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A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea.

A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60 degrees C and the maximum fibrinolytic activity was at 55 degrees C.

Agaricales↗

Rufuslactone, a new antifungal sesquiterpene from the fruiting bodies of the basidiomycete Lactarius rufus.

A new lactarane sesquiterpene, rufuslactone (1), was isolated from the fruiting bodies of the basidiomycete Lactarius rufus. Rufuslactone (1) is an isomer of a previously described lactarane 3,8-oxa-13-hydroxylactar-6-en-5-oic acid gamma-lactone (2) from Lactarius necatar. Its structure,was elucidated on the basis of spectroscopic data. Rufuslactone (1) showed antifungal properties against plant pathogenic fungi.

Antifungal Agents↗

Kinetic characterization of lysine-specific metalloendopeptidases from Grifola frondosa and Pleurotus ostreatus fruiting bodies.

Two zinc-metalloendopeptidases, GFMEP (accession number P81054) and POMEP (accession number P81055), from the fruiting bodies of two edible mushrooms, Grifola frondosa and Pleurotus ostreatus, respectively, specifically hydrolyze peptidyl-lysine bonds (-X-Lys-) in polypeptides. To understand detailed substrate specificities and kinetic characters of these enzymes, we have synthesized various intramolecularly quenched fluorescent peptide substrates and determined their kinetic constants with these substrates. Each synthesized fluorogenic peptide has a fluorescent residue, tryptophan, at its carboxyl terminus and a quenching group, dinitrophenyl (Dnp), at its amino terminus. Quenching of the Trp fluorescence in an intact substrate is relieved on hydrolysis of the -X-Lys- bond, giving rise to a continuous increase in fluorescence. The octapeptide substrate, Dnp-Ser-Thr-Ala-Thr-Lys-Leu-Ser-Trp, was an efficient substrate for both enzymes, the kcat/Km values being 9.8 x 10(6) and 7.0 x 10(5) M-1.s-1 for GF- and POMEP, respectively. Peptides with aspartic acid adjacent to the Lys residue were found to be poor substrates for both enzymes. Neither the shortest peptide, Dnp-Thr-Lys-Trp, nor peptides with substitution of L-Arg, L-ornithine, or D-Lys for Lys were hydrolyzed by either enzyme. These results confirmed the strict specificities of GF- and POMEP toward the peptide bond, -X-Lys-. Substitution of Co2+ for Zn2+ enhanced the activity, while the Km values were comparable. All peptides not hydrolyzed by either enzyme had inhibitory effects on GFMEP activity. The active site structure is discussed in relation to these observations.

2,4-Dinitrophenol↗

Purification of a laccase from fruiting bodies of the mushroom Pleurotus eryngii.

A purification scheme involving ion exchange chromatography on diethylaminoethyl (DEAE) cellulose, carboxymethyl (CM) cellulose, and Q-Sepharose, followed by fast protein liquid chromatography-gel filtration on Superdex 75, was utilized to isolate a laccase from the fruiting bodies of the mushroom Pleurotus eryngii. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose and unadsorbed on CM-cellulose. The molecular mass of the laccase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 34 kDa. The laccase activity was maximal at 70 degrees C and remained high at 80 degrees C. High activity of the laccase was maintained at ph 3 to 5. The activity underwent an abrupt decline at pH 6 and 7 and was indiscernible at ph 8 and 9. The laccase exhibited an inhibitory activity toward HIV-1 reverse transcriptase with an IC50 of 2.2 microM. Its N-terminal sequence was dissimilar to those of laccase isoenzymes previously isolated from cultured mycelia of the same species.

Amino Acid Sequence↗

Effect of a polysaccharide (TAP) from the fruiting bodies of Tremella aurantia on glucose metabolism in mouse liver.

An acidic polysaccharide (TAP) obtained from the fruiting bodies of Tremella aurantia significantly increased the activities of glucokinase, hexokinase, and glucose-6-phosphate dehydrogenase, and decreased the activity of glucose-6-phosphatase in normal and diabetic mouse liver after intraperitoneal administration, while the glycogen content in the liver was reduced. Furthermore, TAP lowered the plasma cholesterol level in normal and diabetic mice.

Animals↗

Immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi).

The immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi, RR), which has been used as a traditional supplement for human health, were investigated in mice. BALB/c mice were administered orally with RR for 3 days at a dose of 50 mg/kg or 500 mg/kg, and interferon-gamma (IFN-gamma) production by splenocytes in response to lipopolysaccharide (LPS) was examined on day 4. The oral administration of 500 mg/kg of RR resulted in a significant increase (p<0.05) in IFN-gamma production. Stimulation of splenic adherent cells from these mice with LPS also resulted in a significant increase (p<0.05) in interleukin-12 (IL-12) production compared with that from the control mice, suggesting that splenic macrophages were activated by RR administration. Furthermore, 500 mg/kg of RR administered for 14 days resulted in a significant increase (p<0.05) in IFN-gamma production by splenocytes in response to both LPS and concanavalin A (Con A). These results suggest that not only splenic macrophages but also T cells were activated by the long-term treatment with RR in vivo. On the other hand, the production of interleukin-4 (IL-4), which is known as an allergic disease-related cytokine, was not affected by the long-term treatment with RR. Our results suggest that the oral administration of RR resulted in Th1-associated immuno-potentiating activities in vivo.

Administration, Oral↗

Effects of ingested fruiting bodies, submerged culture biomass, and acidic polysaccharide glucuronoxylomannan of Tremella mesenterica Retz.:Fr. on glycemic responses in normal and diabetic rats.

Mushroom polysaccharides have been shown to regulate glucose metabolism. Using male Wistar rats injected with saline (normal rats), streptozotocin (STZ-NT rats), or streptozotocin plus nicotinamide (STZ+NT rats), we investigated the hypoglycemic activity of orally ingested fruiting bodies (FB), submerged culture biomass (CM), or the acidic polysaccharide glucuronoxylomannan (GXM) of Tremella mesenterica, an edible jelly mushroom. Our results demonstrated that FB ingestion significantly attenuated the elevated blood glucose levels in an oral glucose tolerance test (OGTT) in STZ-NT rats. However, in STZ+NT rats, FB, CM, and GXM ingestion significantly attenuated the increases in food and water intake, 2-h postprandial blood glucose concentrations, and blood glucose levels in OGTT. Moreover, FB and GXM ingestion significantly decreased serum concentration of fructosamine in STZ+NT rats. Our results indicated that T. mesenterica might be developed as a potential oral hypoglycemic agent or functional food for diabetic patients and for persons with high risk for diabetes mellitus.

Agaricales↗

A novel lectin with potent immunomodulatory activity isolated from both fruiting bodies and cultured mycelia of the edible mushroom Volvariella volvacea.

A novel lectin has been purified from the fruiting bodies as well as cultured mycelia of the edible mushroom Volvariella volvacea. The lectin, designated as VVL, was a homodimeric protein with a molecular weight of 32 kDa as demonstrated by gel filtration and SDS-PAGE. VVL had no carbohydrate moiety, and its hemagglutinating activity was inhibited by thyroglobulin but not by simple carbohydrates such as monomeric or dimeric sugars. The immunomodulatory activity of VVL was demonstrated by its potent stimulatory activity toward murine splenic lymphocytes. VVL was also found to markedly enhance the transcriptional expression of interleukin-2 and interferon-gamma by reverse transcriptase-polymerase chain reaction. As revealed by its N-terminal amino acid sequence, VVL possessed a molecular structure distinct from other immunomodulatory proteins previously reported in the same fungus.

Adjuvants, Immunologic↗

[Distribution of mercury in the fruit-bodies of Boletus edulis and Agaricus species (authors transl)].

The mercury content of various parts of single fruit-bodies of the Yellow Bolete Boletus edulis (n = 26), the Field-Mushroom Agaricus campester (n = 23) and of Agaricus silvicola (n = 17) was determined by flameless atomic absorption spectroscopy. In each species the lowest mercury content was found in the stem (x = 1,96--3,21 mg/kg dry wt.), whereas the caps contained significantly more mercury (x = 4,19--6,97 mg/kg dry wt.). The highest mercury content was found in the gills and tubes (x = 4,85--8,77 mg/kg dry wt.), which contained significantly more mercury then the flesh of the caps (x = 3,11--5,60 mg/kg dry wt.). Young mushrooms seemed to contain more mercury then older ones.

Agaricales↗

On the water-soluble heterogalactan from the fruit bodies of Lentinus edodes.

The chemical structure of a heterogalactan isolated from the trichloracetic acid extract of the fruit bodies of Lentinus edodes is reported. It consists of a main chain of (1 yields 6)-linked alpha-D-galactopyranose residues, part of which are substituted in the 2-position either with single L-fucopyranose or D-mannopyranose residues. However, there is a possible alternative structure of a branched D-galactan in which most of the side-chains are terminated with L-fucose or D-mannose residues.

Basidiomycota↗

Russujaponols A-F, illudoid sesquiterpenes from the fruiting body of Russula japonica.

Six new illudoid sesquiterpenes, russujaponols A-F (1-6), were isolated from the fruiting bodies of Russula japonica Hongo. Their structures were established primarily by 2D NMR experiments, and the structure of the main compound, russujaponol A (1), was confirmed by X-ray crystallographic analysis of its benzoate (1a). Russujaponol A (1) suppressed invasion of human fibrosarcoma (HT1080) cells into Matrigel in a concentration-dependent manner and caused 63% inhibition at 3.73 microM.

Antineoplastic Agents↗

The inhibitory properties and primary structure of a novel serine proteinase inhibitor from the fruiting body of the basidiomycete, Lentinus edodes.

A novel proteinase inhibitor, Lentinus proteinase inhibitor, has been purified from the fruiting bodies of the edible mushroom, Lentinus edodes, by buffer extraction and affinity chromatography on immobilized anhydrotrypsin. The protein simultaneously inhibits bovine beta-trypsin and alpha-chymotrypsin at independent sites, with apparent dissociation constants of 3.5 x 10(-10) M and 4 x 10(-8) M, respectively. The purified protein is eluted as two well-separated peaks on reversed-phase HPLC, one of which is inhibitory-active and the other inactive, and they are interconvertible under folding/unfolding conditions. Among the mammalian and microbial serine proteinases examined, including human enzymes of blood coagulation and fibrinolysis, activated factor XI was inhibited by the Lentinus proteinase inhibitor. Chemical modification studies suggest involvement of one or more arginine residues in the inhibition of trypsin. The complete primary structure composed of 142 amino acids with an acetylated N-terminus was determined by protein analysis. The theoretical molecular mass (15999.2) from the sequence is close to the experimental value of 15999.61 +/- 0.61 determined by mass spectrometry. Although there are no apparently homologous proteinase inhibitors in the protein database, there is a rather striking similarity to the propeptide segment of a microbial serine proteinase, as well as to the N-terminal region of the mature enzyme.

Amino Acid Sequence↗

Polysaccharides in fungi. XXXV. Anti diabetic activity of an acidic polysaccharide from the fruiting bodies of Tremella aurantia.

An acidic polysaccharide (TAP) was isolated from a hot-water extract of the fruiting bodies of Tremella aurantia. It showed remarkable hypoglycemic activity in normal mice and two diabetic mouse models, streptozotocin-induced diabetes and genetic diabetes, following intraperitoneal administration. Continuous oral administration of TAP solution (0.5g/l) for a long period was found to be also effective in hyperglycemia in glucose-loaded mice and no harmful physical effects were found. TAP had an [alpha]D -7 degrees in water, and its molecular weight was estimated to be about 1500000. TAP is composed of mannose, xylose, glucuronic acid and glucose (molar ratio, 4:2:1:0.3), and it contains 2.2% of O-acetyl groups.

Animals↗

[Studies on the ganoderic acid, a new constituents from the fruiting body of Ganoderma lucidum (Fr.) Karst].

Three compounds have been isolated from the dichloromethane soluble fraction of the fruiting body of Ganoderma lucidum (Fr.) Karst. On basis of spectral analyses (UV, IR, MS, 1HNMR, 13CNMR and 2D-NMR), they were identified as 3, 7-dioxo-lanosta-8, 24(E)-dien-26-oic acid (I), 7 beta-15 alpha-dihydroxy-3, 11, 23-trioxo-5 alpha-lanost-8-en-26-oic acid (II) and 3 beta, 7 beta, 15 alpha-trihydroxy-11, 23-dioxo-5 alpha-lanosta-8-en-26-oic acid (III). Compound I is a new compound named ganoderic acid DM.

Drugs, Chinese Herbal↗

A new lanostane-type triterpene from the fruiting bodies of Ganoderma lucidum.

A new lanostane-type triterpene, named ganoderic acid LM2 (5), was isolated from the fruiting bodies of Ganoderma lucidum. Its structure was characterized as (23S) 7beta, -dihydroxy-3, 11, 15-trioxo-5alpha-lanosta-8, 24-dien-26-oic acid by 1D- and 2D-NMR spectra. In addition, a known triterpene, ganoderic acid epsilon (4), was obtained. Both of them exhibited potent enhancement of ConA-induced mice splenocytes proliferation in vitro.

Animals↗

Neuroprotective diterpenes from the fruiting body of Antrodia camphorata.

Three new compounds, 19-hydroxylabda-8(17)-en-16,15-olide (1), 3beta,19-dihydroxylabda-8(17),11E-dien-16,15-olide (2), and 13-epi-3beta,19-dihydroxylabda-8(17),11E-dien-16,15-olide (3), together with four known compounds, 19-hydroxylabda-8(17),13-dien-16,15-olide (4), 14-deoxy-11,12-didehydroandrographolide (5), 14-deoxyandrographolide, and pinusolidic acid, were isolated from the fruiting bodies of Antrodia camphorata. The structures of compounds 1-3 were elucidated by the analysis of their spectroscopic data. The in vitro neuroprotective activity of all compounds was evaluated, and compounds 1-5 protected neurons from Abeta damage by 39.2, 35.0, 36.7, 30.6, and 27.0%, respectively, at concentrations between 5 and 20 microM.

Animals↗