Lignin-degrading enzymes from a pathogenic canker-rot fungus Inonotus obliquus strain IO-B2.

Inonotus obliquus is a pathogenic fungus found in living trees and has been widely used as a traditional medicine for cancer therapy. Although lignocellulose-degrading enzymes are involved in the early stages of host infection, the parasitic life cycle of this fungus has not been fully understood. In this study, we aimed to investigate the activities of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) from I. obliquus cultivated in Kirk's medium. The fungus was subjected to genome sequencing, and genes related to wood degradation were identified. The draft genome sequence of this fungus comprised 21,203 predicted protein-coding genes, of which 134 were estimated to be related to wood degradation. Among these, 47 genes associated with lignin degradation were found to have the highest number of mnp genes. Furthermore, we cloned the cDNA encoding a putative MnP, referred to as IoMnP1, and characterized its molecular structure. The results show that IoMnP1 has catalytic properties analogous to MnP. Phylogenetic analysis also confirmed that IoMnP1 was closely related to the MnPs from Pyrrhoderma noxium, Fomitiporia mediterranea, and Sanghuangporus baumii, which belong to the same family of Hymenochaetaceae. From the above results, we suggest that IoMnP1 is a member of MnPs.

Pectin decomposition at the early stage of brown-rot decay by Fomitopsis palustris.

The sapwood of Japanese cedar (Cryptomeria japonica D. Don) was decayed by the brown-rot fungus Fomitopsis palustris under bright and dark conditions. Scanning electron microscopy revealed the presence of mycelia inside the wood even after 1 week from the start of fungal exposure. Moreover, holes were observed in the torus after fungal exposure. Ruthenium red staining revealed that the pectin in pits was largely absent for 3 weeks. These events occurred before the mass loss of wood samples was confirmed at the early stage. Moreover, FpPG28A was more highly expressed at the hyphal front on a pectin-containing medium under dark conditions compared with bright conditions. This up-regulation under dark conditions indicated that the pectin decomposition ability was promoted inside the wood where light could not reach. In conclusion, we suggest that the brown-rot fungus completed its hyphal expansion within the wood via pectin decomposition in pits before holocellulose decomposition.

The complete mitochondrial genome sequence of the medicinal fungus Inonotus obliquus (Hymenochaetaceae, Basidiomycota).

Inonotus obliquus is a medicinal fungus in the family Hymenochaetaceae and commonly known as chaga. The sclerotium of this fungus has been used as a traditional medicine for long time. In this study, we present the mitochondrial genome sequence of I. obliquus. The mitochondrial DNA (mtDNA) is 119,110 base pairs in length and contained genes for 58 Open reading frames, 2 ribosomal RNAs, and 30 transfer RNAs. Consequently performed phylogenetic analysis indicates that this fungus is closely related to Sanghuangporus sanghuang which belongs to the same family Hymenochaetaceae. We first reported about the complete mitochondrial genome of fungi belonging to the genus Inonotus.

A GH family 28 endo-polygalacturonase from the brown-rot fungus Fomitopsis palustris: Purification, gene cloning, enzymatic characterization and effects of oxalate.

Brown-rot fungi are the wood-decay basidiomycetes and have ability to break down plant cell wall carbohydrates. It has been suggested that degradation of pectin is important for the initial stages of brown rot. We purified an endo-polygalacturonase (FpPG28A) from the brown-rot fungus Fomitopsis palustris, analysis of the predicted amino acid sequence indicated that FpPG28A belongs to GH family 28. The highest activity of purified FpPG28A was observed at 60 °C in 50 mM sodium acetate buffer (pH 5.0); this activity was highly specific for polygalacturonic acid chains. However, calcium polygalacturonate gel was not degraded by FpPG28A under those optimal conditions. We observed that calcium polygalacturonate gel was readily degraded by the enzyme in the oxalate buffer. Furthermore, the thermostability of FpPG28A was elevated in oxalate buffer at pH 3.0. These results indicated that oxalate has an important role in the degradation of woody pectin by FpPG28A.

Identification of mutations through dominant screening for obesity using C57BL/6 substrains.

The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon, and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work.

Expression and purification of cyto-insectotoxin (Cit1a) using silkworm larvae targeting for an antimicrobial therapeutic agent.

Antimicrobial peptides (AMPs), both synthetic and from natural sources, have raised interest recently as potential alternatives to antibiotics. Cyto-insectotoxin (Cit1a) is a 69-amino-acid antimicrobial peptide isolated from the venom of the central Asian spider Lachesana tarabaevi. The synthetic gene Cit1a fused with the enhanced green fluorescent protein (EGFP) gene was expressed as the EGFP-Cit1a fusion protein using a cysteine protease-deleted Bombyx mori nucleopolyhedrovirus (BmNPV-CP(-)) bacmid in silkworm larva and pupa. The antimicrobial effect of the purified protein was assayed using disk diffusion and broth microdilution methods. The minimum inhibitory concentration of EGFP-Cit1a was also measured against several bacterial strains and showed similar antimicrobial activity to that of the synthetic Cit1a reported earlier. The EGFP-Cit1a fusion protein showed antibiotic activity toward gram-positive and gram-negative bacteria at the micromolar concentration level. These results show that active Cit1a can be produced and purified in silkworm, although this peptide is insecticidal. This study demonstrates the potential of active Cit1a purified from silkworms to use as an antimicrobial agent.

An antifungal protein from the culinary-medicinal beech mushroom, Hypsizygus marmoreus (Peck) Bigel. (Agaricomycetideae).

An antifungal protein (HM-af) was purified from the culinary-medicinal mushroom Hypsizygus marmoreus. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HM-af indicated that its molecular mass was 9.5 kDa. The N-terminal amino acid sequence of HM-af showed homology to ribonuclease H from Clostridium thermocellum. HM-af showed the antifungal activity against Flammulina velutipes.

Inhibition of IgE-dependent mouse triphasic cutaneous reaction by a boiling water fraction separated from mycelium of Phellinus linteus.

Phellinus linteus, a mushroom, contains constituents that exhibit potent antitumor effects through activating immune cells. Recently, anti-inflammatory and anti-allergic properties of P. linteus extracts have also been implicated. In the present study, therefore, we separated the constituents of mycelium of P. linteus into five fractions-chloroform-soluble (CF), ethyl acetate-soluble (EA), methanol-soluble (AE), water-soluble (WA) and boiling water-soluble (BW) fractions-and examined their suppressive effects on the IgE-dependent mouse triphasic cutaneous reaction. The triphasic reaction was induced in the ear of BALB/c mice passively sensitized with anti-dinitrophenol IgE by painting with 2,4-dinitrofluorobenzene 24 h later. Ear swelling appeared triphasically with peak responses at 1 h, 24 h and 8 days after the challenge. ME, WA and BW given orally at a dose of 100 mg kg significantly inhibited the first and second phase ear swelling, and BW also inhibited the third phase response. CF only inhibited the second phase. The inhibition by BW was the most potent and almost dose-dependent at doses of 30-300 mg kg. BW also inhibited vascular permeability increase caused by passive cutaneous anaphylaxis and histamine, and ear swelling caused by tumor necrosis factor-alpha. In contrast, BW apparently potentiated the production of interleukin-4 and interferon-gamma from anti-CD3-stimulated mouse splenocytes. These results indicate that BW derived from mycelium of P. linteus contains some constituents with anti-allergic as well as immunopotentiating properties.