charantia) and the fruit bodies of Grifola frondosa.

Alpha-glucosidase inhibitory activities were found in aqueous methanol extracts of the seeds of Momordica charantia and the fruit bodies of Grifola frondosa. An active principle against the enzyme prepared from rat small intestine acetone powders was isolated and characterized. The structure of the isolated compound was identified as D-(+)-trehalose by FDMS, 1H-, 13C-NMR, and [alpha]D measurements. The inhibitory activity of trehalose was compared with 1-deoxynojirimycin. Trehalose showed 45% inhibitory activity at the concentration of 2 x 10(-3) m, but 1-deoxynojirimycin had 52% inhibitory activity at 1 x 10(-7) M.

1-Deoxynojirimycin↗
PubMed2004

A distinctive ribonuclease from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum.

A ribonuclease with an N-terminal sequence distinct from other mushroom ribonucleases was isolated from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. The ribonuclease was adsorbed on DEAE-cellulose and Q-Sepharose, and unadsorbed on CM-Sepharose. It possessed a molecular mass of 42 kDa as judged by gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular mass was similar to that of straw mushroom ribonuclease but much higher compared with those of other mushroom ribonucleases. The ribonuclease was unique among mushroom ribonucleases in that it exhibited the highest potency toward poly(U), followed by poly(A). Its activity toward poly(G) and poly(C) was about one-half of that toward poly(A) and one-quarter of that toward poly(U). A pH of 4.0 and a temperature of 60 degrees C were required for optimal activity of the enzyme. The optimum pH was low compared with those reported for other mushroom ribonucleases.

Agaricales↗
PubMed2002

(3R,6R)-4-methyl-6-(1-methylethyl)-3-phenylmethyl-perhydro-1,4-oxazine-2,5-dione: an apoptosis-inducer from the fruiting bodies of Isaria japonica.

(3R,6R)-4-methyl-6-(1-methylethyl)-3-phenylmethylperhydro-1,4-oxazine-2,5-dione (1) was isolated from the fruiting bodies of Isaria japonica as an apoptosis-inducing agent. The complete structural assignment of the compound was accomplished on the basis of spectroscopic methods and chemical transformations. Compound 1 induced apoptotic cell death of the human leukemia cells (HL-60) in a dose-dependent manner, ranging from 5.0 microg/ml to 100.0 microg/ml.

Apoptosis↗
PubMed1981

Investigation of cell patterning in the asexual fruiting body of Dictyostelium discoideum using haploid and isogenic diploid strains.

The cell patterning (proportion of spores, stalk cells, and basal disk cells) of individual asexual fruiting bodies of haploid and isogenic diploid strains of D. discoideum was examined to test the hypothesis that the patterning mechanism is based on the 'sensing' of only a single parameter, e.g., cell volume, in dividing the aggregate into the three cell types. If cell patterning is based on sensing only a single parameter, there is no reason to predict a change in cell patterning with ploidy change, and thus haploid and isogenic diploid strains should not differ in their cell patterning. The cell patterning of each of the three pairs of haploid and isogenic diploid strains examined was different. Therefore we conclude that the cell patterning mechanism must involve at least two components not changing in the same way with change in ploidy. The cell patterning of both the haploid and the diploid strains was qualitatively similar, i.e., relationships between the three cell types were described by equations of the same form in the haploid and diploid strains. However a quantitative change in cell patterning led to an increased percentage of stalk and basal disk cells in each diploid compared to its parent haploid. The ratio of basal disk to stalk cell was also greater in the diploids than in their parent haploids. We conclude that these are general ploidy-related changes because the cell patterning of each of the three parent haploid strains was different; the average percentage of stalk cells was 11.6% for X22 (12.4% for its diploid DU162), 20.5% for NP73 (27.2% for its diploid DP62), and 24.5% for HU127 (29.1% for its diploid DU310). One possible patterning mechanism could involve a diffusible signal (s), which shows gene dosage, interacting with cell-surface molecules which we predict occupy a limited number of sites per unit area of the cell membrane. The observed change in cell patterning leading to an increased percentage of stalk cells in diploid strains is predicted from such a model involving diffusible signal interacting with cell-surface molecules.

Cell Aggregation↗
PubMed1996

New contributions to the wall polysaccharide structure of vegetative mycelium and fruit body cell walls of Agaricus bisporus.

Significant differences in chemical composition and structure of wall polysaccharides were found in Agaricus bisporus vegetative mycelium in comparison with the fruit body mycelium. Chemical fractionation of the walls demonstrated distinct percentages of the successively solubilized polysaccharides in both walls, and different proportions of their respective sugar monomers. Permethylation of isolated fractions also showed striking differences relative to the sugar linkages between the two types of walls studied. Furthermore, infrared spectrophotometry exhibited a distinct polysaccharidic configuration in several fractions. All the described differences were correlated with the transition of vegetative mycelial walls to the fruiting stage of differentiation.

Agaricus↗
PubMed2000

Antioxidant activity of the extracts from fruiting bodies of cultured Cordyceps sinensis.

Cordyceps sinensis is one of the most valued herbs in traditional Chinese medicine. We investigated the antioxidant activities of the cultured fruiting bodies of Cordyceps sinesis. The water and ethanol extracts of Cordyceps sinensis were found to possess a potent antioxidant activity. The scavenging effects of the extracts on superoxide were very weak, but the extracts moderately inhibited malondialdehyde formation via hydroxyl radical induced by SIN-1, a peroxynitrite generator. Of the extracts examined, the hot water extract (70 degrees C for 5 min) showed the greatest oxygen free radical scavenging activity. Also, when low-density lipoprotein (LDL) was incubated with macrophages in the presence of CuCl2 (1 microM), the hot water extract showed a strong inhibitory effect against lipid peroxidation in the medium and consequent accumulation of cholesteryl ester in macrophages. Their activities were comparable to that of authentic Cu/Zn SOD. These results suggest that the extracts of cultured Cordyceps sinensis possess potent antioxidant and anti-lipid peroxidation activities and inhibit accumulation of cholesteryl ester in macrophages via suppression of LDL oxidation.

Animals↗
PubMed1997

Amino acid sequences of metalloendopeptidases specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies.

The complete amino acid sequences of two lysine-specific zinc metalloendopeptidases (EC 3.4.24), Grifola frondosa metalloendopeptidase (GFMEP) and Pleurotus ostreatus metalloendopeptidase (POMEP), from the fruiting bodies of these two edible mushrooms have been established based on the sequence information of the peptides generated from the reduced and alkylated GFMEP and POMEP by proteolytic digestions using GFMEP, trypsin, and other proteinases as well as by several chemical cleavages. From the sequences, it was found that GFMEP and POMEP were polypeptides composed of 167 and 168 amino acid residues, from which their molecular weights were calculated to be 18,040.5 and 17,921.3 in accord with the observed (M+H)+ values of 18,028 and 17,927, respectively, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two disulfide bonds in GFMEP were found to link Cys5 to Cys75 and Cys77 to Cys97. An unusual post-translational modification of GFMEP was corroborated to be a partial attachment of a single mannose to Thr42. Comparison of the sequences revealed that overall identity between the enzymes was 61.3%. Although a highly homologous sequence was not found in sequence data bases except for a consensus zinc-binding sequence, HEXXH, both metalloendopeptidases somewhat resembled a family of metalloproteinases categorized as deuterolysin. These proteases together with GFMEP and POMEP do not have conserved third and/or fourth liganding amino acid residues seen in metzincin or thermolysin superfamily proteins and belong to a novel zinc metalloendopeptidase superfamily.

Amino Acid Sequence↗
PubMed1993

devRS, an autoregulated and essential genetic locus for fruiting body development in Myxococcus xanthus.

Two Tn5 lac insertions into the Myxococcus genome at sites omega 4414 and omega 4473, which are separated by 550 nucleotides, inactivate fruiting body development. Sporulation is decreased 100- to 10,000-fold. At least two genes, devR and devS, are transcribed in this region, probably as an operon. Expression of devR begins by 6 h after starvation has initiated development. On the basis of their nucleotide sequences, devR and devS are expected to encode proteins of 302 and 214 amino acids, respectively. Dev+ function can be restored by a segment of 7.8 kb cloned from the devRS region of wild-type cells. Two experiments show that devR expression is under strong negative autoregulation. beta-Galactosidase is expressed at a higher level from a transcriptional devR::lacZ fusion when the fused operon is in a dev strain than when it is in the dev/dev+ genetic background of a partial diploid. There is more mRNA accumulation from the devRS region in the dev strain than in a rescued dev/dev+ tandem duplication strain. Sporulation rescue is correlated with some degree of negative autoregulation, even though sporulation is not inversely proportional to beta-galactosidase expression from omega 4414. A second level of regulation is suggested by complementation of dev by dev+ in duplication strains. The expression of devRS, measured by sporulation levels, differs 1,000-fold when devRS+ is moved from a distance of 20 kb to 3 Mb from the mutant devRS locus. Expression of devR is also dependent on the cell density at which development is initiated, a third level of regulation. Multiple levels of regulation suggest that devRS is a switch required to activate completion of aggregation and sporulation.

Amino Acid Sequence↗
PubMed2000

Inhibitory effects of water extracts from fruiting bodies of cultured Cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice.

We investigated the effects of the water extracts of the fruiting bodies of cultured Cordyceps sinensis (WECS) on lipid metabolism in mice fed an atherogenic diet. WECS was orally administered at doses of 50, 100 and 200 mg/kg/day for 12 weeks. WECS showed no toxic effects on the growth rate, liver or kidney weights of the mice. Mice fed the atherogenic diet showed marked increases in serum lipid and lipid peroxide levels and also aortic cholesterol levels, particularly cholesteryl ester level, a major lipid constituent in atherosclerotic lesions. WECS significantly suppressed the increased serum lipid peroxide level but not other lipid levels in a dose-dependent manner. WECS also suppressed the increased aortic cholesteryl ester level in a dose-dependent manner. These results suggest that WECS prevents cholesterol deposition in the aorta by inhibition of LDL oxidation mediated by free radicals rather than by reduction in serum lipid level. WECS may exert beneficial effects on the formation of the atherosclerotic lesion induced by oxidative stress with few side effects.

Animals↗
PubMed1998

Isolation and Characterization of a Type 1 Ribosome-Inactivating Protein from Fruiting Bodies of the Edible Mushroom (Volvariella volvacea).

A novel single-chained ribosome-inactivating protein (RIP) with a molecular weight of approximately 29 000 was purified from fruiting bodies of the edible mushroom Volvariellavolvacea with a procedure involving ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, and gel filtration on Superdex 75. The mushroom RIP, designated volvarin, exhibited a potent inhibitory action on protein synthesis in the rabbit reticulocyte lysate system with an IC(50) value of 0.5 nM. Like most plant RIPs, volvarin acted as an N-glycosidase that depurinated rRNA from rabbit reticulocyte lysate, releasing a characteristic RNA fragment after treatment with aniline. It also exerted a deoxyribonuclease activity on supercoiled SV-40 DNA and demonstrated a strong abortifacient effect in mice.

Journal Article↗
PubMed1991

Mitogenic and colony-stimulating factor-inducing activities of polysaccharide fractions from the fruit bodies of Dictyophora indusiata Fisch.

Biological effects (mitogenic and colony-stimulating factor (CSF)-inducing activities) of five homogeneous polysaccharides and a conjugated polysaccharide fraction isolated from the fruit bodies of Dictyophora indusiata Fisch. were investigated. Fucomammogalactan (T-3-Ad) and conjugated polysaccharide fraction (T-2-A) exhibited significant mitogenic and CSF-inducing activities. Among two beta-(1----6)-branched (1----3)-beta-D-glucans (T-4-N and T-5-N), only T-4-N showed both mitogenic and CSF-inducing effects. Partially O-acetylated (1----3)-alpha-D-mannans (T-2-HN and T-3-M') did not show these effects.

Animals↗
PubMed1994

Complete amino acid sequence of a lectin-related 16.5-kDa protein isolated from fruit bodies of a lectin-deficient strain of Pleurotus cornucopiae.

The complete amino acid sequence of a lectin-related 16.5-kDa protein (PCL-RP) isolated from fruit bodies of a lectin-deficient strain of P. cornucopiae is presented. The sequences of six out of the seven peptides generated by digestion with lysylendopeptidase and four of the five peptides generated by cyanogen bromide cleavage were completely analyzed. Overlapping peptides were obtained by arginylendopeptidase digestion. PCL-RP was a single-chain protein consisting of 144 amino acid residues and its N-terminal serine was blocked with acetate. A proline-rich sequence was found in the carboxyl terminal portion. The N-terminal sequence of PCL-RP showed some homology with those of two known Basidiomycete lectins.

Amino Acid Sequence↗
PubMed2002

Simple method for reversed-phase high-performance liquid chromatographic analysis of fungal pigments in fruit-bodies of Boletales (Fungi).

A reversed-phase HPLC method has been developed for the analysis of hydroxylated pulvinic acid derivatives which are responsible for the pigmentation of fruit-bodies belonging to the order Boletales (Fungi). Variegatic, xerocomic and atromentic acid as well as variegatorubin were detected and separated in methanolic extracts of Boletus permagnificus and Xerocomus parasiticus, and the pigment profile of these species was compared. The identity of the pigments was confirmed by means of LC-atmospheric pressure chemical ionization (APCI) mass spectrometry.

Basidiomycota↗
PubMed2003

Isolation of a ribonuclease from fruiting bodies of the wild mushroom Termitomyces globulus.

A ribonuclease, with a molecular mass of 13 kDa and a ubiquitin-like N-terminal sequence, has been isolated from fruiting bodies of the mushroom Termitomyces globulus. The ribonuclease demonstrated ribonucleolytic activity toward poly A, poly C, poly G and poly U, with the activity toward poly A and poly C being much higher than that toward poly G and poly U. The ribonuclease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-Sepharose. The enzyme required a temperature of 70 degrees C for expression of maximal activity. However, the enzyme expressed nearly the same optimal activity over a wide pH range of 5.0-8.0.

Agaricales↗
PubMed1994

An antitumor, branched (1-->3)-beta-D-glucan from a water extract of fruiting bodies of Cryptoporus volvatus.

A water-soluble, (1-->6)-branched (1-->3)-beta-D-glucan (H-3-B) was isolated from a hot-water extract of the fruiting bodies of the fungus, Cryptoporus volvatus (Basidiomycetes). Enzymatic analysis using exo-(1-->3)-beta-D-glucanase and methylation analysis indicated that this polysaccharide has a main chain composed of beta-(1-->3)-linked D-glucopyranosyl residues, and single, beta-(1-->6)-linked D-glucopyranosyl residues attached as side chains to, on average, every fourth sugar residue of the main chain. This structure was confirmed by 13C NMR spectra of the glucan in Me2SO-d6. The weight-average molecular weight (Mw) of H-3-B was determined to be 44.0 x 10(4) by gel permeation chromatography equipped with a low-angle laser-light-scattering photometer. The electron microscopic observations showed that H-3-B and its sonicated sample (S-H-3-B, Mw = 13.7 x 10(4)) can be described as linear worm-like chains. The mass per unit length for native and sonicated H-3-B was determined to be 1750 and 1780 g mol-1 nm-1, respectively, from the contour lengths obtained by electron microscopy and the molecular weights. These values are in good agreement with that expected for the triple stranded structure. A sample denatured in 0.1 M NaOH and subsequently renatured by neutralization showed a mixture of linear and cyclic structures, and larger aggregates with less well-defined morphology. The H-3-B and S-H-3-B had antitumor activity against the Sarcoma 180 tumor.

Animals↗
PubMed2004

Isolation of a new ribonuclease from fruiting bodies of the silver plate mushroom Clitocybe maxima.

A ribonuclease, with an N-terminal sequence exhibiting some homology to ribonuclease from Pleurotus ostreatus (Family Pleurotaceae), has been purified from fruiting bodies of the silver plate mushroom Clitocybe maxima (Family Tricholomataceae). However, there is little resemblance between the N-terminal sequences of ribonucleases from various Pleurotus species, and a lesser extent of resemblance between ribonucleases from C. maxima and Pleurotus tuber-regium. No structural relationship exists between ribonuclease from C. maxima, and those from Volvariella volvacea, Lentinus edodes and Irpex lacteus. The purification protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose, and fast protein liquid chromatography on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. It exhibited a molecular mass of 17.5 kDa in both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It manifested roughly the same ribonucleolytic potency toward poly A and poly G followed by poly U. Its activity toward poly C was, by comparison, meager. The temperature and pH required for its optimal activity were, respectively, 70 degrees C and 6.5-7.0.

Agaricales↗