An Immunomodulatory Polysaccharide-Protein Complex Isolated from the Polypore Fungus Royoporus badius.

Many wild edible polypore mushrooms have medicinal value. In this study, we investigate the potential medicinal properties of the wild polypore mushroom Royoporus badius collected from north-central British Columbia, Canada. Water extract from R. badius was found to exhibit potent immunomodulatory activity. The extract was purified using DEAE-Sephadex anion-exchange chromatography as well as Sephacryl S-500 and HPLC BioSEC5 size-exclusion chromatography, to yield a novel polysaccharide-protein complex (IMPP-Rb).IMPP-Rb has a peak maxima molecular weight (Mp) of 950 kDa. GC-MS analyses showed that IMPP-Rb is composed predominantly of glucose (49.2%), galactose (11.3%), mannose (10.8%), rhamnose (9.6%), and galacturonic acid (8.2%), with smaller amounts of xylose (5.2%), fucose (2.8%), N-acetyl glucosamine (1.8%), and arabinose (1.2%). IMPP-Rb has multiple linkages, with 4-Glcp, 4-Manp, 6-Manp, 3,4-Manp, 4-Xylp, and 2-Rhap being the most prominent. IMPP-Rb is capable of inducing many cytokines in vitro and the protein component is indispensable for its immunomodulatory activity. IMPP-Rb has potential application as an immuno-stimulatory agent with pharmaceutical value.

Isolation and characterization of an anti-proliferative polysaccharide from the North American fungus Echinodontium tinctorium.

A novel polysaccharide EtGIPL1a was purified from fruiting bodies of Echinodontium tinctorium, a fungus unique to western North America. EtGIPL1a has an estimated weight average molecular weight of 275 kDa and is composed of glucose (54.3%), galactose (19.6%), mannose (11.1%), fucose (10.3%), glucuronic acid (4%), and rhamnose (0.6%). It has multiple glycosidic linkages, with 3-Glcp (28.9%), 6-Glcp (18.3%), 3,6-Glcp (13%), 4-GlcpA (9.2%), 6-Galp (3.9%), 2,6-Galp (2.6%), 3-Fucp (2.5%), 6-Manp (2.4%) being the most prominent, and unsubstituted glucose (15.3%), mannose (1.3%) and fucose (0.9%) as major terminal sugars. EtGIPL1a has a backbone containing mostly 3-substituted ß-glucopyranose with 4-substituted glucopyranosyluronic acid. EtGIPL1a showed anti-proliferative activity against multiple cancer cell lines, with IC50 ranging from 50.6 to 1446 nM. Flow cytometry analyses confirmed that apoptosis induction is one mechanism for its anti-proliferative activity. EtGIPL1a should be further investigated for its potential anti-cancer activity in animal models, and for its possible utility in differentiation cancer therapy.

Structural elucidation and immuno-stimulatory activity of a novel polysaccharide containing glucuronic acid from the fungus Echinodontium tinctorium.

An immuno-stimulatory polysaccharide (EtISPFa) was purified from water extract of the fungus Echinodontium tinctorium. EtISPFa has an estimated weight average molecular weight (Mw) of 1354 kDa and is composed of glucose (66.2 %), glucuronic acid (10.1 %), mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), rhamnose (3.1 %), fucose (1.8 %), and arabinose (0.2 %). It has multiple glycosidic linkages, with 3-Glcp (19.8 %), 4-GlcpA (10.8 %), 6-Glcp (10.7 %), and 3,6-Glcp (8.7 %) being the most prominent. NMR analysis showed that EtISPFa has a backbone containing mostly of 3-substituted ß-glucopyranose with 4-substituted glucopyranosyluronic acid. Short side chains consisting of an average of two ß-glycopyranose residues, connected through 1→6 linkages, are attached to the 6-position of about every 4th or 5th backbone glucose residue. EtISPFa is a novel glucuronic acid-containing ß-glucan capable of significantly inducing the production of cytokines IL-17, IL-16, MIP-2, G-CSF,GM-CSF, LIF, MIP-1α, MIP-1ß, and RANTES in vitro. EtISPFa should be further explored for its immuno-stimulatory activity in vivo.

Grifolin, neogrifolin and confluentin from the terricolous polypore Albatrellus flettii suppress KRAS expression in human colon cancer cells.

In our search for bioactive mushrooms native to British Columbia, we determined that the ethanol extracts from fruiting bodies of the terrestrial polypore Albatrellus flettii had potent anti-cell viability activity. Using bioassay-guided fractionation, mass spectrometry and nuclear magnetic resonance, we successfully isolated three known compounds (grifolin, neogrifolin and confluentin). These compounds represent the major anti-cell viability components from the ethanol extracts of A. flettii. We also identified a novel biological activity for these compounds, specifically in down-regulating KRAS expression in two human colon cancer cell lines. Relatively little is known about the anti-cell viability activity and mechanism of action of confluentin. For the first time, we show the ability of confluentin to induce apoptosis and arrest the cell cycle at the G2/M phase in SW480 human colon cancer cells. The oncogenic insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) has been previously shown to regulate KRAS mRNA expression in colon cancer cells, possibly through its ability to bind to the KRAS transcript. Using a fluorescence polarization assay, we show that confluentin dose-dependently inhibits the physical interaction between KRAS RNA and full-length IMP1. The inhibition also occurs with truncated IMP1 containing the KH1 to KH4 domain (KH1to4 IMP1), but not with the di-domain KH3 and KH4 (KH3&4 IMP1). In addition, unlike the control antibiotic neomycin, grifolin, neogrifolin and confluentin do not bind to KRAS RNA. These results suggest that confluentin inhibits IMP1-KRAS RNA interaction by binding to the KH1&2 di-domains of IMP1. Since the molecular interaction between IMP1 and its target RNAs is a pre-requisite for the oncogenic function of IMP1, confluentin should be further explored as a potential inhibitor of IMP1 in vivo.

Antiproliferative, Immunostimulatory, and Anti-Inflammatory Activities of Extracts Derived from Mushrooms Collected in Haida Gwaii, British Columbia (Canada).

Wild mushrooms, while largely explored for their ecological significance, have not been systematically studied for their medicinal properties. This is the first report of biological activities of mushrooms from Haida Gwaii, British Columbia, Canada. The 17 mushroom species in this study were collected from multiple locations on Haida Gwaii and were screened for antiproliferative, immunostimulatory, and anti-inflammatory activities. Prior to screening, mushrooms were genetically identified and then sequentially fractionated into four crude extracts using 80% ethanol, 50% methanol, water, and 5% sodium hydroxide. We report here the strong antiproliferative and antiinflammatory activities of Amanita augusta, Phellodon atratus, Guepinia helvelloides, Chroogomphus tomentosus, Laetiporus conifericola, and Inocybe sp. In addition, A. augusta, G. helvelloides, and Inocybe sp. showed potent immunostimulatory activity. Two other species (Ganoderma tsugae and Pleurotus ostreatus) displayed strong immunostimulatory activity consistent with previous reports by others, suggesting that similar constituents are present in the same species from Haida Gwaii. For nine species (Russula paludosa, Hygrophoropsis aurantiaca, Tricholomopsis rutilans, Tyromyces chioneus, Hydnum repandum, Hypholoma fasciculare, Clavulina cinerea, P. ostreatus and Ramaria cystidiophora), we describe antiproliferative, immunostimulatory, and/or anti-inflammatory activities that have never been reported before. The new findings serve as a platform for future investigations into the potentially novel bioactive constituents of these mushrooms, as well as an incentive to further study a wider array of wild mushrooms for medicinal properties.

Anti-Inflammatory Activity of the Wild Mushroom, Echinodontium tinctorium, in RAW264.7 Macrophage Cells and Mouse Microcirculation.

The aim of this study was to investigate the anti-inflammatory activity of a previously un-studied wild mushroom, Echinodontium tinctorium, collected from the forests of north-central British Columbia. The lipopolysaccharide (LPS)-induced RAW264.7 macrophage model was used to study the in vitro anti-inflammatory activity. The crude alkaline extract demonstrated potent anti-inflammatory activity, and was further purified using a "bio-activity-guided-purification" approach. The size-exclusion and ion-exchange chromatography yielded a water-soluble anti-inflammatory polysaccharide (AIPetinc). AIPetinc has an average molecular weight of 5 kDa, and is a heteroglucan composed of mainly glucose (88.6%) with a small amount of galactose (4.0%), mannose (4.4%), fucose (0.7%), and xylose (2.3%). In in vivo settings, AIPetinc restored the histamine-induced inflammatory event in mouse gluteus maximus muscle, thus confirming its anti-inflammatory activity in an animal model. This study constitutes the first report on the bioactivity of Echinodontium tinctorium, and highlights the potential medicinal benefits of fungi from the wild forests of northern British Columbia. Furthermore, it also reiterates the need to explore natural resources for alternative treatment to modern world diseases.

Inonotus obliquus attenuates histamine-induced microvascular inflammation.

Cell-to-cell communication is a key element of microvascular blood flow control, including rapidly carrying signals through the vascular endothelium in response to local stimuli. This cell-to-cell communication is negatively impacted during inflammation through the disruption of junctional integrity. Such disruption is associated with promoting the onset of cardiovascular diseases as a result of altered microvascular blood flow regulation. Therefore, understanding the mechanisms how inflammation drives microvascular dysfunction and compounds that mitigate such inflammation and dysfunction are of great interest for development. As such we aimed to investigate extracts of mushrooms as potential novel compounds. Using intravital microscopy, the medicinal mushroom, Inonotus obliquus was observed, to attenuate histamine-induced inflammation conducted vasodilation in second-order arterioles in the gluteus maximus muscle of C57BL/6 mice. Mast cell activation by C48/80 similarly disrupted endothelial junctions and conducted vasodilation but only histamine was blocked by the histamine antagonist, pyrilamine not C48/80 suggesting the importance of mast cell activation. Data presented here supports that histamine induced inflammation is a major disruptor of junctional integrity, and highlights the important anti-inflammatory properties of Inonotus obliquus focusing future assessment of mast cells as putative target for Inonotus obliquus.

Anti-proliferative activity of a purified polysaccharide isolated from the basidiomycete fungus Paxillus involutus.

A growth-inhibitory polysaccharide (GIPinv) was purified using size-exclusion and ion-exchange chromatography from the fourth sodium hydroxide extraction step of a fungus found in British Columbia. The fungus was genetically identified as a member of the Paxillus involutus complex. GIPinv has an average molecular weight of 229kDa and is a heteroglycan composed of glucose (65.9%), galactose (20.8%), mannose (7.8%), fucose (3.2%) and xylose (2.3%). GC-MS methylation analysis suggests that GIPinv has mixed linkages in the backbone containing (1→6)-Gal (25.5%), (1→4)-Glc (18.3%), (1→6)-Glc (8.3%), (1→3)-Glc (5.3%) and (1→2)-Xyl (4.5%). GIPinv has branching points at (1→2, 6)-Man (8.6%) and (1→3, 6)-Man (4.9%) having unsubstituted fucose (8.3%) and glucose (16.3%) as terminal sugars. GIPinv had growth-inhibitory activity against several cancer cell lines and triggered apoptosis. GIPinv should be further explored as a potential anti-cancer agent and a unique polysaccharide.

Growth-Inhibitory and Immunomodulatory Activities of Wild Mushrooms from North-Central British Columbia (Canada).

Wild mushrooms, especially from North America, have not been systematically explored for their medicinal properties. Here we report screening for the growth-inhibitory and immunomodulatory activities of 12 species collected from multiple locations in north-central British Columbia, Canada. Mushrooms were characterized using morphology and DNA sequencing, followed by chemical extraction into 4 fractions using 80% ethanol, 50% methanol, water, and 5% sodium hydroxide. Growth-inhibitory, immunostimulatory, and anti-inflammatory activities of 5 mushrooms (Leucocybe connata, Trichaptum abietinum, Hydnellum sp., Gyromitra esculenta, and Hericium coralloides) are reported here, to our knowledge for the first time. Growth-inhibitory effects were assessed using the cytotoxic MTT assay. Immunostimulatory activity was assessed by tumor necrosis factor-α production in Raw 264.7 macrophages, whereas anti-inflammatory activity was assessed based on the inhibition of lipopolysaccharide-induced tumor necrosis factor-α production. The ethanol and aqueous extracts of Hydnellum sp. were potent growth inhibitors, with a half-maximal inhibitory concentration of 0.6 mg/mL. All 5 fungi displayed strong immunostimulatory activity, whereas only L. connata and T. abietinum showed strong anti-inflammatory activity. For the 7 other fungi investigated, which included well-known medicinal species such as Inonotus obliquus, Phellinus igniarius, and Ganoderma applanatum, the remarkable similarities in the biological activities reported here, and by others for specimens collected elsewhere, suggest that mushrooms can produce similar metabolites regardless of their habitat or ecosystem. This is to our knowledge the first study to explore wild mushrooms from British Columbia for biological activities that are relevant to cancer, and the results provide an initial framework for the selection of mushroom species with the potential for discovery of novel anticancer compounds.

Archaeal ammonia oxidizers respond to soil factors at smaller spatial scales than the overall archaeal community does in a high Arctic polar oasis.

Archaea are ubiquitous and highly abundant in Arctic soils. Because of their oligotrophic nature, archaea play an important role in biogeochemical processes in nutrient-limited Arctic soils. With the existing knowledge of high archaeal abundance and functional potential in Arctic soils, this study employed terminal restriction fragment length polymorphism (t-RFLP) profiling and geostatistical analysis to explore spatial dependency and edaphic determinants of the overall archaeal (ARC) and ammonia-oxidizing archaeal (AOA) communities in a high Arctic polar oasis soil. ARC communities were spatially dependent at the 2-5 m scale (P < 0.05), whereas AOA communities were dependent at the ∼1 m scale (P < 0.0001). Soil moisture, pH, and total carbon content were key edaphic factors driving both the ARC and AOA community structure. However, AOA evenness had simultaneous correlations with dissolved organic nitrogen and mineral nitrogen, indicating a possible niche differentiation for AOA in which dry mineral and wet organic soil microsites support different AOA genotypes. Richness, evenness, and diversity indices of both ARC and AOA communities showed high spatial dependency along the landscape and resembled scaling of edaphic factors. The spatial link between archaeal community structure and soil resources found in this study has implications for predictive understanding of archaea-driven processes in polar oases.

Host and habitat filtering in seedling root-associated fungal communities: taxonomic and functional diversity are altered in 'novel' soils.

Climatic and land use changes have significant consequences for the distribution of tree species, both through natural dispersal processes and following management prescriptions. Responses to these changes will be expressed most strongly in seedlings near current species range boundaries. In northern temperate forest ecosystems, where changes are already being observed, ectomycorrhizal fungi contribute significantly to successful tree establishment. We hypothesised that communities of fungal symbionts might therefore play a role in facilitating, or limiting, host seedling range expansion. To test this hypothesis, ectomycorrhizal communities of interior Douglas-fir and interior lodgepole pine seedlings were analysed in a common greenhouse environment following growth in five soils collected along an ecosystem gradient. Currently, Douglas-fir's natural distribution encompasses three of the five soils, whereas lodgepole pine's extends much further north. Host filtering was evident amongst the 29 fungal species encountered: 7 were shared, 9 exclusive to Douglas-fir and 13 exclusive to lodgepole pine. Seedlings of both host species formed symbioses with each soil fungal community, thus Douglas-fir did so even where those soils came from outside its current distribution. However, these latter communities displayed significant taxonomic and functional differences to those found within the host distribution, indicative of habitat filtering. In contrast, lodgepole pine fungal communities displayed high functional similarity across the soil gradient. Taxonomic and/or functional shifts in Douglas-fir fungal communities may prove ecologically significant during the predicted northward migration of this species; especially in combination with changes in climate and management operations, such as seed transfer across geographical regions for forestry purposes.

Site properties have a stronger influence than fire severity on ectomycorrhizal fungi and associated N-cycling bacteria in regenerating post-beetle-killed lodgepole pine forests.

Following a pine beetle epidemic in British Columbia, Canada, we investigated the effect of fire severity on rhizosphere soil chemistry and ectomycorrhizal fungi (ECM) and associated denitrifying and nitrogen (N)-fixing bacteria in the root systems of regenerating lodgepole pine seedlings at two site types (wet and dry) and three fire severities (low, moderate, and high). The site type was found to have a much larger impact on all measurements than fire severity. Wet and dry sites differed significantly for almost all soil properties measured, with higher values identified from wet types, except for pH and percent sand that were greater on dry sites. Fire severity caused few changes in soil chemical status. Generally, bacterial communities differed little, whereas ECM morphotype analysis revealed ectomycorrhizal diversity was lower on dry sites, with a corresponding division in community structure between wet and dry sites. Molecular profiling of the fungal ITS region confirmed these results, with a clear difference in community structure seen between wet and dry sites. The ability of ECM fungi to colonize seedlings growing in both wet and dry soils may positively contribute to subsequent regeneration. We conclude that despite consecutive landscape disturbances (mountain pine beetle infestation followed by wildfire), the "signature" of moisture on chemistry and ECM community structure remained pronounced.

Long-term experimental warming alters nitrogen-cycling communities but site factors remain the primary drivers of community structure in high arctic tundra soils.

Arctic air temperatures are expected to rise significantly over the next century. Experimental warming of arctic tundra has been shown to increase plant productivity and cause community shifts and may also alter microbial community structure. Hence, the objective of this study was to determine whether experimental warming caused shifts in soil microbial communities by measuring changes in the frequency, relative abundance and/or richness of nosZ and nifH genotypes. Five sites at a high arctic coastal lowland were subjected to a 13-year warming experiment using open-top chambers (OTCs). Sites differed by dominant plant community, soil parent material and/or moisture regimen. Six soil cores were collected from each of four replicate OTC and ambient plots at each site and subdivided into upper and lower samples. Differences in frequency and relative abundance of terminal restriction fragments were assessed graphically by two-way cluster analysis and tested statistically with permutational multivariate analysis of variance (ANOVA). Genotypic richness was compared using factorial ANOVA. The genotype frequency, relative abundance and genotype richness of both nosZ and nifH communities differed significantly by site, and by OTC treatment and/or depth at some sites. The site that showed the most pronounced treatment effect was a wet sedge meadow, where community structure and genotype richness of both nosZ and nifH were significantly affected by warming. Although warming was an important factor affecting these communities at some sites at this high arctic lowland, overall, site factors were the main determinants of community structure.

The effect of experimental warming on the root-associated fungal community of Salix arctica.

The effect of experimental warming on the root-associated fungal community of arctic willow (Salix arctica) was studied in three distinct habitats at a tundra site in the Canadian High Arctic. Plots were passively warmed for 5-7 years using open-top chambers and compared to control plots at ambient temperature. Fungal communities were assessed using terminal restriction fragment length polymorphisms. We found the following: (1) the root-associated fungal community in these high arctic tundra habitats is highly diverse; (2) site and soil characteristics are the most important drivers of community structure and (3) warming increased the density of different genotypes on individual root sections but has not (yet) affected the composition, richness or evenness of the community. The change in genotype density in the warmed plots was associated with an increase in PCR amplification efficiency, suggesting that increased C allocation belowground is increasing the overall biomass of the fungal community.

Molecular diversity of nifH genes from bacteria associated with high arctic dwarf shrubs.

Biological nitrogen fixation is the primary source of new N in terrestrial arctic ecosystems and is fundamental to the long-term productivity of arctic plant communities. Still, relatively little is known about the nitrogen-fixing microbes that inhabit the soils of many dominant vegetation types. Our objective was to determine which diazotrophs are associated with three common, woody, perennial plants in an arctic glacial lowland. Dryas integrifolia, Salix arctica, and Cassiope tetragona plants in soil were collected at Alexandra Fiord, Ellesmere Island, Canada. DNA was extracted from soil and root samples and a 383-bp fragment of the nifH gene amplified by the polymerase chain reaction. Cloned genotypes were screened for similarity by restriction fragment length polymorphism (RFLP) analysis. Nine primary RFLP phylotypes were identified and 42 representative genotypes selected for sequencing. Majority of sequences (33) were type I nitrogenases, whereas the remaining sequences belonged to the divergent, homologous, type IV group. Within the type I nitrogenases, nifH genes from posited members of the Firmicutes were most abundant, and occurred in root and soil samples from all three plant species. nifH genes from posited Pseudomonads were found to be more closely associated with C. tetragona, whereas nifH genes from putative alpha-Proteobacteria were more commonly associated with D. integrifolia and S. arctica. In addition, 12 clones likely representing a unique clade within the type I nitrogenases were identified. To our knowledge, this study is the first to report on the nifH diversity of arctic plant-associated soil microbes.

Impacts of warming and fertilization on nitrogen-fixing microbial communities in the Canadian High Arctic.

The impacts of simulated climate change (warming and fertilization treatments) on diazotroph community structure and activity were investigated at Alexandra Fiord, Ellesmere Island, Canada. Open Top Chambers, which increased growing season temperatures by 1-3 degrees C, were randomly placed in a dwarf-shrub and cushion-plant dominated mesic tundra site in 1995. In 2000 and 2001 20N:20P2O5:20K2O fertilizer was applied at a rate of 5 gm(-2) year(-1). Estimates of nitrogen fixation rates were made in the field by acetylene reduction assays (ARA). Higher rates of N fixation were observed 19-35 days post-fertilization but were otherwise unaffected by treatments. However, moss cover was significantly positively associated with ARA rate. NifH gene variants were amplified from bulk soil DNA and analyzed by terminal restriction fragment length polymorphism analysis. Non-metric multidimensional scaling was used to ordinate treatment plots in nifH genotype space. NifH gene communities were more strongly structured by the warming treatment late in the growing season, suggesting that an annual succession in diazotroph community composition occurs.

Bacterial diversity associated with subalpine fir (Abies lasiocarpa) ectomycorrhizae following wildfire and salvage-logging in central British Columbia.

To assess the effect of fire and salvage logging on the diversity of mycorrhizal-bacterial communities, bacteria associated with Cenococcum, Thelephora, Tomentella, Russulaceae, and E-strain ectomycorrhizae (ECM) of Abies lasiocarpa seedlings were characterized using two approaches. First, bacteria were isolated and characterized by Biolog, gas chromatography fatty acid methyl ester (GC-FAME), and amplified 16S rDNA restriction analysis (ARDRA). The bacterial communities retrieved from ECM from both sites were dominated by Proteobacteria (groups gamma and beta). Pseudomonas was the most common genus isolated, followed by Variovorax, Burkholderia, and Xanthomonas. Gram-positive isolates (mostly high-G+C Gram-positive bacteria) were more frequently retrieved on the burned-salvaged site, many commonly associated with the two ascomycete ECM, Cenococcum and E-strain. Pseudomonas species were retrieved more frequently from Thelephora. Although actinomycetes were isolated from all sites, almost no actinomycetes or other Gram-positive bacteria were isolated from either Thelephora or Tomentella. Second, amplified 16S rRNA gene sequences were amplified directly from root tips and then cloned into the plasmid vector pAMP1, followed by restriction analysis. This technique distinguished more genotypes than isolates retrieved by culturing methods, but generally, results were similar in that the largest proportion of the bacteria were putatively Gram-negative; putative Gram-positive bacteria were fewer and most were from the burned-salvaged site. Direct cloning resulted in many patterns that did not match any identified isolates, suggesting that a large proportion of clones were unique or not culturable by the methods used. Analysis for both protocols showed no significant difference in bacterial diversity between the burned-salvaged and unburned sites.