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.

Effects of Dietary Intake of Japanese Mushrooms on Visceral Fat Accumulation and Gut Microbiota in Mice.

A lot of Japanese people are generally known for having a healthy diet, and consume a variety of mushrooms daily. Many studies have reported anti-obesity effects of mushrooms, but few have investigated the effects of consuming a variety of edible mushroom types together in realistic quantities. In this study, we investigated whether supplementation with a variety of mushroom types affects visceral fat accumulation and gut microbiota in mice. The most popular mushroom varieties in Japan were lyophilized and mixed according to their local production ratios. C57BL/6J mice were fed a normal diet, high-fat (HF) diet, HF with 0.5% mushroom mixture (equivalent to 100 g mushrooms/day in humans) or HF with 3% mushroom mixture (equivalent to 600 g mushrooms/day in humans) for 4 weeks. The mice were then sacrificed, and blood samples, tissue samples and feces were collected. Our results show that mushroom intake suppressed visceral fat accumulation and increased the relative abundance of some short chain fatty acid- and lactic acid-producing gut bacteria. These findings suggest that mushroom intake is an effective strategy for obesity prevention.

Agarol, an ergosterol derivative from Agaricus blazei, induces caspase-independent apoptosis in human cancer cells.

Agaricus blazei (A. blazei) is a mushroom with many biological effects and active ingredients. We purified a tumoricidal substance from A. blazei, an ergosterol derivative, and named it 'Agarol'. Cytotoxic effects of Agarol were determined by the MTT assay using A549, MKN45, HSC-3, and HSC-4 human carcinoma cell lines treated with Agarol. Apoptosis was detected by flow cytometry analysis. Reactive oxygen species (ROS) levels and mitochondria membrane potential (∆ψm) were also determined by flow cytometry. Western blot analysis was used to quantify the expression of apoptosis-related proteins. Agarol predominantly induced apoptosis in two p53-wild cell lines (A549 and MKN45) compared to the other p53-mutant cell lines (HSC-3 and HSC-4). Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of ROS, reduced ∆ψm, release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol, upregulation of Bax, and downregulation of Bcl-2. Caspase-3 activities did not increase, and z-VAD-fmk, a caspase inhibitor, did not inhibit the Agarol-induced apoptosis. These findings indicate that Agarol induces caspase-independent apoptosis in human carcinoma cells through a mitochondrial pathway. The in vivo anticancer activity of Agarol was confirmed in a xenograft murine model. This study suggests a molecular mechanism by which Agarol induces apoptosis in human carcinoma cells and indicates the potential use of Agarol as an anticancer agent.

The Anti-Inflammatory Effects of Lion's Mane Culinary-Medicinal Mushroom, Hericium erinaceus (Higher Basidiomycetes) in a Coculture System of 3T3-L1 Adipocytes and RAW264 Macrophages.

Chronic low-grade inflammation in the adipose tissue accompanying obesity is thought to be an underlying driver of metabolic diseases. In this study, we aimed to investigate the efficacy of Hericium erinaceus on adipose tissue inflammation. The anti-inflammatory effects of the ethyl acetate soluble fraction of H. erinaceus (EAHE) were examined using cocultures of 3T3-L1 adipocytes and RAW264 macrophages. EAHE significantly suppressed tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in cultured RAW264 macrophages stimulated by lipopolysaccharide (LPS). EAHE also caused notable inhibition of c-Jun N-terminal kinase (JNK) activation, which is thought to be involved in the suppression of proinflammatory cytokines by EAHE. In a coculture system with 3T3-L1 and RAW264 cells stimulated with LPS, EAHE reduced TNF-α and IL-6 concentrations in the conditioned medium and lowered the gene expression levels of these cytokines in 3T3-L1 adipocytes. Furthermore, EAHE suppressed the LPS-induced reduction of adiponectin mRNA levels in 3T3-L1 adipocytes cocultured with RAW264 macrophages. However, in 3T3-L1 adipocytes cultured alone, the concentration of LPS used in this study did not affect the gene expression levels of these adipokines. We attributed the anti-inflammatory effects of EAHE on 3T3-L1 adipocytes cocultured with RAW264 macrophages to the suppression of Toll-like receptor 4 (TLR4) signaling and subsequent proinflammatory cytokine secretion in RAW264 cells. Our findings indicate the possibility that H. erinaceus exerts anti-inflammatory effects on macrophages through the inhibition of TLR4-JNK signaling and prevents or ameliorates adipose tissue inflammation associated with obesity.

Breakdown of continuum fracture mechanics at the nanoscale.

Materials fail by the nucleation and propagation of a crack, the critical condition of which is quantitatively described by fracture mechanics that uses an intensity of singular stress field characteristically formed near the crack-tip. However, the continuum assumption basing fracture mechanics obscures the prediction of failure of materials at the nanoscale due to discreteness of atoms. Here, we demonstrate the ultimate dimensional limit of fracture mechanics at the nanoscale, where only a small number of atoms are included in a singular field of continuum stress formed near a crack tip. Surprisingly, a singular stress field of only several nanometers still governs fracture as successfully as that at the macroscale, whereas both the stress intensity factor and the energy release rate fail to describe fracture below a critically confined singular field of 2-3 nm, i.e., breakdown of fracture mechanics within the framework of the continuum theory. We further propose an energy-based theory that explicitly accounts for the discrete nature of atoms, and demonstrate that our theory not only successfully describes fracture even below the critical size but also seamlessly connects the atomic to macroscales. It thus provides a more universal fracture criterion, and novel atomistic insights into fracture.

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.