Heterologous expression of the mutated melon ethylene receptor gene Cm-ERS1/H70A produces stable sterility in transgenic lettuce (Lactuca sativa)

The mutated melon ethylene receptor gene Cm-ERS1/H70A was introduced into tobacco and induced stable sterility in transgenic lines. This gene contains a missense mutation that converts the His(70) residue to Ala in the melon ethylene receptor gene Cm-ERS1. To test the applicability of this inducible sterility system to other plants, lettuce (Lactuca sativa) was transformed with the gene using Agrobacterium, and putative transformants containing Cm-ERS1/H70A were obtained. Thirteen randomly selected putative transformants were grown in a growth room under constant conditions, and seven of the lines showed sterility or significantly reduced fertility. DNA gel blot analysis confirmed the integration of the Cm-ERS1/H70A gene into the genomes of the putative transformants, and RT-PCR and protein gel blot analysis confirmed the expression of Cm-ERS1/H70A mRNA and protein in all of the transformants. Five transformants showing sterility or reduced fertility when grown in a growth room under constant conditions were randomly selected to be grown in an open-air greenhouse under various environmental conditions. All five showed stable sterility under the various conditions. These results suggest that Cm-ERS1/H70A can induce sterility in heterologous transgenic plants.     

Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice

To ascertain an optimum condition for detecting micronuclei in the liver caused by numerical aberration inducers, either carbendazim (125-1000mg/kg, p.o.), colchicine (0.375-1.5mg/kg, i.v.), cytochalasin B (2.5-20mg/kg, i.v.), diazepam (3.13-25mg/kg, i.v.), noscapine (7.8-62.5mg/kg, i.v.), paclitaxel (1-100mg/kg, i.v.) or trichlorfon (18.75-150mg/kg, i.v.) was administered once to male Slc:ddY mice 1 day before or after partial hepatectomy (PH, Day 1). Five days after PH (on Day 6), hepatic micronuclei were determined in conjunction with classifications of the main nuclei and relative liver weights as a proliferative indicator or a dysfunction marker of cell division. Additionally, hepatocyte proliferation index (HPI) was calculated by using mono-, bi- and multinucleated cell counts. Treatment of mice with six compounds, except for colchicine, after PH showed higher incidence of micronucleated hepatocytes (MNH) than that before PH, and also increases in binucleated and multinucleated cells. Especially for carbendazim, diazepam, noscapine and trichlorfon, the dosing after PH was essential for the detecting numerical aberration. Colchicine evidently increased HPI and decreased relative liver weights without MNH induction on Day 6. On Day 8 when HPI and relative liver weights almost returned to the basal range, a significant increase in MNH was noted. This implied that the strong inhibition of colchicine on hepatocyte proliferation may obstruct the induction of MNH on Day 6. In conclusion, to detect the potential numerical aberration, exposure of mice to test chemicals should be performed 1 day after PH, during which enhanced proliferation of hepatocytes was seen, and it would be better to analyze the liver specimens on Day 6 or more post-PH.     

Ecophysiology of Thioploca ingrica as revealed by the complete genome sequence supplemented with proteomic evidence

Large sulfur-oxidizing bacteria, which accumulate a high concentration of nitrate, are important constituents of aquatic sediment ecosystems. No representative of this group has been isolated in pure culture, and only fragmented draft genome sequences are available for these microorganisms. In this study, we successfully reconstituted the genome of Thioploca ingrica from metagenomic sequences, thereby generating the first complete genome sequence from this group. The Thioploca samples for the metagenomic analysis were obtained from a freshwater lake in Japan. A PCR-free paired-end library was constructed from the DNA extracted from the samples and was sequenced on the Illumina MiSeq platform. By closing gaps within and between the scaffolds, we obtained a circular chromosome and a plasmid-like element. The reconstituted chromosome was 4.8 Mbp in length with a 41.2% GC content. A sulfur oxidation pathway identical to that suggested for the closest relatives of Thioploca was deduced from the reconstituted genome. A full set of genes required for respiratory nitrate reduction to dinitrogen gas was also identified. We further performed a proteomic analysis of the Thioploca sample and detected many enzymes/proteins involved in sulfur oxidation, nitrate respiration and inorganic carbon fixation as major components of the protein extracts from the sample, suggesting that these metabolic activities are strongly associated with the physiology of T. ingrica in lake sediment.     

Influence of position of substituent groups on removal of chlorophenols and cresols by horseradish peroxidase and determination of optimum conditions

Enzymatic treatment of o-, m-, and p-chlorophenols and o-, m-, and p-cresols from artificial wastewater was undertaken through the enzymatic conversion into the corresponding phenoxy radicals with horseradish peroxidase (HRP) and nonenzymatic radical coupling reaction. The concentration of chlorophenols and cresols decreased sharply over the reaction time and water-insoluble oligomer precipitates were generated. The optimum conditions were determined to be the H2O2 concentration of 2.5 mM and poly(ethylene glycol) with molecular mass of 1.0 x 10(4) (10K-PEG) of 0.10 mg/cm3 at 30 degrees C for treatment of p-chlorophenol at 2.5 mM. The optimum pH values depended on the relative position of a chlorine atom for chlorophenols and on a methyl group for cresols. Concentrations of HRP and 10K-PEG were increased to 1.0 U/cm3 and 1.0 mg/cm3 respectively to treat m-chlorophenol highly. For o-chlorophenol, a decrease in the pH value to 3.0 after the enzymatic treatment led to the enhancement of the aggregation of oligomer precipitates. The % residual value for o-cresol effectively decreased by absorbing water-soluble intermediates on the chitosan films. These results indicate that chlorophenols and cresols were removed to a great degree by this technique, although the detailed procedure depended on the position of substituent groups of chlorophenols and cresols.     

Evidence of local antibody response against Alloiococcus otitidis in the middle ear cavity of children with otitis media

Alloiococcus otitidis is a recently discovered bacterium frequently associated with otitis media. However, no study is available as to whether A. otitidis has a pathogenic role and induces local immune response in the middle ear as a true pathogen. Whole bacterial sonicate of A. otitidis was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. Then, Western blot analysis was performed with supernatant of the middle ear effusions from children with A. otitidis-positive otitis media. SDS-PAGE of the bacterial sonicate showed several protein bands, designated A1-A11. Western blot analysis revealed the presence of IgG, secretory IgA, IgG2, and IgM against A. otitidis in the middle ear effusions. Absorption of the specimens with sonicates of other major middle ear pathogens did not alter the reactivity of antibodies against the alloiococcal antigens. The results suggest that specific local immune response against A. otitidis is induced during middle ear infection of the organism as a true pathogen. A5, A6 or A11 is expected to be a main antigenic determinant. This is the first report to show evidence of local antibody response against A. otitidis and to disclose antigenic components of A. otitidis.     

Localized PEM mRNA and protein are involved in cleavage-plane orientation and unequal cell divisions in ascidians

BACKGROUND: Orientation and positioning of the cell division plane are essential for generation of invariant cleavage patterns and for unequal cell divisions during development. Precise control of the division plane is important for appropriate partitioning of localized factors, spatial arrangement of cells for proper intercellular interactions, and size control of daughter cells. Ascidian embryos show complex but invariant cleavage patterns mainly due to three rounds of unequal cleavage at the posterior pole. RESULTS: The ascidian embryo is an emerging model for studies of developmental and cellular processes. The maternal Posterior End Mark (PEM) mRNA is localized within the egg and embryo to the posterior region. PEM is a novel protein that has no known domain. Immunostaining showed that the protein is also present in the posterior cortex and the in centrosome-attracting body (CAB) and that the localization is extraction-resistant. Here we show that PEM of Halocynthia roretzi is required for correct orientation of early-cleavage planes and subsequent unequal cell divisions because it repeatedly pulls a centrosome toward the posterior cortex and the CAB, respectively, where PEM mRNA and protein are localized. When PEM activity is suppressed, formation of the microtubule bundle linking the centrosome and the posterior cortex did not occur. PEM possibly plays a role in anchoring microtubule ends to the cortex. In our model of orientation of the early-cleavage planes, we also amend the allocation of the conventional animal-vegetal axis in ascidian embryos, and discuss how the newly proposed A-V axis provides the rationale for various developmental events and the fate map of this animal. CONCLUSIONS: The complex cleavage pattern in ascidian embryos can be explained by a simple rule of centrosome attraction mediated by localized PEM activity. PEM is the first gene identified in ascidians that is required for multiple spindle-positioning events.     

Distribution and fluxes of fluorescent whitening agents discharged from domestic wastewater into small rivers with seasonal changes of flow rates

Distributions of fluorescent whitening agents (FWAs) in the waters of a small river system in Japan were surveyed in summer when the water volume rose following high precipitation and in winter when it subsided as precipitation declined. The main source of FWAs in the river system was domestic wastewater, and fluxes in the tributaries depended on the size of the residential population and the elimination rates of sewage treatment systems in their catchments, although FWA concentrations in the river itself fluctuated, largely as a consequence of dilution by heavy precipitation and unstable water flows in the small-sized river system. The FWA concentrations in the river waterduring summer decreased not only by the dilution of river water but also by photodegradation because of more prolonged exposure to sunlight.