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A lectin was isolated from the mushroom Hericium erinaceum. This lectin is composed of two different subunits of 15 and 16 kDa and the molecular mass of the intact lectin was estimated to be 54 kDa by gel filtration. It exhibits specificity towards sialic acids, especially N-glycolylneuraminic acid.
1. A lectin has been purified from the mushroom Boletus satanas Lenz. 2. The protein, called bolesatine, is mitogenic for human T lymphocytes in a dose- and time-dependent manner. 3. Optimal mitogenic doses induce the release of interleukin-1 alpha and interleukin-2 from mononuclear cell cultures.
The specificity of mushroom tyrosinase in displacing 3H from estradiol and catechol estrogens labeled at C-1, C-2, C-4 or C-6,7 was investigated under various conditions. [2-3H]E2 Yielded significant amounts of 3H2O, in the presence of NADH, and the rate of 3H loss from the steroid paralleled that of the radioactivity remaining in the aqueous fraction after extraction with organic solvents. NADH had little effect on the release of 3H from [1-3H]E2 or [4-3H]E2 but glutathione was highly active in this respect, with considerable differences being observed between lyophilizable 3H2O and yields of water-soluble products. It is proposed that 3H losses from C-2 of estradiol reflects oxidative displacement of this isotope by tyrosinase while the loss observed from C-1 and C-4 is the result of non-enzymatic conjugation with glutathione after the formation of the catechol estrogen. The difference between lyophilizable 3H2O and the yield of water-soluble products obtained with [1-3H]E2 and [4-3H]E2 provided a measure of the relative amount of conjugation occurring at C-1 and C-4. These findings were confirmed by double label experiments with 3H- and 14C-labeled estrogens and the isolation of the glutathionyl derivatives. The catechol estrogens did not serve as substrates for further hydroxylation by the enzyme even when C-2 was available for this reaction. These experiments give further information about the specificity of tyrosinase in its reaction with aromatic steroids and provide a simple and rapid method for confirming the distribution of 3H at C-2 or C-4 of estradiol.
The presence of catechol in a reaction mixture has been shown previously to promote oxidation of 2-hydroxyestradiol by mushroom tyrosinase. It was now found that the oxidized products of the catecholesterogen are incorporated into melanin under the influence of the enzyme. Whether the oxidation is restricted to tyrosinase or to enzymes with specific steroid oxidizing properties was examined by separating tyrosinase on agarose gel followed by hydroxylapatite. The effectiveness of separation was monitored electrophoretically. Two bands of enzyme activity of 127 kDa were found. One of these bands could be cleanly separated from the other. The fraction which contained the single band, as well as the one which contained both bands, had similar apparent Km values; i.e. 1.5 x 10(-4) and 2.1 x 10(-4) M. They both catalyzed oxidation of 2-hydroxyestradiol but only in the presence of catechol. All enzyme fractions showed the same pattern of activity towards the estrogen. HPLC analysis of reaction products of catechol indicated that not all of the substrate was consumed during the reaction. About 26% remained unreacted at an initial concentration of 100-400 microM of catechol. This remaining catechol, rather than its reaction products, appears to function as activator of the steroid reaction. The data are consistent with the presence on the enzyme of an allosteric activator site specific for catechol and an active site with a much lower structural specificity occupied by the catecholestrogen.
A polysaccharide-peptide complex (PSPC) with immunomodulatory and antitumor activities was obtained from a submerged mycelial culture of Tricholoma sp., a local edible mushroom. The polysaccharide-peptide complex exhibited a molecular weight of 17 K in gel filtration and a single band after SDS-polyacrylamide gel electrophoresis. It was characterized by non-adsorption on both DEAE-Sepharose CL-6B and CM-cellulose. It could activate the macrophages, stimulate the proliferation of T-cells, and inhibit the growth of sarcoma 180 in mice. It possessed more potent immunomodulatory and antitumor activities than Coriolus versicolor polysaccharopeptide (PSP) and deserves to be studied as a potential agent for immunomodulation and cancer therapy.
Two antitumor polysaccharide-protein complexes, PSPC and PSK from mushrooms, were compared for their modulating effect on cytokine and cytokine receptor gene expression. RNA samples were isolated from the splenocytes and peritoneal exudate cells of the untreated or treated mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the cytokine gene expression. Nine out of 17 cytokine mRNAs and 5 out of 6 cytokine receptor mRNAs were detected in the splenocytes and peritoneal exudate cells from both untreated and treated mice. However, IL-4 was only detected in the splenocytes while IL-7 and IL-1R(typeI) were only detected in the peritoneal exudate cells. Among the 9 cytokine genes, the expression level of M-CSF was up-regulated in splenocytes and peritoneal exudate cells of the mice by PSPC and PSK. The expression level of TNF-alpha was only up-regulated in the peritoneal exudate cells by PSK, but not by PSPC.
The wild mushroom, Dermocybe sanguinea, contains several anthraquinone pigments, of which emodin (1,3,8-trihydroxy-6-methylanthraquinone) is quantitatively the most important. In our preliminary tests, Dermocybe sanguinea extracts were genotoxic without metabolic activation. The ethanol extract of Dermocybe sanguinea was fractionated by flash chromatography, and the emodin contents of the fractions were determined by HPLC. Their genotoxicities were assayed using a bacterial repair assay and sister-chromatid exchange analysis. The cytotoxicity of the fractions was assayed with mouse hepatoma cells using growth inhibition as the endpoint. The results of the biological tests were compared with those obtained with pure emodin. It was concluded that, in addition to emodin, Dermocybe sanguinea contains several other geno- and cytotoxic compounds.
A novel pyradine-derivative was isolated from the mushroom Albatrellus confluens. This compound promoted melanin synthesis by B16 melanoma cells.
A fetuin derivative of alpha-amanitin was prepared and used as an antigen in rabbits. The antigen was superior to previous bovine serum albumin derivatives of beta-amanitin by its lower toxicity and high immunogenicity. On the other hand, the antibodies raised with the alpha-amanitin derivative did not show full crossreactivity with the other amatoxins, as did the immunoglobulins induced by protein derivatives of beta-amanitin. The procedure for activating nylon surfaces and coupling proteins onto them was improved with respect to surface charge and homogeneity. A partially purified IgG-fraction derived from the sera of rabbits immunized against amatoxins was covalently attached to the activated nylon surfaces. The covalently coupled immunoglobulins were complexed with a tritiated amatoxin. Then small pieces of the nylon sheet were punched out and incubated with the amatoxin solution to be analyzed. This procedure represents a method for dosing, in one step and without pipetting, the immunoglobulins and the labelled hapten. Determination of amatoxin concentrations was achieved by counting the radioactivity in the incubation fluid. The limit of detection was about 3 ng of amatoxins per ml. The radioimmunoassay was used to measure amatoxin concentrations in serum, urine, duodenal fluid, and gastric juice of patients with Amanita poisoning. Since such assays can be performed in 2-3 hr, the results can be used to determine the therapeutic protocol. The assay was likewise used to determine the concentration of amatoxins in mushroom tissue. For Amanita phalloides, for example, we found that the amatoxin concentration (mg/g dry weight) is 4.5 times higher in the gills than in the bulb.
Quantitative structure-activity relationships are established based upon the toxicity of 21 substituted pyridines. They are used as a predictive tool to calculate the toxicity of orellanine, a toxin from the mushroom Cortinarius orellanus (Fries). The actual toxicity of orellanine is quite different from the values calculated based upon the postulated bipyridine structure. The exact structure of orellanine may be questioned. However, our results show that even slight variations of the substituents of pyridines may cause dramatic changes of toxicity.
A hemolytic protein which causes diarrhea and death to mice was purified from the fruit bodies of a poisonous mushroom species Rhodophyllus rhodopolius (Fries) by DEAE-cellulose column chromatography, ammonium sulfate precipitation, gel filtration on Sephadex G-200, and DEAE-Sephadex A-25 column chromatography. The mol. wt of the purified hemolysin was estimated to be 40,000 by SDS-polyacrylamide slab gel electrophoresis. The hemolytic activity of the purified hemolysin was destroyed by heating at 60 degrees C for 10 min, and partially reduced by pepsin, papain and 2-mercaptoethanol. Cholesterol and phosphatidylcholine did not inhibit the activity. The hemolysin was unstable below pH 7.0 but stable at pH 8.0. The optimal pH for hemolysis was 6.0. Hemolysis did not occur below 4 degrees C even though the hemolysin bound to the erythrocyte. Mouse, chicken, rat, horse and human erythrocytes were sensitive in this order, but sheep and cow erythrocytes were not lysed by the toxin.
Polysaccharopeptide (PSP) is a substance produced by an edible mushroom, Coriolus versicolor which has been claimed to possess antitumor activity. However, neither tumoricidal activity nor cytotoxicity was observed when five tumor cell lines and mouse peritoneal macrophages were cultured in vitro in the presence of 2.5-10 micrograms/ml PSP. An increase in the production of reactive nitrogen intermediates, reactive oxygen intermediates (superoxide anions) and tumor necrosis factor was measured in peritoneal macrophages collected from inbred C57 mice which had received PSP in the drinking water for 2 weeks. Northern blot analysis also demonstrated that PSP activated the transcription of tumor necrosis factor gene in these cells, indicating that PSP exerted an immunomodulatory effect on the defensive cells.
TML-1 and TML-2 were two lectins isolated from the mushroom Tricholoma mongolicum. They did not differ appreciably in their pH stability and cationic requirement for hemagglutinating activity. They both stimulated the production of nitrite ions and activated the macrophages in mice. The two lectins were able to inhibit the growth of implanted sarcoma 180 cells by 68.84% and 92.39% respectively. The growth of tumor cells in the mouse peritoneal cavity was also inhibited by the two lectins with TML-2 expressing a greater potency.
The mutagenic activity of 'necatorin', a strongly mutagenic compound isolated from the wild edible mushroom Lactarius necator, was studied with the Ames test, the bacterial intraperitoneal host-mediated assay and the micronucleus test. Whereas the results of the Ames tests were clearly positive, no evidence of mutagenicity was detectable in host-mediated assays or micronucleus tests.
Chemical modification and NMR studies on a beta-galactosyl-specific lectin which was isolated from the fruiting bodies of a mushroom, Ischnoderma resinosum, has been carried out in order to investigate the amino acid residues involved in its sugar-binding sites. Modification of amino groups with succinic anhydride greatly affected the hemagglutinating activity. Inhibitory sugar lactulose could prevent the loss of the activity. Modification of carboxyl groups with glycine ethyl ester led to a 75% loss of the activity, the presence of inhibitory sugar being protective against the modification. Treatment with cyclohexane-1,2-dione for modification of arginine residues was accompanied by a complete loss of the activity. The arginine residues modification could also be protected by the inhibitory sugar. N-Bromosuccinimide treatment for modification of tryptophan residues caused a loss of the activity, although the inhibitory sugar exhibited no protective effect against this treatment. Modification of thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in a 50% loss of the activity. Modification of histidine residues with ethoxyformic anhydride led to a complete loss of the activity. The loss of the activity could be protected by the inhibitory sugar. Treatment with N-acetylimidazole for modification of tyrosine residues was accompanied by a loss of the activity. This modification was completely prevented in the presence of the inhibitory sugar. The activity of the tyrosine-modified lectin was recovered by the treatment with hydroxylamine. Furthermore, in the NOESY spectrum of the mixture of IRA and its inhibitory sugar, methyl beta-galactoside, an NOE cross peak between H-3 and/or 5 of the p-hydroxyphenyl group of a tyrosine in the lectin, and H-5 of the galactoside could be observed. These results indicate that a tyrosine residue is involved in the carbohydrate-binding site of the lectin. In addition, line broadening and down-field shifts of the galactoside-protons were observed in the presence of the lectin.
A fucose-specific lectin with a unique sugar recognizing property was purified from an orange peel mushroom, Aleuria aurantia, by using a specific affinity adsorbent prepared from L-fucose and starch. From 100 g of fruiting bodies, 145 mg of pure lectin was obtained. The lectin was crystallized and the crystals showed hexagonal bipyramid in shape. Distribution of hydrophobic and hydrophilic regions in the molecule of this lectin was predicted from the amino acid sequence deduced from the previously reported nucleotide sequence of the lectin cDNA. Circular dichroism spectra revealed a very low content of alpha-helical and beta-sheet structures and a relatively high content of turns in this lectin. From the spectrum observed in the presence of L-fucose, a hapten sugar of this lectin, certain conformational change was assumed to occur.
A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.
A study was performed on the hepatic reaction of mice to acute intoxication with virotoxins (alaviroidin, viroisin, deoxoviroisin, viroidin, deoxoviroidin) and phalloidin, cyclic peptides isolated from Amanita virosa mushrooms. Purified fractions were administered intraperitoneally at various dosages to determine the LD50 which ranged from 1.0 to 5.3 mg/kg, with viroidin, phalloidin, and viroisin being the most potent. The virotoxins and phalloidin induced hemorrhagic necrosis of the liver. The development of hepatic lesions was followed by enhanced serum alanine aminotransferase (ALT) activity as well as by light and electron microscopic changes. In additional groups, bile ducts were cannulated and bile was collected for 2 hr after injection of the peptides (1 mg/kg) to determine their cholestatic potential. The earliest changes in hepatocytes were plasma membrane invagination and cytoplasmic vacuole formation. At later time periods, erythrocyte accumulation was evident in vacuoles and in the cytoplasm. The severity of hepatic damage, as judged by morphologic analysis, was correlated with serum ALT activity. Two of the peptides tested (viroisin and phalloidin) decreased bile flow by more than 50% over control values. Mild ultrastructural alterations in the bile canalicular pole of hepatocytes were observed during the development of cholestasis. Since virotoxins, like phalloidin, are bound to actin, it is possible that their affinity to cellular actin may be responsible for their hepatotoxicity.