Lobular distribution of enhanced expression levels of heat shock proteins using in-situ hybridization in the mouse liver treated with a single administration of CCl4.

This study was conducted to visualize the lobular distribution of enhanced mRNA expression levels of heat shock proteins (HSPs) in liver samples from carbon tetra chloride (CCl4)-treated mice using in-situ hybridization (ISH). Male BALB/c mice given a single oral administration of CCl4 were euthanized 6 hours or 1 day after the administration (6 h or 1 day). Paraffin-embedded liver samples were obtained, ISH for HSPs was conducted, as well as hematoxylin-eosin staining and immunohistochemistry (IHC). At 6 h, centrilobular hepatocellular vacuolization was observed, and increased signals for Hspa1a, Hspa1b, and Grp78, which are HSPs, were noted in the centrilobular area using ISH. At 1 day, zonal hepatocellular necrosis was observed in the centrilobular area, but mRNA signal increases for HSPs were no longer observed there. Some discrepancies between ISH and IHC for HSPs were observed, and they might be partly caused by post-transcriptional gene regulation, including the ribosome quality control mechanisms. It is known that CCl4 damages centrilobular hepatocytes through metabolization by cytochrome P450, mainly located in the centrilobular region, and HSPs are induced under cellular stress. Therefore, our ISH results visualized increased mRNA expression levels of HSPs in the centrilobular hepatocytes of mice 6 hours after a single administration of CCl4 as a response to cellular stress, and it disappeared 1 day after the treatment when remarkable necrosis was observed there.

The dominance of Suillus species in ectomycorrhizal fungal communities on Larix gmelinii in a post-fire forest in the Russian Far East.

Wildfires can negatively affect ectomycorrhizal (EM) fungal communities. However, potential shifts in community structures due to wildfires have rarely been evaluated in the forests of eastern Eurasia, where surface fires are frequent. We investigated EM fungal communities in a Larix gmelinii-dominated forest that burned in 2003 in Zeya, in the Russian Far East. A total of 120 soil samples were collected from burned and adjacent unburned forest sites. The EM fungal root tips were morphotyped and internal transcribed spacer (ITS) sequences were obtained for fungal identification. We detected 147 EM fungal operational taxonomic units, and EM fungal richness was 25% lower at the burned site than at the unburned site. EM fungal composition was characterized by the occurrence of disturbance-adapted fungi (Amphinema and Wilcoxina) at the burned site and late-successional fungi (Lactarius, Russula and Cortinarius) at the unburned site. These findings suggest that the EM fungal communities did not recover to pre-fire levels 16 years after the fire. Suillus species were the dominant EM fungi on L. gmelinii, with greater richness and frequency at the burned site. Both Larix and Suillus exhibit adaptive traits to quickly colonize fire-disturbed habitats. Frequent surface fires common to eastern Eurasia are likely to play important roles in maintaining Larix forests, concomitantly with their closely associated EM fungi.

Temperature niche position and breadth of ectomycorrhizal fungi: Reduced diversity under warming predicted by a nested community structure.

Species with narrow niche breadths are assumed to be more susceptible to environmental changes than those with wide niche breadths. Although information on niche properties is necessary for predicting biological responses to environmental changes, such information is largely missing for soil microbes. In this study, we present the temperature niche positions and breadths of a functionally important group of eukaryotic soil microbes, ectomycorrhizal (EM) fungi. We compiled high-quality EM fungal sequence data from 26 forested sites in Japan (with mean annual temperatures ranging from 1.6 to 23.6°C) to create temperature niche profiles for each individual fungal species. Nested theory and a newly developed weighted-randomization null model were applied to 75 fungal operational taxonomic units (OTUs) with high occurrence records to examine potential preferences for certain temperature positions and breadths. Our analyses revealed that (a) many EM fungal OTUs were restricted to habitats with low mean annual temperatures, (b) fungal OTUs observed at colder sites exhibited narrower temperature breadths than expected by chance, (c) the composition of EM fungal OTUs exhibited a nested pattern along the temperature gradient, and (d) EM fungal richness was highest at colder sites, where the greatest degree of overlap in OTU occurrence was observed. These findings imply that future warming may limit the distribution of many EM fungal species that are currently adapted to only cold climates. This could eventually reduce EM fungal biodiversity, which is linked to forest function through symbiotic associations with trees. This study demonstrates the distribution and environmental ranges of various EM fungal species and can contribute to develop species distribution models with the aim of conserving microbes in the face of climate change.

Soil propagule banks of ectomycorrhizal fungi share many common species along an elevation gradient.

We conducted bioassay experiments to investigate the soil propagule banks of ectomycorrhizal (EM) fungi in old-growth forests along an elevation gradient and compared the elevation pattern with the composition of EM fungi on existing roots in the field. In total, 150 soil cores were collected from three forests on Mt. Ishizuchi, western Japan, and subjected to bioassays using Pinus densiflora and Betula maximowicziana. Using molecular analyses, we recorded 23 EM fungal species in the assayed propagule banks. Eight species (34.8 %) were shared across the three sites, which ranged from a warm-temperate evergreen mixed forest to a subalpine conifer forest. The elevation pattern of the assayed propagule banks differed dramatically from that of EM fungi on existing roots along the same gradient, where only a small proportion of EM fungal species (3.5 %) were shared across sites. The EM fungal species found in the assayed propagule banks included many pioneer fungal species and composition differed significantly from that on existing roots. Furthermore, only 4 of 23 species were shared between the two host species, indicating a strong effect of bioassay host identity in determining the propagule banks of EM fungi. These results imply that the assayed propagule bank is less affected by climate compared to EM fungal communities on existing roots. The dominance of disturbance-dependent fungal species in the assayed propagule banks may result in higher ecosystem resilience to disturbance even in old-growth temperate forests.

Expression of steroidogenic factor 1 and pituitary specific transcription factor 1 in rat pituitary adenomas.

The protein expressions of steroidogenic factor l (SF-l) and pituitary-specific transcription factor 1 (Pit-1) were investigated immunohistochemically for 53 spontaneous pituitary adenomas of the pars distalis from male Crl:CD(SD) rats. Luteinizing hormone (LH)-positive/prolactin (PRL)-negative and LH-negative/PRL-positive adenomas showed that the expression of SF-1 and Pit-1 was exclusively related to the immunoreactivity of LH and PRL, respectively. All double-positive adenomas (positive for both LH and PRL) were positive for Pit-1 and were supposed to be derived from PRL cells, although some of them also showed SF-1 immunoreactivity. In addition, all null cell adenomas (negative for all anterior pituitary hormones) were positive for SF-1 and negative for Pit-1, indicating that they originated from the gonadotroph cell lineage. This is the first report focusing on the application of transcription factors for the classification of rat pituitary adenomas.

Soil propagule banks of ectomycorrhizal fungi associated with Larix cajanderi above the treeline in the Siberian Arctic.

Microbial symbionts are essential for plant niche expansion into novel habitats. Dormant propagules of ectomycorrhizal (EM) fungi are thought to play an important role in seedling establishment in invasion fronts; however, propagule bank communities above the treeline are poorly understood in the Eurasian Arctic, where treelines are expected to advance under rapid climate change. To investigate the availability of EM fungal propagules, we collected 100 soil samples from Arctic tundra sites and applied bioassay experiments using Larix cajanderi as bait seedlings. We detected 11 EM fungal operational taxonomic units (OTUs) by obtaining entire ITS regions. Suillus clintonianus was the most frequently observed OTU, followed by Cenococcum geophilum and Sebacinales OTU1. Three Suillus and one Rhizopogon species were detected in the bioassay seedlings, indicating the availability of Larix-specific suilloid spores at least 30 km from the contemporary treeline. Spores of S. clintonianus and S. spectabilis remained infective after preservation for 14 mo and heat treatment at 60 °C, implying the durability of the spores. Long-distance dispersal capability and spore resistance to adverse conditions may represent ecological strategies employed by suilloid fungi to quickly associate with emerging seedlings of compatible hosts in treeless habitats.

The abdominal skin of female Sprague-Dawley rats is more sensitive than the back skin to drug-induced phototoxicity.

In vivo phototoxicity studies are important to predict drug-induced phototoxicity in humans; however, a standard methodology has not established. To determine differences in sensitivity to drug-induced phototoxicity among various skin sites, we evaluated phototoxic reactions in the back and abdominal skin of female Sprague-Dawley rats orally dosed with phototoxic drugs (pirfenidone, 8-methoxysoraren, doxycycline, and lomefloxacin) or a non-phototoxic drug (gatifloxacin) followed by solar-simulated light irradiation comprising 18J/cm2 ultraviolet A. Tissue reactions were evaluated by macroscopic and microscopic examination and immunohistochemistry for γ-H2AX, and tissue concentrations of pirfenidone, doxycycline, and lomefloxacin were measured by tandem mass spectrometry. In addition, the thicknesses of the skin layers at both sites were measured in drug-naïve rats. The abdominal skin showed more severe reactions to all phototoxic drugs than the back skin, whereas the minimal erythema dose in drug-naïve rats and skin concentrations of each drug were comparable between the sites. Furthermore, histopathological lesions and γ-H2AX-positive cells in the abdominal skin were detected in deeper layers than in the back skin. The stratum corneum and dermis in the abdominal skin were significantly thinner than in the back skin, indicating a difference in the depth of light penetration and potentially contributing to the site differences observed in sensitivity to phototoxicity. Gatifloxacin did not induce any phototoxic reactions at either site. In conclusion, the abdominal skin is more sensitive to drug-induced phototoxicity than the back skin and may represent a preferable site for irradiation in this rat phototoxicity model.

Strong effect of climate on ectomycorrhizal fungal composition: evidence from range overlap between two mountains.

Separating the effects of environmental factors and spatial distance on microbial composition is difficult when these factors covary. We examined the composition of ectomycorrhizal (EM) fungi along elevation gradients on geographically distant mountains to clarify the effect of climate at the regional scale. Soil cores were collected from various forest types along an elevation gradient in southwestern Japan. Fungal species were identified by the internal transcribed spacer regions of the rDNA using direct sequencing. The occurrence of fungal species in this study was compared with a previous study conducted on a mountain separated by ∼550 km. In total, we recorded 454 EM fungi from 330 of 350 soil cores. Forty-seven fungal species (∼20% of the total excluding singletons) were shared between two mountains, mostly between similar forest types on both mountains. Variation partitioning in redundancy analysis revealed that climate explained the largest variance in EM fungal composition. The similarity of forest tree composition, which is usually determined by climatic conditions, was positively correlated with the similarity of the EM fungal composition. However, the lack of large host effects implied that communities of forest trees and EM fungi may be determined independently by climate. Our data provide important insights that host plants and mutualistic fungi may respond to climate change idiosyncratically, potentially altering carbon and nutrient cycles in relation to the plant-fungus associations.

The mid-domain effect in ectomycorrhizal fungi: range overlap along an elevation gradient on Mount Fuji, Japan.

Mid-domain effect (MDE) models predict that the random placement of species' ranges within a bounded geographical area leads to increased range overlap and species richness in the center of the bounded area. These models are frequently applied to study species-richness patterns of macroorganisms, but the MDE in relation to microorganisms is poorly understood. In this study, we examined the characteristics of the MDE in richness patterns of ectomycorrhizal (EM) fungi, an ecologically important group of soil symbionts. We conducted intensive soil sampling to investigate overlap among species ranges and the applicability of the MDE to EM fungi in four temperate forest stands along an elevation gradient on Mount Fuji, Japan. Molecular analyses using direct sequencing revealed 302 EM fungal species. Of 73 EM fungal species found in multiple stands, 72 inhabited a continuous range along the elevation gradient. The maximum overlap in species range and the highest species richness occurred at elevations in the middle of the gradient. The observed richness pattern also fit within the 95% confidence interval of the mid-domain null model, supporting the role of the MDE in EM fungal richness. Deviation in observed richness from the mean of the mid-domain null estimation was negatively correlated with some environmental factors, including precipitation and soil C/N, indicating that unexplained richness patterns could be driven by these environmental factors. Our results clearly support the existence of microbial species' ranges along environmental gradients and the potential applicability of the MDE to better understand microbial diversity patterns.