Improved method for in situ biolistic transformation to analyze barley–powdery mildew interactions

Although the use of stable transformants is indispensable to elucidate mechanisms underlying molecular plant–pathogen interactions, this approach remains difficult to apply to crops. Alternatively, biolistic transformation has often been used as a transient expression method in various plants. In this study, we developed a method for in situ biolistic transformation without separating leaves from barley seedlings by using a hand-held particle bombardment system because unwounded leaves are preferable for analyzing interactions between barley and Blumeria graminis f. sp. hordei which requires healthy living cells. As a result, we found that the infection rate in intact leaves was higher than in separated leaves and that the transformation efficiencies in leaves were higher when plants were grown in vermiculite rather than in culture soil. Furthermore, we determined the appropriate inoculation time after bombardment to analyze the incompatible interaction and successfully monitored the gradual occurrence of cell death over time. Our system was suitable for relatively long-term follow-up analysis of the fate of each single cell during plant–pathogen interactions.     

cDNA cloning and expression analysis of two heat shock protein genes,Hsp90 and Hsp60, from a sterile Ulva pertusa (Ulvales,Chlorophyta)

Molecular chaperones are synthesized and accumulated under a variety of unfavorable conditions in all organisms. Heat shock protein 90 (Hsp90) and Hsp60, which are classified into the major classes of molecular chaperones, play important roles in cellular stress responses. In this study, we characterized sterile Ulva pertusa Hsp90 (UpHsp90) and UpHsp60 genes which may be involved in tolerance to thermal and heavy metal stresses in this alga. The UpHsp90 cDNA consisting of 2,118 nucleotides encoded a polypeptide of 705 amino acids (AA). On the other hand, the UpHsp60 cDNA consisting of 1,722 nucleotides encoded a protein whose predicted length was 573 AA. The AA sequence alignment and phylogenetic analyses showed that the UpHsp90 and UpHsp60 proteins were more similar to cytoplasmic Hsp90s and mitochondrial Hsp60s, respectively, than to other types of the respective Hsps. Southern blot analysis indicated that the sterile U. pertusa genome had at least two cytoplasmic Hsp90-encoding genes and two mitochondrial Hsp60-encoding genes. The UpHsp90 and UpHsp60 mRNA levels were significantly affected by diurnal and temperature changes, and slightly affected by exposure to heavy metals. These results suggest that UpHsp90 and UpHsp60 genes play particularly important roles in adaptation to diurnal and temperature changes.     

Purification and functional characterization of complement C3 and a novel zymosan-binding protein in tilapia serum

Zymosan, a yeast cell wall preparation that binds activated forms of complement C3, is a useful model target to activate the complement system. In our trial to analyze C3 diversity in Nile tilapia at the protein level using zymosan, we found that a novel 240-kDa serum protein (ZBP-240) also bound to zymosan in addition to C3-derived fragments. In the present study, we aimed to characterize tilapia C3 and ZBP-240, focusing on their immune-related functions. Four distinct C3 isoforms were purified from tilapia serum and shown to possess an intrachain thioester bond. ZBP-240 was also isolated from tilapia serum and examined for its binding properties to various microbial targets. As a result, ZBP-240 showed a wide spectrum of binding to Gram-positive and Gram-negative bacteria and yeasts. Amino acid sequence analysis of CNBr fragments of ZBP-240 suggested that this is a novel protein with no homologous sequence in protein databases. It was also suggested that the binding of ZBP-240 to microbes largely depends on hydrophobic interactions in a divalent-cation-independent manner, and that there may be a divalent-cation-dependent factor that enhances the binding of ZBP-240 in tilapia serum. Interestingly, ZBP-240 showed opsonic activity for tilapia kidney phagocytes at a level comparable to that of C3, implying that ZBP-240 is a novel teleost opsonic serum protein.     

Evaluation of available silicon content and its determining factors of agricultural soils in Japan

To evaluate the available silicon (Si) content in agricultural soils in Japan and to investigate the determining factors of this content, we collected 180 soil samples from the surface layer of paddies and upland fields in Japan and determined their available Si contents. A phosphate buffer (PB; 0.02 M, pH 6.9) or an acetate buffer (AB; 0.1 M, pH 4.0) was used to extract available Si from the soil samples, and the Si concentrations in the extracts were determined by inductively coupled plasma-atomic emissions spectroscopy (ICP-AES). The total Si content and selected physicochemical properties were also determined for the soil samples. The median values of the available Si contents by the PB and AB methods were 48.8 and 79.7 mg kg^?1 and corresponded to 0.017% and 0.027% of the total Si content, respectively. The overall data showed log-normal distributions. The available Si content of the upland soils was significantly higher than that of the paddy soils by both the PB (p < 0.01) and AB methods (p < 0.05). The available Si contents by the PB and AB methods had a significant positive correlation (p < 0.01) and they had significant negative correlation with the total Si content (p < 0.01). The values of the available Si contents by the PB and AB methods correlated positively with the pH, total carbon (C) content, and dithionite-citrate bicarbonate extractable iron (Fed) and aluminum (Ald), acid oxalate extractable iron (Feo) and aluminum (Alo), Fed-Feo and Alo+1/2Feo values (p < 0.01). A multi-regression analysis indicated that pH, amorphous minerals and crystalline iron (Fe) oxides were the dominant determining factors of available Si in the soils, and these three variables explained approximately two thirds of the variation of available Si content in agricultural soils in Japan. In terms of soil type, Terrestrial Regosols, Dark Red soils and Andosols had relatively high available Si contents, whereas Sand-dune Regosols, Red soils and Gray Lowland soils had relatively low contents. In terms of region, the soils in the Kanto and Okinawa regions had relatively high available Si contents and those in the Kinki, Shikoku and Chugoku regions had relatively low contents. In conclusion, the available Si content and its determining factors for agricultural soils in Japan were quantitatively elucidated, and this will contribute to the establishment of rational soil management?—including the application of silicate materials, taking into account the Si-supplying power of the relevant soils—for sustainable and productive agriculture in Japan.     

Enthalpy relaxation behavior of dry wood detected by temperature-modulated differential scanning calorimetry

Enthalpy relaxation of dry wood has been investigated by temperature-modulated differential scanning calorimetry. The reversing and non-reversing heat flow changes revealed that enthalpy relaxation occurred in dry wood, which did not exhibit any clear glass transitions. This enthalpy relaxation behavior seemed to differ significantly from those of previously reported isolated lignins, which implies that the microstructure of dry wood possesses a rigid amorphous state derived from interactions among wood components. The observed enthalpy relaxation is considered to be related to other components besides lignin, and the time-dependent physical properties due to unstable states or physical aging of wood originate not only from lignin but also from other components, such as cellulose and hemicellulose and the interactions between them.     

Gross nitrogen retranslocation within a canopy of Quercus serrata saplings

Nitrogen (N) retranslocation within tree canopies has been intensively studied and assumed to function as a one-way process (e.g., from older to newer leaves). However, recent studies have found that both N output and input occur in individual leaves, suggesting that 'gross' N retranslocation exists behind 'net' N retranslocation. In the present study, the amount and direction of gross N retranslocation within a canopy of deciduous oak Quercus serrata Thunb. ex. Murray saplings were investigated. Labeling was conducted with leaves of Q. serrata saplings cultivated under conditions of low-N (LN) or high-N (HN) fertility. Subsequently, N movement within the canopy was traced. Leaves at two different positions in the canopy (top and lateral) were labeled to determine the direction of gross N retranslocation. To detect seasonal differences, the leaf-labeling experiment was conducted twice during the early and late phases of the growing season. In addition, to compare the quantitative importance of gross N retranslocation and root N uptake, the latter was determined by labeling Q. serrata roots. The N-labeling experiment revealed gross N retranslocation among leaves, i.e., from top to lateral, lateral to top and lateral to lateral positions. Gross N retranslocation was quantitatively more important than root uptake, especially for plants cultivated at LN fertility. Season also affected the amount of gross N retranslocation, and these effects differed between LN and HN fertilities. These findings suggest that N allocation within a canopy is controlled dynamically by both gross N output and input. The mechanisms controlling gross N output and input likely function as key determinants of N allocation within a tree canopy.     

Spontaneously occurring intracranial lipomatous hamartoma in a young BALB/c mouse and a literature review

An intracranial lipomatous hamartoma was found in the third ventricle of a 7-week-old female BALB/cAnNCrlCrlj mouse. The nodule was composed of mature white adipose cells, which contained one large fat droplet, and there was no evidence of cytological atypia. The brain parenchyma at the retrosplenial granular cortex and the hippocampus in the cerebrum were slightly compressed, and the choroid plexus was dislocated downward. Scattered capillary vessels penetrated the nodule from the surrounding tissue. Based on these findings, the lesion was diagnosed as a lipomatous hamartoma that occurred from the roof of the third ventricle. This extremely rare tumor-like nodule represents an overgrowth of the mature adipocyte population as a malformation rather than a true neoplasm.     

Flow cytometric immune profiling of specific-pathogen-free chickens before and after infectious challenges

Broilers and layer chickens have been intensively selected for production parameters. This selection has affected immune capacity. Consequently, the fine-tuning of immune responses is becoming important for maximum productivity. Flow cytometry is a recurrent technology used for the immunophenotyping of birds. Studies, however, have focused on the mechanism of specific diseases or have used animals whose immunological condition could be biased-by vaccination or environmental stressors, for example. The aim of this study was to evaluate the immune status of specific-pathogen-free birds across different age ranges to characterize the natural changes that occur over time. Additionally, specific-pathogen-free chickens were challenged with four infectious agents, allowing identification of the subpopulations of peripheral blood immune cells that are consistently altered under various conditions. Several lymphocyte subsets vary naturally with aging, so the interpretation of results using animals of different age ranges must proceed with care. Parameters such as CD8(+)CD28(-), CD8αα(+), CD4(+)CD8(+), and CD8(+)TCRVβ1(+) have been shown to be valuable in understanding immune changes during disease. The use of these data allows a determination of the consistency of cytometric parameters under various conditions, which should ease the interpretation of immunophenotyping and the future application of cytometric analysis in the poultry industry.     

Effects of bisphenol A administration to pregnant mice on serum Ca and intestinal Ca absorption

Bisphenol A (BPA) is a xenoestrogen commonly used in food storage plastics. The present study was conducted to clarify the effects of BPA administration to pregnant mice on serum calcium (Ca) and Ca metabolism of the gut and kidney. From 6.5 to 16.5 days post coitus (dpc), pregnant mice were administered at 2 mg or 20 mg/kg body weight/day of BPA. Serum Ca was decreased in mice treated with 20 mg BPA at 17.5 dpc, but no remarkable differences were detected in the alkaline phosphatase activity and vitamin D receptor protein expression in the duodenum and jejunum. The messenger RNA (mRNA) expressions of calcium binding protein (CaBP‐9k) and active vitamin D synthesis enzyme (CYP27B1) in the kidney were increased in mice treated with 20 mg BPA. The mRNA expressions of occludin and junction adherence molecular A (JAM‐A) in the duodenum and ileum, which regulate paracellular transport, were increased in mice treated with 20 mg BPA. However, the administration of 2 mg BPA had no effect on serum Ca and mRNA expressions of relative genes in Ca metabolism. These results imply that BPA administration at 20 mg/kg body weight/day during pregnancy decreases serum Ca in pre‐delivery mice, which may be partly due to decreased paracellular Ca absorption.     

Hydroxy-Al polymers block the frayed edge sites of illitic minerals in acid soils: studies in southwestern Japan at various weathering stages

The transformation of hydroxy-interlayered vermiculite (HIV) to vermiculite in humid temperate soils may involve the gradual alteration of illitic minerals. However, it is difficult to detect such minor and progressive changes using conventional methods. We measured the amount of the frayed edge site (i.e. the weathering front of illitic minerals) in soil clays using Radiocaesium Interception Potential (RIP) methodology, and elucidated the effect of hydroxy-Al polymers on the frayed edge site that may be occluded within HIV structures in the acidic soils of southwestern Japan. X-ray diffraction patterns showed the progressive transformation of HIV to vermiculite, or further, to smectite in soil clays located in upper horizons and therefore subjected to more intensive podzolization. In this process, the amount of hydroxy-Al polymers (represented by hot-citrate Al) gradually decreased, while the vermiculitic charges increased (represented by Cs-fixing capacity). However, the amount of the frayed edge site (represented by the RIP) firstly increased but then reversed and decreased in the upper layers of podzolic soils. After hot-citrate extraction to remove the hydroxy-Al polymers from HIV, the amount of the frayed edge site increased in HIV-dominated clays, whereas there was a negligible increase in vermiculitic- or smectitic-clays, thus indicating the blockage effect of hydroxy-Al polymers on the frayed edge site. The sequential increase followed by the decrease that we documented in the amount of the frayed edge site along with the HIV-vermiculite-smectite transformation, suggested that the weathering front of illitic minerals was exposed as the HIV layers lose hydroxy-Al polymers. Thereafter, the charges in the exposed frayed edge site might decline under the very acidic conditions of the upper layers of podzolic soils.