Reactive oxygen species (ROS) generation and ROS-induced lipid peroxidation are associated with plasma membrane modifications in host cells in response to AK-toxin I from Alternaria alternata Japanese pear pathotype

AK-toxin I caused plasma membrane modifications with plasma membrane-derived membrane fragments only in sensitive Japanese pear tissues. H₂O₂ generation was abundant in both the membrane fragments and the plasma membranes of the toxin-treated sensitive tissues. Whether lipid peroxidation was induced in plasma membranes of the toxin-treated sensitive tissues was examined biochemically and histochemically. Lipid peroxidation was caused only in the toxin-treated sensitive tissues or the toxin-treated plasma membrane-enriched fractions from sensitive young pear fruits. The results indicated that the peroxidation was probably induced by reactive oxygen species in the modified plasma membranes by action of toxin, suggesting that peroxidation is closely associated with plasma membrane modifications.     

Factors associated with antimicrobial-resistant Escherichia coli in zoo animals

Factors associated with the carriage of antimicrobial-resistant Escherichia coli isolates were analysed among zoo animals. An association was observed between selection of amoxicillin as the first-line therapy and a significantly higher percentage of resistance to ampicillin (54.5%) from 11 animals treated with antimicrobials, compared with isolates from 32 untreated animals (9.4%). In addition, the percentage resistance to kanamycin (36.4%), gentamicin (27.3%), trimethoprim (27.3%) and tetracycline (63.6%) from 11 treated animals was significantly higher than those from 32 untreated animals (3.1%, 3.1%, 3.1% and 25%, respectively), although these antimicrobials were rarely used. All kanamycin-, gentamicin- and trimethoprim-resistant isolates and more than half of the tetracycline-resistant isolates from treated animals were also resistant to ampicillin. Co-resistance to other antimicrobials with ampicillin was suggested to contribute to an increasing of resistance towards antimicrobials that were rarely administered. The present investigation revealed an association of antimicrobial treatment with the spread of antimicrobial-resistant bacteria among zoo animals.     

Chondrulelike objects in short-period comet 81P/Wild 2

The Stardust spacecraft returned cometary samples that contain crystalline material, but the origin of the material is not yet well understood. We found four crystalline particles from comet 81P/Wild 2 that were apparently formed by flash-melting at a high temperature and are texturally, mineralogically, and compositionally similar to chondrules. Chondrules are submillimeter particles that dominate chondrites and are believed to have formed in the inner solar nebula. The comet particles show oxygen isotope compositions similar to chondrules in carbonaceous chondrites that compose the middle-to-outer asteroid belt. The presence of the chondrulelike objects in the comet suggests that chondrules have been transported out to the cold outer solar nebula and spread widely over the early solar system.     

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

Background

The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis.

Results

A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1.

Conclusions

A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome.     

Growing porcine oocyte-granulosa cell complexes acquired meiotic competence during in vitro culture

The aim of this study was to establish a culture system to improve the meiotic competence of porcine oocyte-granulosa cell complexes (OGCs) obtained from preantral or early antral follicles. Porcine OGCs were recovered from follicles with diameters of 230-300 (preantral follicles), 300-500, and 500-700 mum (early antral follicles) using scalpels. The OGCs were cultured for 2 weeks in culture medium. We examined the effects of the sizes of the follicles from which OGCs were recovered, the concentrations of polyvinylpyrrolidone (PVP, 0-8%) in the culture medium, and 2 types of culture dish (Falcon 3002 vs 1007) on formation of the antrum of OGCs. After culture, the oocytes were matured for 44 h to assess their meiotic competence. OGCs recovered from small follicles (230-500 microm) required longer (P<0.05) than larger follicles to form the antrum structure. The percentage of OGCs forming the antrum structure that were cultured in 2% PVP (31%) was higher (P<0.05) than for those cultured in other PVP concentrations (0-11%). The percentages of antrum-structure formation for OGCs cultured on Falcon 3002 (83% for 2% PVP and 60% for 4% PVP) were higher (P<0.05) than those cultured on Falcon 1007 (47% for 2% PVP and 9% for 4% PVP). Furthermore, all of the intact oocytes that were obtained from culture of OGCs and that formed an antrum were in the GV stage (n=28). When these immature oocytes were cultured for 44 h, the percentage of oocytes that reached the metaphase II stage (25%, n=68) was higher (P<0.0001) than that of oocytes matured without culture (0.7%, n=137). The results of the present study show that porcine OGCs obtained from preantral or early antral follicles acquire meiotic competence in vitro.     

Enhanced freezability of goat spermatozoa collected into tubes containing extender supplemented with bovine serum albumin (BSA)

The purpose of this study was to investigate the effects of semen collection into tubes containing extender supplemented with BSA on the cryosurvival of goat spermatozoa. Semen was collected from two goats into empty tubes or tubes containing 10 ml extender supplemented with 0, 0.1, 1, or 5% BSA, and the washed spermatozoa were frozen as pellets in egg yolk-trehalose extender with the addition of 0.04% SDS and 4% glycerol. Sperm motion parameters were evaluated after post-thawing and during a thermal resistance test. The acrosome status of frozen-thawed spermatozoa was also observed using FITC-PNA staining. In frozen semen that was collected into tubes containing extender supplemented with 5% BSA, the post-thawed spermatozoa exhibited a significant improvement in motion parameters and maintained high motility throughout incubation and acrosome integrity, as compared with semen collected into tubes containing extender supplemented with lower concentrations of BSA. In conclusion, semen collection into tubes with a large volume of extender containing high concentrations of BSA dramatically improves the motility and acrosome integrity of frozen-thawed spermatozoa. This suggests that the in vitro functional freezability of spermatozoa is abruptly modified by reducing contact with seminal plasma and by flash contact with BSA at ejaculation.     

Soil respiration and methane flux in adjacent forest,grassland, and cornfield soils in Hokkaido,Japan

Soil respiration and methane flux from adjacent forest, grassland, and cornfield were measured by using the closed chamber method from June to November, 1999 in Shizunai, Hokkaido, Japan, where the soil was an Aquic Humic Udivitrands derived from volcanic ash. The forest soil absorbed methane, at arate ranging from -0.12 to -0.02 mg C m_-2 h_-1, while the grassland soil emitted methane, at a rate ranging from undetectable levels to 0.18 mg C m_-2 h_-1. In the cornfield soil methane flux ranged from -0.01 to 0.04 mg C m_-2 h_-1. The soil respiration rate varied from 3 to 230 mg C m_-2 h_-1, 27 to 372 mg C m_-2 h_-1, and 29 to 156 mg C m_-2 h_-1 for the cornfield, grassland, and forest soils, respectively. Linear regression analysis demonstrated that the methane flux rate was positively correlated with the soil water-filled pore space (WFPS), and negatively correlated with the relative gas diffusion coefficient (D/D _o) and air-filled pore space (AFPS). Soil respiration rates were positively correlated with the soil temperature at all the sites. The Q ₁₀ value was 4.8, 3.3, and 1.9 for the cornfield, grassland, and forest soils, respectively.