Developmental ability of somatic cell nuclear transferred embryos aggregated at the 8-cell stage or 16- to 32-cell stage in cattle

Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.     

Contribution of the type II secretion system in systemic infectivity of <Emphasis Type="Italic">Ralstonia </Emphasis><Emphasis Type="Italic">solanacearum</Emphasis> through xylem vessels

Ralstonia solanacearum strain OE1-1 (OE1-1) systemically invades tobacco plants and causes bacterial wilt. A type II secretion system (T2SS)-deficient mutant of OE1-1, derived from EZ::TN<KAN-2>transposon-insertion, retained the ability of the parent strain to produce exopolysaccharide in vitro and grow in intercellular spaces immediately after invasion of host plants, but lost the ability to systemically infect the host. With transmission electron microscopy, the mutant was not observed in xylem vessels. These findings suggest that the T2SS contributes to systemic infection by enabling the bacteria to invade xylem vessels.     

Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens,as revealed by a combination of species‐specific detection methods

Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.     

Isolation and some properties of methane-oxidizing bacteria from a subtropical paddy field

Methane is the second most important greenhouse gas which contributes to global warming. As an important source of methane, rice paddy fields contribute an estimated lO% to the global methane emissions (IPCe 1992). Land use and agricultural practices significantly affect atmospheric methane fluxes (Bouwman 1989; Hütsch et al. 1994). Microbial oxidation of atmospheric methane in terrestrial environments is the only known net biological methane sink and the process consumes the equivalent of 1–l0% of the total global emission (Adamsen and King 1993). Methane-oxidizing bacteria (MOB, methanotrophic bacteria) are considered to be obligately or facultatively aerobic respiratory bacteria that can utilize methane as the sole source of carbon and energy for growth (Hanson et al. 1992; Roslev and King 1994). As a result, they are important regulators of atmospheric methane fluxes in nature (Mancinelli 1995). MOB have been isolated from a variety of environments including freshwater lakes, wetlands, and the open ocean (Whittenbury et al. 197Gb; Saralov et al. 1984; Holzapfel-Pschorn et al. 1985; Hanson et al. 1992; Omelchenko et al. 1993; Bowman et al. 1993a; Mancinelli 1995). However, reports on the isolation of MOB from rice paddy fields are limited. More information is needed on the ecology and taxonomy of MOB in paddy fields.     

Physicochemical properties of native and recombinant mungbean (Vigna radiata L. Wilczek) 8S globulins and the effects of the N-linked glycans

We have previously cloned and characterized the cDNAs of three isoforms of the 8S globulin of mungbean, expressed the major 8Salpha isoform in Escherichia coli, and purified and successfully crystallized it (Bernardo, A. E. N.; Garcia, R. N.; Adachi, M.; Angeles, J. G. C.; Kaga, A; Ishimoto, M.; Utsumi, S.; Tecson-Mendoza, E. M. J. Agric. Food Chem. 2004, 52, 2552-2560). Herein, we report the physicochemical and emulsifying properties of the native 8S and recombinant 8Salpha globulin or vicilin. The circular dichroism spectra analysis of the native 8S and recombinant 8Salpha globulins revealed that the recombinant 8Salpha formed a secondary structure close to that of the native 8S. Further, gel filtration analysis showed that 8Salpha was able to assemble into trimers. The native 8S and recombinant 8Salpha globulins were soluble at pH 3.4 and at pH 7.4-9.0 at low ionic strength, mu = 0.08. Interestingly, the native 8S was more soluble at pH 7.0 and pH 7.4 than the recombinant 8Salpha at mu = 0.08. Both forms were very soluble at pH 3.4-9.0 at high ionic strength, mu = 0.50. The native form exhibited a higher T(m) (69.2, 79.5, and 83.8 degrees C) than the recombinant form (65.6, 71.6, 77.5 degrees C) at mu = 0.1, 0.2, and 0.5, respectively. The recombinant form was found to have greater surface hydrophobicity than the native form. There was little difference in the emulsifying ability between the native 8S and 8Salpha at pH 3.4 and pH 7.6. The results indicate that the presence of N-linked glycans is not essential in the assembly and stable conformation of the mungbean vicilin. However, the N-linked glycans might have contributed to the higher solubility at low ionic strength, greater thermal stability, and decreased surface hydrophobicity of the native vicilin as compared to the recombinant 8Salpha. On the other hand, the N-linked glycans showed little effect on the emulsifying ability of the protein.     

Use of beer bran as an adsorbent for the removal of organic compounds from wastewater

Beer bran was found to effectively adsorb several organic compounds, such as dichloromethane, chloroform, trichloroethylene, benzene, pretilachlor, and esprocarb. Equilibrium adsorption isotherms conformed to the Freundlich isotherm (log-log linear). Adsorption of these organic compounds by beer bran was observed in the pH range of 1-11. At equilibrium, the adsorption efficiency of beer bran for benzene, chloroform, and dichiloromethane was higher than that of activated carbon. The removal of these organic compounds by beer bran was attributed to the uptake by intracellular particles called spherosomes. The object of this work was to investigate several adsorbents for the effective removal of organic compounds from wastewater.     

Properties of extracts from defatted rice bran by its subcritical water treatment

Defatted rice bran was extracted with water and subcritical water at 50-250 degrees C for 5 min. The highest extract yield was achieved at 200 degrees C, at which the maximum amounts of protein and carbohydrate were also obtained. The total phenolic and furfural contents, radical scavenging activity, and antioxidative activity for the autoxidation of linoleic acid increased with increasing treatment temperature. The bran extracts exhibited emulsifying activity except for the extract prepared at 250 degrees C, which was concomitant with the disappearance of its high-molecular-mass substances. The extract prepared at 200 degrees C also had the highest emulsion-stabilizing activity.     

Studies on defatted seed removal efficiency for organochlorine compounds

Defatted seeds were evaluated for effective adsorption of organochlorine compounds such as chloroform, dichloromethane, and trichloroethylene. The amounts of these compounds adsorbed were plotted against the equilibrium concentration of substances in solution on a logarithmic scale. A linear relationship was obtained, indicating that the adsorption reactions were of the Freundlich type. The removal of these organochlorine compounds by defatted seed was attributed to the uptake by intracellular particles called spherosomes.