Testing the possibility of horizontal transfer of introduced neomycin phosphotransferase (nptⅡ) gene of transgenic Eucalyptus camaldulensis into soil bacteria

The possible horizontal transfer of transgenes is of great concern when the transgenic plants are released into the field. To test the possible transfer of nptⅡ of transgenic trees into soil bacteria, we have used a stool DNA preparation kit to isolate the DNA from the soils in the rhizospheres of two non- and eight transgenic Eucalyptus camaldulensis trees. All the samples have provided the corresponding PCR products in the amplification with bacterial 16S RNA specific sequences, which indicates that the quality of the isolated DNA is adequate for amplification. The nptⅡ specific band has been amplified in three soil samples from the transgenic trees and even treated with filtration before the DNA isolation. This indicates that nptlI DNA exists in the soil, although it is still unclear whether the DNA was in the soil particles, in the soil bacteria or in the Agrobaeterium contamination which was used for the E.camaldulensis transformation. Two approaches on isolation of bacterial DNA have been suggested for testing the possibility of this event in the future.     

Effects of Saccharomyces cerevisiae supplementation on growth performance,plasma metabolites and hormones,and rumen fermentation in Holstein calves during pre‐ and post‐weaning periods

This study aimed to evaluate the effects of supplementing Saccharomyces cerevisiae (SC) during the pre‐ and post‐weaning periods on growth, metabolic and hormonal responses, and rumen fermentation in calves. Three‐week‐old Holstein calves were assigned to either control (n = 12) or SC group (n = 12), the latter of which received 2 × 10⁹ cfu/day of SC. The experiment was conducted over a period of 7 weeks around weaning. Daily gain (DG) in the SC group was higher (p < .05) than that in the control group. In the SC group, plasma glucose, insulin, and growth hormone (GH) concentrations were higher (p < .05) and concentrations of glucagon and insulin‐like growth factor 1 (IGF‐1) tended to be higher (p < .1) than in the control group. Proportion of rumen propionate and concentration of rumen ammonia nitrogen at 10 weeks of age were greater (p < .05) in the SC group than that in the control group. Supplementation of SC around weaning may improve dietary nutrient and energy availability and increase plasma GH and IGF‐1 concentrations. These changes observed in SC‐supplemented calves could be closely related to the improvement of DG.     

Nondestructive measurement of cross-sectional shape of a tree trunk

To evaluate windthrow resistance with respect to stem breakage, a nondestructive method for determining the shape of trunk cross sections was developed. In this method, the coordinates of multiple gauge points set on the perimeter of a trunk are calculated by measuring the distances between them. The shape between the gauge points is generated with the use of a profile gauge placed between them. Measurement tests were conducted using profile gauges with lengths of 300 and 900 mm on model specimens with four shape patterns and four different diameters. The accuracy of the estimation was verified by comparing the section modulus calculated for the generated image and for the photograph. The average ratio of section modulus (generated/photo) for all specimens was 0.994, which indicates that the proposed method is highly accurate. The section moduli of hollow trunks can be evaluated using the profile method together with the drill resistance technique on the condition that 26% of the trunk diameter could be drilled without skew.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Susceptibilities of some aquatic ferns to paddy herbicide bensulfuron methyl

Paddy herbicides have the potential to cause adverse effects on non-target plants. Susceptibilities of some aquatic ferns ( Azolla japonica Franch. et Savat., Isoetes japonica A. Braun, Marsilea quadrifolia L. and Salvinia natans All.) and duckweeds ( Lemna minor L. and Spirodela polyrhiza Schleid.) to paddy herbicide bensulfuron methyl (BSM) were evaluated with a 20 day exposure experiment using 200 cm² pots. The BSM concentrations in the surface water of monitoring pots with no plants dissipated exponentially with half lives of 3.5 and 3.9 days at application rates of 15 and 150 g ha⁻¹, respectively. The BSM concentrations in the surface water 1 day after application in the culture pots were comparable among plant species, and were lower than those in the monitoring pots. Bensulfuron methyl reduced the plant growth in all species. I . japonica showed the lowest intrinsic relative growth rate (RGR) and the lowest susceptibility with an effective dose resulting in 50% growth inhibition (ED₅₀) of 21 g ha⁻¹. Except for I . japonica , the RGR of the duckweeds was similar to the ferns, and ED₅₀ for the duckweeds was higher than the ferns. ED₅₀ for Sa . natans , A . japonica and M . quadrifolia were 1.1, 1.8 and 1.2 g ha⁻¹, respectively, which were smaller than 1/20 of the recommended field dose (51–75 g ha⁻¹) and ranged from 1/2 to 1/6 of ED₅₀ for L . minor and Sp . polyrhiza (6.5 and 3.2 g ha⁻¹, respectively). These results suggest that BSM application in paddy fields and its runoff in some localities is expected to have adverse effects on the growth of Sa . natans , A . japonica and M . quadrifolia .     

Generation and characterization of anti-leptin antisera against synthetic peptides and recombinant protein

The objective of this study was to generate antisera against recombinant bovine leptin and synthetic oligopeptides corresponding to the amino acid sequence 21-40 and 91-110 of bovine leptin. Recombinant bovine leptin was raised in the 293 cells and purified from 10 L of conditioned medium and utilized for immunization. The synthetic peptides were conjugated with keyhole limpet hemocyanin and inoculated into rabbits for antibody generation. Antibody titer was monitored by enzymeimmunoassay, immunoblotting and sandwich binding assay techniques. Each of the antisera, against three different antigens, was found to react with bovine leptin. The titers of anti-peptide antisera were lower than that of anti-recombinant leptin antiserum. Since anti-recombinant leptin antiserum was not neutralized by the leptin peptides 21-40 and 91-110, it is suggested that each antiserum recognizes a distinct epitope. In immunoblot analyses, all antisera exhibited cross-reactivity with human and mouse leptins. However, in the sandwich binding assay, the combination of anti-peptide antisera and anti-recombinant leptin antiserum, originated from bovine leptin, did not cross-react with either human or mouse leptin. The discrepancy of antigenic recognition between the immunoblot analyses and sandwich assay is thought to be dependent on the conformational status of leptin molecules between the species. The antisera generated in this study, which recognized distinct epitopes of bovine leptin, will provide a useful tool for studies of bovine leptin functions.     

Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia

The aim of this study was to compare the osteogenic and proliferative potential of canine mesenchymal stromal cells (cMSCs) derived from bone marrow (BM-cMSCs) and adipose tissue (AT-cMSCs). Proliferation potential was determined under varying oxygen tensions (1%, 5%, and 21% O(2)). Effects of reduced oxygen levels on the osteogenic differentiation of AT-cMSCs were also investigated. AT-cMSCs proliferated at a significantly faster rate than BM-cMSCs, although both cell types showed robust osteogenic differentiation. Culture in 5% and 1% O(2) impaired proliferation in cMSC from both sources and osteogenic differentiation in AT-cMSCs. Our data suggests that AT-cMSCs might be more suitable for use in a clinical situation, where large cell numbers are required for bone repair, due to their rapid proliferation combined with robust osteogenic potential. Our data also suggests that the inhibitory effects of hypoxia on both cell proliferation and differentiation should be considered when using MSCs in a potentially hypoxic environment such as a fracture site.