Novel acid trehalase belonging to glycoside hydrolase family 37 from Pleurotus sp.: cloning, expression and characterization.

The N-terminal amino acid sequence of the Pleurotus sp. 90 kDa protein was in good agreement with the corresponding sequence of the glycoside hydrolase (GH) family 37 protein (trehalase) from P. ostreatus PC 15 v2.0. The length of the Pleurotus sp. trehalase gene was 2247 bp, encoding a protein of 749 amino acids with a predicted molecular mass of 81.2 kDa. The molecular mass of the recombinant enzyme was estimated to be about 117 kDa by SDS-PAGE. We found that the recombinant enzyme comprised an N-glycosylated sugar chain and that its optimum pH and temperature were 4.5 and 40 ºC, respectively. Moreover, this enzyme exhibited high activity against trehalose exclusively. We found that the enzyme is novel acid trehalase belonging to GH family 37.

Genetically Independent Tetranucleotide to Hexanucleotide Core Motif SSR Markers for Identifying Lentinula edodes Cultivars.

For the purpose of protecting the rights of Lentinula edodes breeders, we developed a new simple sequence repeat (SSR) marker set consisting only of genetically independent tetranucleotide or longer core motifs. Using available genome sequences for five L. edodes strains, we designed primers for 13 SSR markers that amplified polymorphic sequences in 20 L. edodes cultivars. We evaluated the independence of every possible marker pair based on genotype data. Consequently, eight genetically independent markers were selected. The polymorphic information content values of the markers ranged from 0.269 to 0.764, with an average of 0.409. The markers could distinguish among 20 L. edodes cultivars and produced highly repeatable and reproducible results. The markers developed in this study will enable the precise identification of L. edodes cultivars, and may be useful for protecting breeders' rights.

Pleurotus eryngii Ameliorates Lipopolysaccharide-Induced Lung Inflammation in Mice.

Pleurotus eryngii (P. eryngii) is consumed as a fresh cultivated mushroom worldwide and demonstrated to have multiple beneficial effects. We investigated the anti-inflammatory effect of P. eryngii in mice with acute lung injury (ALI). Intranasal instillation of lipopolysaccharide (LPS) (10 µ g/site/mouse) induced marked lung inflammation (increase in the number of inflammatory cells, protein leakage, and production of nitric oxide in bronchoalveolar lavage fluid) as well as histopathological damage in the lung, 6 h after treatment. Mice administered heat-treated P. eryngii (0.3-1 g/kg, p.o. (HTPE)) 1 h before LPS challenge showed decreased pulmonary inflammation and ameliorated histopathological damage. These results suggest that HTPE has anti-inflammatory effects against ALI. Thus, P. eryngii itself may also have anti-inflammatory effects and could be a beneficial food for the prevention of ALI induced by bacterial infection.

Characterization of a gene coding for a putative adenosine deaminase-related growth factor by RNA interference in the basidiomycete Flammulina velutipes.

A full-length cDNA coding for a putative adenosine deaminase (Fv-ada) was isolated from the basidiomycete Flammulina velutipes. Fv-ada encodes a polypeptide consisting of 537 amino acid residues, which has a consensus sequence conserved among adenosine deaminase-related growth factors (ADGF) found in several metazoa, including chordates and insects. Fv-ada transcript was detected at all stages of growth in dikaryotic F. velutipes cells, with a peak at the primordial stage. Heterologous expression of Fv-ada in the yeast Pichia pastoris produced recombinant Fv-ADA that catalyzed the conversion of adenosine to inosine. Dikaryotic mycelia from F. velutipes were transformed with the binary plasmid pFungiway-Fv-ada, which was designed to suppress the expression of Fv-ada through RNA interference. The growth rates of the resulting transformants were retarded in response to the degree of suppression, indicating that Fv-ada plays an important role in the mycelial growth of F. velutipes. These results suggested that ADGF could function as growth factors in fungi, as is seen in other eukaryotes.

Effects of Hericium erinaceus on amyloid ß(25-35) peptide-induced learning and memory deficits in mice.

The mushroom Hericium erinaceus has been used as a food and herbal medicine since ancient times in East Asia. It has been reported that H. erinaceus promotes nerve growth factor secretion in vitro and in vivo. Nerve growth factor is involved in maintaining and organizing cholinergic neurons in the central nervous system. These findings suggest that H. erinaceus may be appropriate for the prevention or treatment of dementia. In the present study, we examined the effects of H. erinaceus on amyloid ß(25-35) peptide-induced learning and memory deficits in mice. Mice were administered 10 µg of amyloid ß(25-35) peptide intracerebroventricularly on days 7 and 14, and fed a diet containing H. erinaceus over a 23-d experimental period. Memory and learning function was examined using behavioral pharmacological methods including the Y-maze test and the novel-object recognition test. The results revealed that H. erinaceus prevented impairments of spatial short-term and visual recognition memory induced by amyloid ß(25-35) peptide. This finding indicates that H. erinaceus may be useful in the prevention of cognitive dysfunction.

Agrobacterium tumefaciens-mediated transformation of the vegetative dikaryotic mycelium of the cultivated mushroom Flammulina velutipes.

Agrobacterium tumefaciens was used to transform the vegetative dikaryotic mycelium of Flammulina velutipes using a hygromycin B resistance gene as selectable marker. The gene coding for urogen III methyltransferase (cob) was introduced into F. velutipes dikaryotic cells. The resulting transformant cells generated a bright red fluorescence, indicating that cob is promising as a reporter gene in F. velutipes.

Inhibitory effect of hericenone B from Hericium erinaceus on collagen-induced platelet aggregation.

Platelet aggregation in the blood vessel causes thrombosis. Therefore, inhibitors of platelet aggregation promise to be preventive or therapeutic agents of various vascular diseases, including myocardial infarction and stroke. In the present study, we found that hericenone B had a strong anti-platelet activity and it might be a novel compound for antithrombotic therapy possessing a novel mechanism. Prior to this study, we examined anti-platelet aggregation activity of ethanol extracts of several species of mushrooms, and found that extract of Hericium erinaceus potently inhibited platelet aggregation induced by collagen. Therefore, we first fractionated the ethanol extract of H. erinaceus to identify the active substances. The anti-platelet activity of each fraction was determined using washed rabbit platelets. As a result, an active component was isolated and identified as hericenone B. Hericenone B selectively inhibited collagen-induced platelet aggregation, but it did not suppress the aggregation induced by U46619 (TXA2 analogue), ADP, thrombin, or adrenaline. Furthermore, hericenone B did not inhibit arachidonic acid- or convulxin (GPVI agonist)-induced platelet aggregation. Therefore, hericenone B was considered to block collagen signaling from integrin α2/ß1 to arachidonic acid release. Moreover, we found that collagen-induced aggregation was inhibited by hericenone B in human platelets, similar to in rabbit platelets.

Hypolipidemic effect of Pleurotus eryngii extract in fat-loaded mice.

Pleurotus eryngii water extract (PEE), which showed the most significant inhibitory activity against pancreatic lipase in vitro among eight edible mushrooms, was investigated to determine the mechanism of its anti-lipase activity in vitro and its hypolipidemic effect in fat-loaded mice. The inhibitory effects of mushroom extracts on pancreatic lipase activity were examined using 4-methylumbelliferyl oleate (4-MUO) or trioleoylglycerol emulsified with lecithin, gum arabic or Triton X-100 as a substrate. For in vivo experiments, blood samples were taken after oral administration of corn oil and [(3)H]trioleoylglycerol with or without PEE to food-deprived mice. PEE inhibited hydrolysis of 4-MUO and trioleoylglycerol emulsified with lecithin or Triton X-100, but not that of trioleoylglycerol emulsified with gum arabic. PEE suppressed the elevations of plasma and chylomicron triacylglycerol levels after oral administration of corn oil, but had no effect on lipoprotein lipase activity. [(3)H]Trioleoylglycerol absorption was also decreased by administration of PEE. The results of in vitro studies suggest that PEE may prevent interactions between lipid emulsions and pancreatic lipase. The hypolipidemic effect of PEE in fat-loaded mice may be due to low absorption of fat caused by the inhibition of pancreatic lipase.

Rat medium-term multi-organ carcinogenesis bioassay of Agaricus blazei Murrill fruit-body extract.

The modifying potential of Agaricus blazei Murrill fruit-body extract (ABFE) on tumor development was investigated in a medium-term multi-organ carcinogenesis bioassay. Male 6-week-old F344 rats were treated with N-nitrosodiethylamine (DEN), N-methyl-N-nitrosourea (MNU), 1,2-dimethylhydrazine dihydrochloride (DMH), N-butyl-N-(hydroxybutyl)-nitrosamine (BBN), and diisopropanolnitrosamine (DHPN) for initiation (DMBDD treatment). After a 1-week withdrawal period, the animals received distilled water (vehicle control) or ABFE A, gamma-amino butyric acid (GABA) at 0.8 mg/kg, ABFE B (GABA level of 3.0mg/kg) or ABFE C (GABA level of 12.0mg/kg) by gavage for 24 weeks. There were no effects of ABFE on survival rate, general condition, body weight, food and water consumption, and organ weights. The multiplicity of large intestinal nodules, smaller than 2mm was significantly increased in the ABFE C group with DMBDD treatment. However, there were no significantly inter-group differences in incidences of hyperplastic or neoplastic lesions in colon or other organs, or in immunohistochemically identified preneoplastic lesions in the liver. In conclusion, A. blazei Murrill fruit-body extract, even at a GABA level up to 12 mg/kg, did not exert modifying potential in the present medium-term multi-organ carcinogenesis bioassay in male F344 rats (DMBDD method).

Improving effects of the mushroom Yamabushitake (Hericium erinaceus) on mild cognitive impairment: a double-blind placebo-controlled clinical trial.

A double-blind, parallel-group, placebo-controlled trial was performed on 50- to 80-year-old Japanese men and women diagnosed with mild cognitive impairment in order to examine the efficacy of oral administration of Yamabushitake (Hericium erinaceus), an edible mushroom, for improving cognitive impairment, using a cognitive function scale based on the Revised Hasegawa Dementia Scale (HDS-R). After 2 weeks of preliminary examination, 30 subjects were randomized into two 15-person groups, one of which was given Yamabushitake and the other given a placebo. The subjects of the Yamabushitake group took four 250 mg tablets containing 96% of Yamabushitake dry powder three times a day for 16 weeks. After termination of the intake, the subjects were observed for the next 4 weeks. At weeks 8, 12 and 16 of the trial, the Yamabushitake group showed significantly increased scores on the cognitive function scale compared with the placebo group. The Yamabushitake group's scores increased with the duration of intake, but at week 4 after the termination of the 16 weeks intake, the scores decreased significantly. Laboratory tests showed no adverse effect of Yamabushitake. The results obtained in this study suggest that Yamabushitake is effective in improving mild cognitive impairment.

Isolation and characterization of a gene coding for chitin deacetylase specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes and its expression in the yeast Pichia pastoris.

Fv-pda, a gene coding for chitin deacetylase (CDA), was isolated from the basidiomycete Flammulina velutipes by differential display targeted for genes specifically expressed during fruiting body development. The fv-pda ORF comprises 250 amino acid residues and is interrupted by 10 introns. The fv-pda cDNA was expressed in the yeast Pichia pastoris, and the resulting recombinant FV-PDA was used for enzymatic characterization. The recombinant FV-PDA catalyses deacetylation of N-acetyl-chitooligomers, from dimer to pentamer, glycol chitin and colloidal chitin. The fv-pda was specifically expressed through the entire stage of fruiting body development, and the transcript was abundant in stipes of mature fruiting bodies. These results suggest that CDA plays an important role in the process of fruiting of F. velutipes.

Antiatherosclerotic effect of the edible mushrooms Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji) in apolipoprotein E-deficient mice.

In the present study, we examined the antiatherosclerotic effects of 3 edible mushrooms, Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji), in atherosclerosis-susceptible C57BL/6J, apolipoprotein E-deficient (apoE(-/-)) mice. Male apoE(-/-) mice (6 weeks of age) were fed a normal diet (cholesterol concentration <66 mg/100 g) or a normal diet containing 3% dried Eringi, Maitake, or Bunashimeji mushroom powder for 10 weeks. Food intake, body weight, serum total cholesterol (TC), and serum triacylglycerols (TG) were measured periodically during the experimental period. At the end of the experiment (at 16 weeks of age), the atherosclerotic lesion area was measured in cross-sections of the aortic root. Serum TC concentrations in the Bunashimeji group were significantly lower than that in the control group at 8, 10, 12, 14, and 16 weeks of age. Serum TC concentrations in the Eringi, and Maitake groups were significantly lower than that in the control group only at 12 weeks of age. There was no significant difference in the serum TG concentrations in all groups during the experimental period. The atherosclerotic lesions were significantly decreased in the Eringi, Maitake, and Bunashimeji groups than that in the control group at the end of the experiment. Dietary supplementation with the Bunashimeji mushroom powder had the strongest antiatherosclerotic effect among 3 mushrooms. In conclusion, supplementation of the 3 edible mushrooms prevents the development of atherosclerosis, even normal diet. Antiatherosclerotic effect is partly via lowering of serum TC concentrations; further mechanisms should be investigated.

Nerve growth factor-inducing activity of Hericium erinaceus in 1321N1 human astrocytoma cells.

Neurotrophic factors are essential to maintain and organize neurons functionally; thereby neurotrophic factor-like substances or their inducers are expected to be applied to the treatment of neurodegenerative diseases such as Alzheimer's disease. In the present study, we firstly examined the effects of ethanol extracts of four edible mushrooms, Hericium erinaceus (Yamabushitake), Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Agaricus blazei (Himematsutake), on nerve growth factor (NGF) gene expression in 1321N1 human astrocytoma cells. Among the four mushroom extracts, only H. erinaceus extract promoted NGF mRNA expression in a concentration-dependent manner. In addition, secretion of NGF protein from 1321N1 cells was enhanced by H. erinaceus extracts, and the conditioned medium of 1321N1 cells incubated with H. erinaceus extract enhanced the neurite outgrowth of PC12 cells. However, hericenones C, D and E, constituents of H. erinaceus, failed to promote NGF gene expression in 1321N1 cells. The enhancement of NGF gene expression by H. erinaceus extracts was inhibited by the c-jun N-terminal kinase (JNK) inhibitor SP600125. In addition, H. erinaceus extracts induced phosphorylation of JNK and its downstream substrate c-Jun, and increased c-fos expression, suggesting that H. erinaceus promotes NGF gene expression via JNK signaling. Furthermore we examined the efficacy of H. erinaceus in vivo. ddY mice given feed containing 5% H. erinaceus dry powder for 7 d showed an increase in the level of NGF mRNA expression in the hippocampus. In conclusion, H. erinaceus contains active compounds that stimulate NGF synthesis via activation of the JNK pathway; these compounds are not hericenones.

Isolation and analysis of genes specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes by fluorescence differential display.

Using fluorescence differential display, cDNAs specifically expressed at the primordial stage of fruiting body development were isolated from the basidiomycete, Flammulina velutipes. Seventy-five cDNAs were sequenced and compared with the amino-acid sequences of proteins in the database by BLASTX search. Significant similarity was found for 29 cDNAs coding for proteins with known function, GTP-binding protein, growth factor, ubiquitin-proteasome, cytochrome P450 and hydrophobin, all of which would be associated with fruiting body development. Seventeen cDNAs were not similar to proteins in the database and may represent unique genes that play specific roles in the process of fruiting in F. velutipes.

Cloning and characterization of a gene coding for a hydrophobin, Fv-hyd1, specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes.

Hydrophobin cDNA (fv-hyd1), which is specifically expressed during fruiting body development, was isolated from the basidiomycete Flammulina velutipes by differential display screening. Analysis of the genomic structure of fv-hyd1 revealed an open reading frame (ORF) composed of 363 nucleotides and interrupted by three introns. The deduced amino acid sequence of FV-HYD1 showed a similarity to those of other fungal class I hydrophobins and contained eight cysteine residues highly conserved among hydrophobin proteins. The pattern of the hydropathy plot of FV-HYD1 was similar to those of class I hydrophobins. Southern blot analysis of genomic DNA showed that fv-hyd1 existed as a single copy. Northern blot analysis indicated that the fv-hyd1 transcript was not present in vegetative mycelia but markedly increased in level at the primordial stage. Moreover, the fv-hyd1 transcript was abundant even at the mature fruiting body stage. This result indicates that fv-hyd1 could encode a hydrophobin closely associated with fruiting body development.

A novel carbon skeletal trichothecane, tenuipesine A, isolated from an entomopathogenic fungus, Paecilomyces tenuipes.

Tenuipesine A (1), a novel trichothecane with an unprecedented carbon-migrated skeleton that embodies of a cyclopropane ring, was isolated from cultivated fruiting bodies of Paecilomyces tenuipes (Isaria japonica), a popular entomopathogenic fungi employed in folk medicine and health foods in China, Korea, and Japan. The structure was determined on the basis of two-dimensional NMR data. Its stereochemistry was elucidated by spectroscopic data and the chemical transformation of the coexisting trichothecene, 4beta-acetoxy-12,13-epoxytrichothec-9-ene-3alpha,15-diol (2). [structure: see text]

Novel spirocyclic trichothecanes, spirotenuipesine A and B, isolated from entomopathogenic fungus, Paecilomyces tenuipes.

Entomopathogenic fungi forming fruiting bodies have been employed as tonics and antitussives from ancient times. Paecilomyces tenuipes, which is also called Isaria japonica, is a very popular entomopathogenic fungus and is often considered a health food in northeast Asian countries such as China, Korea, and Japan. We cultivated the fruiting bodies of Paecilomyces tenuipes. Among the large-scale cultivations, fruiting body grown in barley grain contained two novel spirocyclic trichothecane derivatives, spirotenuipesine A (1) and B (2), and known trichothecane mycotoxins. Compounds 1 and 2 showed potent activity in neurotrophic factor biosynthesis in glial cells. The isolation of these compounds indicated that P. tenuipes is a promising source for producing various biologically active substances including trichothecanes. It is noteworthy that trichothecane mycotoxins are present in Paecilomyces tenuipes, which is typically used in medicinal health food.