Isolation, structural elucidation, and biological activity of a novel isocoumarin from the dark septate endophytic fungus Phialocephala fortinii.

A novel isocoumarin was isolated from the mycelia of the dark septate endophytic fungus Phialocephala fortinii. The chemical structure was determined to be 8-hydroxy-6-methoxy-3,7-dimethyl-1H-2-benzopyran-1-one based on mass spectrometry, 1H-nuclear magnetic resonance (NMR), and 13C-NMR spectroscopic analyses, including 2D-NMR experiments. The isolated compound inhibited root growth of Arabidopsis thaliana, suggesting its potential as a plant growth regulator.

Effects of hemicelluloses on dehydrogenative polymerization of monolignols with cationic cell wall-bound peroxidase.

To elucidate the influence of polysaccharides on hardwood lignification, dehydrogenative polymerization of monolignols, coniferyl alcohol (CA) and sinapyl alcohol (SA), was attempted with recombinant cationic cell wall-bound peroxidase (rCWPO-C) and horseradish peroxidase (HRP) in measurement cells of a quartz crystal microbalance with dissipation (QCM-D). Hardwood cellulose nanofibers were anchored; hemicelluloses, xylan, partially acetylated xylan (AcXY), galactoglucomannan, and xyloglucan, and the enzymes were subsequently adsorbed onto the QCM-D sensor surface, enabling fabrication of artificial polysaccharide matrices. The largest amount of rCWPO-C is found to be adsorbed onto AcXY among all the polysaccharides, which affords the largest amount and size of spherical dehydrogenation polymers (DHPs) from both CA and SA. In contrast, no DHP and a small amount of DHPs are formed from SA and CA, respectively, by HRP catalysis in all of the polysaccharide matrices. This study demonstrates important functions of a real tree-derived peroxidase, rCWPO-C, and AcXY for hardwood lignification.

Phylogenetic placements and cultural characteristics of Tuber species isolated from ectomycorrhizas.

Pure cultures of Tuber were isolated from ectomycorrhizal root tips in Abies sachalinensis plantations in Hokkaido, Japan. Their phylogenetic relationships as well as vegetative hyphal characteristics on culture media were reported. Phylogenetic analysis based on the internal transcribed spacer within ribosomal DNA settled well-supported eight lineages within Puberulum, Latisporum, and Maculatum clades in Tuber. Three and one lineages were grouped with undescribed species of Puberulum clade in Japan and that of the Latisporum group in China, respectively. Two lineages were closely associated to but distinct from an undescribed species of Puberulum clade in Japan. One lineage did not group with any sequences in the International Nucleotide Sequence Database (INSD), proposing a new taxon in the Latisporum group. One lineage was grouped with T. foetidum in Maculatum clade. All strains in each lineage displayed yellowish white, thin, filamentous colonies on Melin-Norkrans agar medium. Various differences in morphological characteristics of hyphae on pure cultures of various strains were noted, but they were frequently uncommon among strains of the same taxa. Isolation from ectomycorrhizal root tips can be among the effective ways to acquire pure cultures of Tuber strains.

Effects of simulated nitrogen deposition on ectomycorrhizae community structure in hybrid larch and its parents grown in volcanic ash soil: The role of phosphorous.

With the rapid industrial development and modern agricultural practices, increasing nitrogen (N) deposition can cause nutrient imbalance in immature volcanic ash soil commonly found in Japan. Larch species, widely distributed in northeast Eurasia, are associated with ectomycorrhizal (ECM) fungi which play a critical role in nutrient acquisition for their hosts. In this study, we investigated species richness and diversity of ECM fungi associated with a hybrid larch (F1) and its parents, Dahurian larch (Larix gmelinii var. japonica) and Japanese larch (L. kaempferi), under simulated N deposition (0 and 100kgha-1yr-1) with/without phosphorous (P) (0 and 50kgha-1yr-1). Seedlings planted in immature volcanic ash with low nutrient availability were subjected to the N and P treatments for fifteen months. We found that response of ECM community structure to the increased nutrient availability depended on host genotypes. Nutrient addition significantly affected ECM structure in Japanese larch, but no such significant effect was found for Dahurian larch. Effects of the nutrient addition to ECM fungal community in F1 were intermediate. F1 was tolerant to high N loading, which was due to consistent, relatively high association with Suillus sp. and Hebeloma sp. F1 showed heterosis in relative biomass, which was most apparent under high N treatments. This co-variation of ECM fungal community structure and F1 biomass in response to N loading suggest that ECM community structure might play an important role in host growth. The present findings indicate effects of N deposition on ECM fungal community structure can depend on larch species, thus it is challenging to predict general trends.

Optimization of simultaneous saccharification and fermentation conditions with amphipathic lignin derivatives for concentrated bioethanol production.

Amphipathic lignin derivatives (A-LDs) prepared from the black liquor of soda pulping of Japanese cedar are strong accelerators for bioethanol production under a fed-batch simultaneous enzymatic saccharification and fermentation (SSF) process. To improve the bioethanol production concentration, conditions such as reaction temperature, stirring program, and A-LDs loadings were optimized in both small scale and large scale fed-batch SSF. The fed-batch SSF in the presence of 3.0g/L A-LDs at 38°C gave the maximum ethanol production and a high enzyme recovery rate. Furthermore, a jar-fermenter equipped with a powerful mechanical stirrer was designed for 1.5L-scale fed-batch SSF to achieve rigorous mixing during high substrate loading. Finally, the 1.5L fed-batch SSF with a substrate loading of 30% (w/v) produced a high ethanol concentration of 87.9g/L in the presence of A-LDs under optimized conditions.

Dehydrogenative polymerization of coniferyl alcohol in artificial polysaccharides matrices: effects of xylan on the polymerization.

To elucidate the influence of wood polysaccharide components on lignin formation in vitro, models for polysaccharide matrix in wood secondary cell wall were fabricated from two types of bacterial cellulosic films, flat film (FBC) and honeycomb-patterned film (HPBC), as basic frameworks by depositing xylan onto the films. An endwise type of dehydrogenative polymerization, "Zutropfverfahren", of coniferyl alcohol was attempted in the films with/without xylan. The resultant dehydrogenation polymer (DHP) was generated inside and outside xylan-deposited films, whereas DHP was deposited only outside the films without xylan. The amount of the generated DHP in the xylan-deposited films was larger than that in the films without xylan. The frequency of 8-O-4' interunitary linkage in DHP was also increased by the xylan deposition. These results suggest that xylan acts as a scaffold for DHP deposition in polysaccharides matrix and as a structure regulator for the formation of the 8-O-4' linkage. In addition, mechanical properties, i.e., tensile strength and modulus of elasticity (MOE), of both cellulosic films were found to be augmented by the deposition of xylan and DHP. Especially, DHP deposition remarkably enhanced MOE. Such effects of xylan on DHP formation and augmentation of mechanical strength were clearly observed for HPBC, revealing that HPBC is a promising framework model to investigate wood cell wall formation in vitro.

The analgesic efficacy of ultrasound-guided transversus abdominis plane block with mid-axillary approach after gynecologic laparoscopic surgery: A randomized controlled trial.

BACKGROUND AND AIMS: The transversus abdominis plane (TAP) block is widely used in clinical practice as a part of the multimodal analgesic regimen after abdominal surgery. The analgesic efficacy of ultrasound (US)-guided TAP block with the mid-axillary approach was investigated in patients undergoing laparoscopic gynecologic surgery in a randomized controlled clinical trial. MATERIALS AND METHODS: Adult patients (n = 119) undergoing laparoscopic gynecologic surgery were randomized to undergo either TAP block with ropivacaine (Group A, n = 60) or that with saline (Group B, n = 59), in a blinded manner. Following general anesthesia, TAP block was performed bilaterally by infusion of either 20-mL 0.375% ropivacaine/one side in Group A or 20-mL saline/one side in Group B, under US guidance with a mid-axillary approach. Patient-controlled analgesia (PCA) was performed postoperatively with tramadol. The analgesic effect was postoperatively evaluated using a four-grade pain score and the prince Henry pain scale (PHS) at 0, 6, 12, and 24 h. Postoperative tramadol PCA consumption and vomiting/nausea were recorded. Statistical analyses were performed using the Mann-Whitney U-test or Fisher's exact probability test. A P < 0.05 was considered significant. RESULTS: There was no statistically significant difference in background characteristics. The dose of remifentanil was significantly higher in Group B (P = 0.01). The pain score (P = 0.02) and PHS (P = 0.01) were significantly lower in Group A at 0 h. Tramadol PCA consumption in the period of 0-6 h (P = 0.01) and postoperative nausea (P = 0.04) were significantly less in Group A. CONCLUSION: Postoperative pain/nausea and PCA consumption were significantly lower in patients with TAP block in the early postoperative stage. TAP block with a mid-axillary approach holds considerable promise as a part of a balanced postoperative analgesic regimen following laparoscopic gynecologic surgery.

Ectomycorrhizal colonization and growth of the hybrid larch F1 under elevated CO2 and O3.

We studied the colonization of ectomycorrhizal fungi and species abundance of a hybrid larch (F1) under elevated CO2 and O3. Two-year-old seedlings were planted in an Open-Top-Chamber system with treatments: Control (O3 < 6 nmol/mol), O3 (60 nmol/mol), CO2 (600 µmol/mol), and CO2 + O3. After two growing seasons, ectomycorrhiza (ECM) colonization and root biomass increased under elevated CO2. Additionally, O3 impaired ECM colonization and species richness, and reduced stem biomass. However, there was no clear inhibition of photosynthetic capacity by O3. Concentrations of Al, Fe, Mo, and P in needles were reduced by O3, while K and Mg in the roots increased. This might explain the distinct change in ECM colonization rate and diversity. No effects of combined fumigation were observed in any parameters except the P concentration in needles. The tolerance of F1 to O3 might potentially be related to a shift in ECM community structure.

Amphipathic lignin derivatives to accelerate simultaneous saccharification and fermentation of unbleached softwood pulp for bioethanol production.

Amphipathic lignin derivatives (A-LDs) were already demonstrated to improve enzymatic saccharification of lignocellulose. Based on this knowledge, two kinds of A-LDs prepared from black liquor of soda pulping of Japanese cedar were applied to a fed-batch simultaneous saccharification and fermentation (SSF) process for unbleached soda pulp of Japanese cedar to produce bioethanol. Both lignin derivatives slightly accelerated yeast fermentation of glucose but not inhibited it. In addition, ethanol yields based on the theoretical maximum ethanol production in the fed-batch SSF process was increased from 49% without A-LDs to 64% in the presence of A-LDs.

Biobleaching of Acacia kraft pulp with extracellular enzymes secreted by Irpex lacteus KB-1.1 and Lentinus tigrinus LP-7 using low-cost media.

The white-rot fungi Irpex lacteus KB-1.1 and Lentinus tigrinus LP-7 have been shown in previous studies to have high biobleaching activity in vivo. The aim of this study was to investigate the activities and stabilities of extracellular enzymes, prepared from I. lacteus and L. tigrinus culture grown in three types of economical media of agricultural and forestry wastes, for biobleaching of Acacia oxygen-delignified kraft pulp using kappa number reduction as an indicator of delignification. After 3 days of incubation, the extracellular enzymes preparations from I. lacteus and L. tigrinus cultures in media of Acacia mangium wood powder supplemented with rice bran and addition 1 % glucose (WRBG), resulted in significant decrease of 4.4 and 6.7 %, respectively. A slightly higher kappa number reduction (7.4 %) was achieved with the combine extracellular enzymes from I. lacteus and L. tigrinus. One of the strategies for reducing the cost of enzyme production for treatment processes in the pulp and paper industry is the utilization of agricultural and forestry waste. Thus, WRBG has potential as a culture medium for producing stable lignolytic enzymes simply and economically.

Morphological and genetic characteristics of the entomopathogenic fungus Ophiocordyceps nutans and its host insects.

We examined the morphology, genetic variation, and insect host species of the fungus Ophiocordyceps nutans. Fifty-two fruit bodies collected in Hokkaido, Japan, were grouped by host species, and 19 samples were analysed. The ranges of the lengths and breadths of all fruit body parts were similar among host groups. In the genetic analysis, the 5.8S rDNA region showed completely identical sequences, although differences of up to six nucleotides were recognized in the ITS regions. The distance values between our samples using the Kimura two-parameter model were lower than 0.007. Thus, the O. nutans examined here were concluded to form a closely related group with little detectable variability that parasitized nine hemipteran species.

Mycorrhizal associations in woody plant species at the Mt. Usu volcano, Japan.

We investigated the association between ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) fungi and pioneer woody plant species in areas devastated by the eruption of Mt. Usu, Japan, in 2000. We observed eight woody plant species at the research site, most of which were associated with ECM and/or AM fungi. In particular, dominant woody plant species Populus maximowiczii, Salix hultenii var. angustifolia and Salix sachalinensis were consistently associated with ECM fungi and erratically associated with AM fungi. We found one to six morphotypes in the roots of each ECM host and, on average, two in the roots of each seedling, indicating low ECM fungal diversity. ECM colonization ranged from 17 to 42% of root tips. Using morphotyping and molecular analyses, 15 ECM fungi were identified. ECM fungi differed greatly between hosts. However, Laccaria amethystea, Hebeloma mesophaeum, Thelephora terrestris and other Thelephoraceae had high relative colonization, constituting the majority of the ECM colonization in the roots of each plant species. These ECM fungi may be important for the establishment of pioneer woody plant species and further revegetation at Mt. Usu volcano.

Pileus differentiation and pileus-specific protein expression in Flammulina velutipes.

Fruiting bodies of Flammulina velutipes formed under complete darkness had a poorly developed pileus on top (pinhead fruiting body), and lacked a hymenium. Upon light stimulation, the pileus immediately began to develop on the apical region of the pinhead fruiting body. Swelling of the apical region caused by cell division was observed 2 days after light treatment; at day 4, the junction fracture between the pileus and stipe, and formation of the hymenium primordia were observed; at 6 days, gills were observed. We identified a cell wall-associated protein (PSH) that was specifically induced in the pileus, but not in the stipe, following light treatment of the pinhead fruiting body. Cloning and sequence analysis of the gene encoding PSH (psh) revealed a motif in the C-terminal region of the predicted amino acid sequence that was similar to hydrophobin. The level of transcription of psh was low in the stipe, but it was expressed at a high level in the pileus of the normal fruiting body. Transcription was also low in pinhead fruiting bodies, but increased after light treatment. These results indicate that psh is specifically expressed during pileus differentiation induced by light stimulation.

Arbuscular mycorrhizal fungi increased early growth of two nontimber forest product species Dyera polyphylla and Aquilaria filaria under greenhouse conditions.

Nontimber forest products (NTFPs) represent an important source of income to millions of people in tropical forest regions, but some NTFP species have decreased in number and become endangered due to overexploitation. There is increasing concern that the planting stocks of Dyera polyphylla and Aquilaria filaria are not sufficient to sustain the yield of NTFPs and promote forest conservation. The objective of this study was to determine the effect of two arbuscular mycorrhizal (AM) fungi, Glomus clarum and Gigaspora decipiens, on the early growth of two NTFP species, D. polyphylla and A. filaria, under greenhouse conditions. The seedlings of both species were inoculated with G. clarum or G. decipiens, or uninoculated (control) under greenhouse conditions. Percentage of AM colonization, plant growth, survival rate, and nitrogen (N) and phosphorus (P) concentrations were measured after 180 days of growth. The percentage of AM colonization of D. polyphylla and A. filaria ranged from 87 to 93% and from 22 to 39%, respectively. Colonization by G. clarum and G. decipiens increased plant height, diameter, and shoot and root dry weights. Shoot N and P concentrations of the seedlings were increased by AM colonization by as much as 70-153% and 135-360%, respectively. Survival rates were higher in the AM-colonized seedlings at 180 days after transplantation than in the control seedlings. The results suggest that AM fungi can accelerate the establishment of the planting stocks of D. polyphylla and A. filaria, thereby promoting their conservation ecologically and sustaining the production of these NTFPs economically.

A phytochemical in the edible Tamogi-take mushroom (Pleurotus cornucopiae), D-mannitol, inhibits ACE activity and lowers the blood pressure of spontaneously hypertensive rats.

D-Mannitol, one of the main phytochemicals of the edible Tamogi-take mushroom (Pleurotus cornucopiae), was found to inhibit an angiotensin I converting enzyme (ACE). The antihypertensive effect of D-mannitol and a hot water extract of Tamogi-take mushroom was demonstrated in spontaneously hypertensive rats (SHR) by oral administration.

Isolation of vegetable wasps and plant worms, Cordyceps nutans, from fruit-body tissue.

We isolated Cordyceps nutans from the stipe and abdominal tissues of fruit bodies using a surface sterilization method. Hyphal growth was observed in inocula from both the stipe and abdominal tissue. Some strains from discharged ascospores were obtained and colony characteristics were compared to the strains isolated from the tissues. Colonies of isolates from ascospores grew quite slowly. Isolates of 43 from the 52 examined fruit bodies formed colonies similar to those from ascospores. To confirm the success of isolation, we analyzed by PCR-RFLP of the ITS regions of rDNA samples from fruit bodies, isolates from fruit bodies, and isolates from ascospores. All the isolates obtained from stipe and abdominal tissues presented identical patterns. In this study, we report the first successful isolation of C. nutans from fruit-body tissue using a surface sterilization method.

[The usefulness of the processed nasal airway for fiberscope guided nasotracheal intubation].

BACKGROUND: The fiberscopy has become an essential tool for tracheal intubation. As we realized that nasal airway acts like a conduit to guide a fiberscope toward the larynx, we have devised a unique nasal airway named "Osaka airway" which is suitable for fiberoptic naso-tracheal intubation. This airway has two pre-cut line from the head to the tip which can be peeled off easily after the insertion of fiberscope. To verify this advantages, we measured the length of nares-vocal cords and assessed the fiberoptic visibility of vocal cords under the aid of nasal airway. METHODS: At the end of operation, 54 patients were extubated and nasal airway was inserted to measure the length of nares-vocal cords and to observe vocal cords and epiglottis. Then, in another series, two beginners conducted fiberoptic intubation on several cases with the aid of Osaka airway. RESULTS: The length of nares-vocal cords of male was 20.2 +/- 1.0 S.D.cm, and that of female 17.3 +/- 1.1 S.D.cm. The length correlated with the body height. In 34 patient, we could see vocal cords just below the airway tip. Two beginners could complete the intubation within 2 min in almost all cases. CONCLUSION: Osaka airway was a help for fiberoptic naso-tracheal intubation for beginners.