Abnormal distribution of chromosomes in the first division of nuclear transferred mouse embryos

The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.     

Histochemical and immunohistochemical characterization of chordoma in ferrets

Chordomas of the tip of the tail in 6 ferrets were examined using histopathological, histochemical and immunohistochemical procedures. Histopathologically, round neoplastic cells containing numerous cytoplasmic vacuoles of varying sizes, categorized as “physaliphorous cells”, were observed in the amorphous eosinophilic or pale basophilic myxoid stroma. Physaliphorous cells were arranged in lobules and in a “chordoid” or “cobblestone” manner. The neoplasms were diagnosed as benign chordoma without local invasion and metastasis. Histochemically, the cytoplasm of small neoplastic cells was positive for periodic acid-Schiff stain and alcian blue (AB) pH 2.5 and pH 1.0 stains, but negative for hyaluronidase digestion-AB pH 2.5 stain. All neoplastic cells were strongly stained with colloidal ion, negative for high iron diamine AB pH 2.5 and toluidine blue pH 2.5 stains, and positive for Mayer’s mucicarmine stain. Immunohistochemistry using antibodies directed against low-molecular-weight cytokeratins (CK18, CK19 and CK20), vimentin and mucin core protein (MUC5AC) revealed that neoplastic cells had both epithelial and mesenchymal elements. The expression of low-molecular-weight cytokeratins suggests that neoplastic cells acquired the properties of glandular epithelial cells and produced epithelial mucus. Furthermore, the expression of cytokeratins, vimentin, S100 protein, brachyury and epithelial membrane antigen indicates that the neoplasms were equivalent to the classic type of human chordoma. Therefore, immunohistochemistry using these antibodies can be useful for the characterization of ferret chordoma.     

Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi Nuclear Power Plant

The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium (¹³⁷Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a ¹³⁷Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood ¹³⁷Cs and muscle ¹³⁷Cs (Y = 28.0X, R² = 0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high‐risk cattle before they enter the market, and will contribute to food safety.     

Expression analysis of defense-related genes in wild kiwifruit (Actinidia rufa) tolerant to bacterial canker

Journal of General Plant Pathology - Kiwifruit bacterial canker, which is caused by Pseudomonas syringae pv. actinidiae biovar 3 (Psa3), is found throughout kiwifruit-growing countries. Here, we...     

Comparison of acute inhalation toxicity of sulfuric acid by the inhalation and intratracheal instillation methods

Recently, intratracheal instillation has been focused on as a simple, low-cost alternative to the inhalation method. In this study, intratracheal instillation of sulfuric acid, a typical acidic compound, was performed to compare the acute toxicity of acidic compounds that could cause damage to the respiratory system between intratracheal instillation and inhalation. Sulfuric acid was administered to male rats at doses of 0.7, 2, 7, 20, and 60 mg/kg by dividing the total dose into four doses. General condition and body weight were examined up to 14 days after administration, and macropathological and histopathological examinations were performed. The half-lethal dose was then estimated. All animals administered 20 and 60 mg/kg sulfuric acid and one animal administered 2 mg/kg sulfuric acid died within 4 h after administration. No abnormalities were observed in other animals. At 20 and 60 mg/kg, multiple red foci or diffuse red areas were macroscopically observed in the lungs. In these lesions, histopathologically, clefts between the mucosal epithelium and basement membrane and necrosis of the alveolar epithelium were observed. Deaths in these groups may have resulted from lung injury. No notable changes were observed in other animals. Therefore, the half-lethal dose of sulfuric acid by intratracheal instillation was estimated as 7–20 mg/kg. The acute toxicity by intratracheal instillation was evaluated with two-fold sensitivity since the exposure at the half-lethal sulfuric acid concentration in inhalation studies was calculated as 43.2 mg/kg.     

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Effect of nitrate fertilization of gametophytes of the kelp Undaria pinnatifida on growth and maturation of the sporophytes cultivated in Matsushima Bay, northern Honshu, Japan

In northern Japan, the early production of high-quality specimens of kelps Undaria pinnatifida prior to the outbreak of pinhole disease is needed. To address this need, we tested the effect of nitrate fertilization of gametophytes on the growth and maturation of sporophytes using experimental and control gametophytes derived from sporophytes cultivated in Matsushima Bay, northern Honshu, Japan. From mid-August to October 2008, experimental gametophytes were cultured on a rope indoors at a nitrate concentration of 1 mg/L at the optimal seawater temperature of 20 °C, whereas control gametophytes were cultured without the addition of nitrate in normal seawater at an average temperature of 23.5 °C during this period in the past 30 years. Next, the seedlings were cultivated in Matsushima Bay until March 2009. For the experimental kelps, the total length, stipe length, stipe width, sporophyll length, sporophyll width, and dry weight of the blade, midrib, and sporophyll increased significantly from December to January compared to those of control kelps. Matured sporophylls of the experimental kelps were more than those of the control kelps from early December to late January. Significant higher photosynthesis occurred at seawater temperatures of 10 and 15 °C, as well as higher uptake rates of NO₃–N of the experimental kelps in December and January. These results show that nitrate fertilization of the gametophytes makes it possible to harvest the high-priced blade, midrib, and sporophyll in January, which is earlier than the traditional harvest date.     

Transactivational property of chemicals via medaka glucocorticoid receptor 1b using a stable reporter gene assay

The transactivational property of natural and synthetic chemicals via medaka GR1b was investigated after development of a stable cell line for the reporter gene assay. In our study, cortisol was the most potent agonist among the natural corticoids assayed for potency [EC₅₀ (concentration of agonist provoking a response halfway between the baseline and maximum response) 68 nM] and efficacy. Three artificial corticosteroids, namely, dexamethasone [EC₅₀ 16 nM, relative agonistic activity to cortisol (RAA) 144 %], prednisolone (EC₅₀ 81 nM, RAA 116 %) and clobetasol propionate (EC₅₀ 0.10 nM, RAA 220 %), showed strong agonistic activity and were more potent than the original corticoid, F. All synthetic corticoids used in our study were full agonists. Interestingly, melengestrol acetate, a synthetic progestogen, induced luciferase activity in a dose-dependent manner. Based on its EC₅₀ value and RAA of 29 nM and 57 %, respectively, this molecule was assessed as a partial agonist. None of the other steroids and chemicals assayed in our study induced an agonistic response. In conclusion, we successfully developed a stable reporter gene assay that can be used to assess the transactivational property of glucocorticoid-like chemicals toward medaka GR1b.     

Spatial variations in nitrous oxide and nitric oxide emission potential on a slope of Japanese cedar (Cryptomeria japonica) forest

To quantify the spatial variation and spatial structure of nitrous oxide (N₂O) and nitric oxide (NO) emission from forest soils, we measured N₂O and NO emission rates from surface soil cores taken at 1 m intervals on a cross-line transect (65 m × 20 m) on a slope of Japanese cedar ( Cryptomeria japonica ) forest in a temperate region of central Japan and analyzed the spatial dependency of N oxide gas emissions using geostatistics. We divided N₂O emission into N₂O from denitrification and N₂O from nitrification using the acetylene inhibition method. According to the geostatistical analysis, N₂O emission rates on the slope had large spatial variation and weak spatial dependency. This weak spatial dependency was caused by the inordinately high N₂O emissions on the slope, which were derived mainly from denitrification. In contrast, NO emission rate on the slope had large spatial variation, but strong spatial dependency and a distinct spatial distribution related to slope position, that is, high in the middle of the slope and low in the shoulder and the foot of the slope. The CN ratio and water-filled pore space were the dominant factors controlling NO emission rate on a slope. Our results suggest that spatial information about topographic factors helps to improve the estimation of both N₂O emission and NO emission from forest soils.     

Relationship between N2O and NO emission potentials and soil properties in Japanese forest soils

To determine the relationship between nitrous oxide (N₂O) and nitric oxide (NO) emission rates and soil properties in forest soils, N₂O and NO emission rates in soils were measured in incubation experiments under standardized temperature and water conditions (water content at a water-holding capacity of 60%) using soils packed into a cylindrical core, and variations in the soil properties were also determined. The N₂O emission rates from nitrification and from denitrification were determined separately using a nitrification inhibitor (10 Pa acetylene). Soil samples were taken from 25 forest stands in a central temperate area of Japan. The N₂O and NO emission rates were highly variable, even under the standardized temperature and water-holding capacity (60%) conditions. According to a partial least squared regression model analysis, the C:N ratio and pH strongly affected the N₂O emission rate, whereas     , water-soluble Al and the C:N ratio strongly affected the NO emission rate. The C:N ratio negatively affected the emission rate of both N oxide gases, suggesting that N mineralization is an important factor in the rates of N oxide gas emission. The acetylene inhibition experiment showed that N₂O emission from denitrification was positively affected by pH, water-filled pore space and filling density, and negatively affected by the C:N ratio, total carbon and total nitrogen.