In the 1970s, mycoviruses were identified that infected the edible mushroom Lentinula edodes (shiitake), but they were not regarded as causal agents for mushroom diseases. None of their genes has been sequenced. In this study, the dsRNA genome of a mycovirus recently found in a shiitake commercial strain was sequenced and its molecular structure was characterized.
A cDNA library was constructed from a dsRNA purified from the fruiting body of L. edodes. The virus was tentatively named L. edodes mycovirus HKB (LeV). Based on the deduced RNA-dependent RNA polymerase (RdRp) sequence, phylogenetic analysis of LeV was conducted. Because no virion particles associated with the dsRNA were observed by electron microscopic observation, atomic force microscopy (AFM) observation was chosen for achieving molecular imaging of the virus.
The 11,282-bp genome of LeV was obtained. The genome encoded two open reading frames (ORFs). ORF1 coded for a hypothetical protein and ORF2 for a putative RdRp, respectively. In addition, a region coding for a NUDIX domain was present in ORF1. There was a 62-bp intergenic region between ORF1 and RdRp. Similarity with coat protein of mycoviruses was not found within the whole sequence. Based on phylogenetic analysis of the putative RdRp sequence, LeV grouped into a clade with dsRNA found in the basidiomycetes Phlebiopsis gigantea and Helicobasidium mompa. The clade was placed apart from the Totiviridae and Chrysoviridae families. As suggested from the genome sequence, AFM revealed that the structure of LeV was linear unencapsidated dsRNA.
The results suggest that LeV represents a novel family of mycoviruses, found thus far only among the basidiomycetes.
Mycovirus; dsRNA; AFM; Lentinula edodes; Mushroom; NUDIX domain
Excessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses.
Capsicum; climacteric; fruit softening; N-glycans; non-climacteric; RNAi; α-mannosidase; β-D-N-acetylhexosaminidase
Genetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system.
Shiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB.
We used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly.
Assembly; Basidiomycota; Mapping; Mushroom; NGS; Pyrosequencing; QTL; Shiitake
To obtain basic information on the biochemical property of basidiospores of shiitake mushroom (Lentinula edodes), the ability of producing extracellular enzyme was assessed using a chromogenic plate-based assay. For the aim, amylase, avicelase, β-glucosidase, CM-cellulase, pectinase, proteinase, and xylanase were tested against monokaryotic strains generated from forty basidiospores of two different parental dikaryotic strains of shiitake mushroom, Sanjo-101Ho and Sanjo-108Ho. These two parental strains showed different degree of extracellular enzyme activity. No identical patterns of the degree of enzyme activity were observed between monokaryotic strains and parental strains of the two shiitake cultivars. The degree of extracellular enzyme activity also varied among monokaryotic strains of the two shiitake cultivars. Our results showed that dikaryotic parental strains of shiitake mushroom produce monokaryotic basidiospores having very diverse biochemical properties.
Basidiospores; Chromogenic media; Extracellular enzyme activity; Shiitake mushroom
Trichoderma spp. cause large crop losses of the cultivated shiitake mushroom, Lentinula edodes. We bred several shiitake strains that are resistant to Trichoderma spp. using di-mon mating to establish a useful method for controlling the greenmold disease. We examined the competitive ability of L. edodes against Trichoderma spp. using a dual culture system to select resistant strains. By screening Trichoderma-resistant strains, we found that among 11 parental strains, 4 strains, including KFRI 36, were confirmed resistant strains. They showed especially strong resistance to T. harzianum, which formed deadlock after mycelial contact and then invaded into the territory of T. harzianum. KFRI 171 also showed resistance to T. atroviride strains. Among 13 strains, which were made by hybridization of shiitake strains, 5 were confirmed to be resistant to Trichoderma, including KFRI 58-1. Their resistance was not correlated to the resistant activity of their parents' strains. Two strains lose resistance and two strains acquire resistance compared to their parents' strains. In SEM observation, the mycelium of L. edodes at the interaction zone of Lentinula-Trichoderma was rugged and swollen by T. harzianum.
Hybrid strains; Lentinula edodes; Screening of resistant strains; Trichoderma species
The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
Insect β-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human β-N-acetyl-D-hexosaminidase A/B (HsHex A/ B). These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human β-N-acetyl-D-hexosaminidase subunits α/β toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing β1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and β1-4 linked GlcNAc from chitooligosaccharides as well as β1-3 linked or β1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa), and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final) instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.
β-N-acetyl-D-hexosaminidase; insect; glycoside hydrolase; N-glycan; Ostrinia furnacalis.
A lectin (LEL) was isolated from the fresh fruiting bodies of the shiitake mushroom Lentinula edodes by a combination of gel filtration chromatography on Sephadex G-150 and affinity chromatography on an N-acetyl-Dgalactosamine-Sepharose 4B column. Its molecular mass, as determined by gel filtration, was estimated to be 71, 000 Daltons and its structure is homotetrameric with subunit molecular weight of approximately 18,000 Daltons. LEL agglutinated non-specifically red blood cells from the human ABO system as well as rabbit erythrocytes and in haemagglutination inhibition assays, exhibited sugar-binding specificity toward N-acetyl-D-galactosamine. EDTA had no inhibitory effect on its haemagglutinating activity, which was stable up to 70°C and was not affected by changes in pH. The lectin had no covalently-linked carbohydrate and amino acid composition analysis revealed that it contained 124 amino acid residues and was rich in tyrosine, proline, phenylalanine, arginine, glutamic acid and cysteine. LEL did not cause mortality neither was it observed to alter the morphology of key organs when administered intraperitoneally at concentrations up to 10,000 mg kg-1 body weight of mice.
agglutination; fruiting bodies; Lectin; lentinula edodes; N-acetyl-D-galactosamine; shiitake mushroom
The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) in in vitro caries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential.
A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM.
Lentinula edodes, known as shiitake, has been utilized as food, as well as, in popular medicine, moreover, compounds isolated from its mycelium and fruiting body have shown several therapeutic properties. The aim of this study was to determine the antiviral activity of aqueous (AqE) and ethanol (EtOHE) extracts and polysaccharide (LeP) from Lentinula edodes in the replication of poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BoHV-1).
The time-of-addition assay was performed at the times -2, -1, 0, 1 and 2 h of the infection. The virucidal activity and the inhibition of viral adsorption were also evaluated. Plaque assay was used to monitor antiviral activity throughout.
The AqE and LeP were more effective when added at 0 h of infection, however, EtOHE was more effective at the times 1 h and 2 h of the infection. AqE, EtOHE and LeP showed low virucidal activity, and the inhibition of viral adsorption was not significant.
The results allowed us to conclude that AqE, EtOHE and LeP act on the initial processes of the replication of both strains of virus.
Lentinula edodes; Antiviral activity; Poliovirus; Bovine herpesvirus
In spite of the global consumption of mushrooms, only two epidemiological studies demonstrated an inverse correlation between mushroom intake and the risk of cancer. Therefore, in the present study we evaluated whether extracts from edible mushrooms Agaricus bisporus (portabella), Flammulina velutipes (enoki), Lentinula edodes (shiitake) and Pleurotus ostreatus (oyster) affect the growth of breast and colon cancer cells. Here, we identified as the most potent, P. ostreatus (oyster mushroom) which suppressed proliferation of breast cancer (MCF-7, MDA-MB-231) and colon cancer (HT-29, HCT-116) cells, without affecting proliferation of epithelial mammary MCF-10A and normal colon FHC cells. Flow cytometry revealed that the inhibition of cell proliferation by P. ostreatus was associated with the cell cycle arrest at G0/G1 phase in MCF-7 and HT-29 cells. Moreover, P. ostreatus induced the expression of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21(CIP1/WAF1), whereas inhibited the phosphorylation of retinoblastoma Rb protein in MCF-7 cells. In addition, P. ostreatus also up-regulated expression of p21 and inhibited Rb phosphorylation in HT-29 cells, suggesting that that P. ostreatus suppresses the proliferation of breast and colon cancer cells via p53-dependent as well as p53-independent pathway. In conclusion, our results indicated that the edible oyster mushroom has potential therapeutic/preventive effects on breast and colon cancer.
mushroom; breast cancer; prostate cancer; cell proliferation
Fungal β-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-N-acetylhexosaminidase. The fungal β-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.
The complete primary structure of the fungal β-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.
Whereas the intracellular bacterial β-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.
Three experiments were performed to determine the effect of selected saccharides on mushroom yield and basidiome size of shiitake (Lentinula edodes) when grown on a synthetic substrate. Substrate formulations of sawdust, wheat bran, and millet were nonamended or amended with sucrose, fructose, or glucose. Addition of sucrose (0.6 to 1.2% [dry weight]) to the substrate stimulated mushroom yield by 11 to 20% or more. Addition of fructose at 1.2% and glucose at 0.6% resulted in similar yield increases. Most of the yield increase occurred on the first break. The substrate amended with 1.2% sucrose tended to have a more synchronous maturation for the second break resulting in fewer days when mushrooms were harvested.
A green mold species that has not previously been reported in Korea was isolated from oak log beds used for shiitake (Lentinula edodes) cultivation that were infested by mushroom flies. In this study, we identify the mold species as Gliocladium viride (an anamorph of Hypocrea lutea) and describe its mycological properties. The fungus was cottony on both potato dextrose agar (PDA) and Czapek yeast extract agar (CYA), but was colored white on PDA and became yellowish green and brown on CYA. Mycelial growth on PDA attained a diameter of 73 mm at 30℃ after 5 days. The fungus grew faster on malt extract agar (> 80 mm, 5 days at 25℃) compared to CYA and PDA (< 68 mm, 5 days at 25℃). Penicillate conidiophores of the fungus are hyaline, smooth walled, branching above typically in four stages, and 120~240 µm in length. Club-shaped or slender phialides are formed on the metulae. Conidia of the fungus were ovate and elliptic, yellowish brown and green, and 2.5~3.0 µm × 1.8~2.3 µm in size. Typically, slimy conidia are formed in a mass and colored brown to dark green to almost black. The internal transcribed spacer rDNA and translation elongation factor 1 alpha gene sequences of the fungus isolated here show 99% identity with previously identified G. viride strains.
Gliocladium viride; ITS rDNA; Mushroom fly; Shiitake mushroom; tef1-α gene
Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 126.96.36.199). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.
The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.
The cultivation of shiitake mushroom (Lentinus edodes) is increasing rapidly in Bangladesh due to its nutritional and medicinal importance with excellent flavor and longer shelf life. With the aim of increased production, we have cultivated L. edodes on saw dust (SD) supplemented with different levels (10%, 15%, 20%, 25%, 30%, 35% and 40%) of wheat bran (WB), rice bran (RB), maize powder (MP) and their combination (WB+RB+MP = 1:1:1) to investigate the growth, yield and quality of this mushroom. Most of the growth, yield and quality parameters varied significantly when mushrooms were cultivated with different levels of supplementation. The yield of mushroom was increased with the level of each supplementation upto a certain level, and then decreased. SD supplemented with 25% WB produced the highest number of fruiting bodies (34.8/500 g packet), highest biological yield (153.3/500 g packet), and biological efficiency (76.6%) of L. edodes. But the yield of the best quality mushroom was observed on SD with 40% WB supplementation; however, the qualities were not always supplementation dose dependent. In this study, we report that 25% WB supplementation with SD may be very effective for higher yield and 40% WB supplementation for better quality of L. edodes.
Lentinus edodes; Cultivation; Wheat bran; Rice bran; Maize powder; Supplementation
In many invertebrates and plants, the N-glycosylation profile is dominated by truncated paucimannosidic N-glycans, i.e., glycans consisting of a simple trimannosylchitobiosyl core often modified by core fucose residues. Even though they lack antennal N-acetylglucosamine residues, the biosynthesis of these glycans requires the sequential action of GlcNAc transferase I, Golgi mannosidase II and, finally, β-N-acetylglucosaminidases. In Drosophila, the recently characterised enzyme encoded by the fused lobes (fdl) gene specifically removes the non-reducing N-acetylglucosamine residue from the α1,3-antenna of N-glycans. In the present study, we examined the products of five β-N-acetylhexosaminidase genes from Caenorhabditis elegans (hex-1 to hex-5, corresponding to reading frames T14F9.3, C14C11.3, Y39A1C.4, Y51F10.5 and Y70D2A.2) in addition to three from Arabidopsis thaliana (AtHEX1, AtHEX2 and AtHEX3, corresponding to reading frames At1g65590, At3g55260 and At1g05590). Based on homology, the Caenorhabditis HEX-1 and all three Arabidopsis enzymes are members of the same sub-family as the aforementioned Drosophila fused lobes enzyme, but either act as chitotriosidases or non-specifically remove N-acetylglucosamine from both N-glycan antennae. The other four Caenorhabditis enzymes a members of a distinct sub-family; nevertheless, two of these enzymes displayed the same α1,3-antennal specificity as the fused lobes enzyme. Furthermore, a deletion of part of the Caenorhabditis hex-2 gene drastically reduces the native N-glycan-specific hexosaminidase activity in mutant worm extracts and results in a shift in the N-glycan profile, which is a demonstration of its in vivo enzymatic relevance. Based on these data, it is hypothesised that the genetic origin of paucimannosidic glycans in nematodes, plants and insects involves highly-divergent members of the same hexosaminidase gene family.
Arabidopsis; Caenorhabditis; hexosaminidase; paucimannosidic N-glycans
To produce a potent antidementia β-secretase inhibitor from a mushroom, the β-secretase inhibitory activities of various mushroom extracts were determined. Methanol extracts of Lentinula edodes exhibited the highest inhibitory activity (40.1%). The inhibitor was maximally extracted when a fruiting body of L. edodes was treated with 50% methanol at 40℃ for 24 h.
Antidementia; BACE1; β-Secretase inhibitor; Lentinula edodes
We investigated diet supplementation with shiitake mushroom fruiting bodies on biochemical and histological changes in hypercholesterolemic rats. Six-wk old female Sprague-Dawley albino rats were divided into three groups of 10 rats each. A diet containing 5% Lentinus edodes fruiting bodies given to hypercholesterolemic rats reduced plasma total cholesterol, triglyceride, low-density lipoprotein (LDL), total lipid, phospholipids, and the LDL/high-density lipoprotein ratio by 34.33, 53.21, 75.00, 34.66, 25.73, and 71.43%, respectively. Feeding mushroom also significantly reduced body weight in hypercholesterolemic rats. However, it had no detrimental effects on plasma albumin, total bilirubin, direct bilirubin, creatinine, blood urea nitrogen, uric acid, glucose, total protein, calcium, sodium, potassium, chloride, inorganic phosphate, magnesium, or enzyme profiles. Feeding mushroom increased total lipid and cholesterol excretion in feces. The plasma lipoprotein fraction, separated by agarose gel electrophoresis, indicated that L. edodes significantly reduced plasma β and pre-β-lipoprotein but increased α-lipoprotein. A histological study of hepatic cells by conventional hematoxylin-eosin and oil red-O staining showed normal findings for mushroom-fed hypercholesterolemic rats. These results suggest that shiitake mushrooms could be recommended as a natural cholesterol lowering substance in the diet.
Agarose gel electrophoresis; Atherogenic lipid profile; Histopathology; Hypercholesterolemic rats; Lentinus edodes
Shiitake Dermatitis is often presented as papules and erythemato-violaceous linear
streaks. It can be associated with bleomycin treatment, dermatomyositis and shiitake
mushroom (Lentinus edodes). There is not any previous report concerning this rare
etiology in our country. Shiitake is the second most consumed mushroom worldwide and
it can cause flagellate erythema when ingested raw or half cooked. It has a higher
incidence in Oriental countries because of their eating habits, this is the first
case reported in Brazil, in a male patient that presented a cutaneous rash after
consuming this raw mushroom.
Dermatitis; Erythema; Shiitake mushrooms
A variety of fungi produce the hydrolytic enzyme β-N-acetylhexosaminidase (HexNAcase), which can be readily detected in assays by using p-nitrophenyl-N-acetyl-β-d-glucosaminide as a substrate. In the present study we developed a microtiter plate-based HexNAcase assay for distinguishing Candida albicans and Candida dubliniensis strains from other yeast species. HexNAcase activity was detected in 89 of 92 (97%) C. albicans strains and 4 of 4 C. dubliniensis strains but not in 28 strains of eight other Candida species, 4 Saccharomyces cerevisiae strains, or 2 Cryptococcus neoformans strains. The HexNAcase activity in C. albicans and C. dubliniensis was strain specific. All except three clinical C. albicans isolates among the C. albicans strains tested produced enzyme activity within 24 h. These strains did produce enzyme activity, however, after a prolonged incubation period. For two of these atypical strains, genomic DNA at the C. albicans HEX1 gene locus, which encodes HexNAcase, showed nucleotide differences from the sequence of control strains. Among the other Candida species tested, only C. dubliniensis had a DNA sequence that hybridized with the HEX1 probe under low-stringency conditions. The microtiter plate-based assay used in the present study for the detection of HexNAcase activity is a simple, relatively inexpensive method useful for the presumptive identification of C. albicans and C. dubliniensis.
GM2 gangliosidoses are a group of panethnic lysosomal storage diseases in which GM2 ganglioside accumulates in the lysosome due to a defect in one of three genes, two of which encode the α- or β-subunits of β-N-acetylhexosaminidase (Hex) A. A small inframe deletion mutation in the catalytic domain of the α-subunit of Hex has been found in five Turkish patients with infantile Tay–Sachs disease. To date it has not been detected in other populations and is the only mutation to be found in exon 10. It results in detectable levels of inactive α-protein in its precursor form. Because the α- and β-subunits share 60% sequence identity, the Hex A and Hex B genes are believed to have arisen from a common ancestral gene. Thus the subunits must share very similar three-dimensional structures with conserved functional domains. Hex B, the β-subunit homodimer is more stable than the heterodimeric Hex A, and much more stable than Hex S, the α homodimer. Thus, mutations that completely destabilize the α-subunit can often be partially rescued if expressed in the aligned positions in the β-subunit. To better understand the severity of the Turkish HEXA mutation, we reproduced the 12 bp deletion mutation (1267–1278) in the β-subunit cDNA. Western blot analysis of permanently transfected CHO cells expressing the mutant detected only the pro-form of the β-subunit coupled with a total lack of detectable Hex B activity. These data indicate that the deletion of the four amino acids severely affects the folding of even the more stable β-subunit, causing its retention in the endoplasmic reticulum and ultimate degradation.
PMID: 15505380 CAMSID: cams1390
Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22°C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-β-d-glucosidase, β-N-acetyl-d-glucosaminidase, α-d-galactosidase, β-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting.