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.     

Growth of the collembolan Folsomia candida Willem in soil supplemented with glucose

Freely available glucose improves the conditions for soil microorganisms which are utilized as food by Collembola. We examined the effects of glucose application on collembolan (Folsomia candida Willem) growth and on several biotic and abiotic soil parameters (microbial biomass, soil respiration, qCO₂, dissolved organic carbon, inorganic nitrogen, and Olson-P) in an artificial system without predatory pressure on Collembola. Glucose addition increased soil respiration and qCO₂, and decreased nutrient levels in the soil. Collembolan growth increased with increasing glucose doses. We conclude that the availability of carbon substrates can sustain collembolan growth via an improvement of microbial growth conditions.     

Pressure-induced denaturation of monomer beta-lactoglobulin is partially irreversible: comparison of monomer form (highly acidic pH) with dimer form (neutral pH)

This study was conducted to assess the effect of high hydrostatic pressure on monomer beta-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy under pressure and by circular dichroism (CD) and (1)H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of center of spectral mass or ANS fluorescence was almost complete upon pressure release. No difference in (1)H NMR spectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the conformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost, although its retinol binding ability was the same as its unpressurized one. Furthermore, CD spectra of the far-UV region and 2D NMR spectra evidently revealed the difference in the conformation of the molecule between unpressurized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.     

Effect of intermittent drainage in reduction of methane emission from paddy soils in Hokkaido,northern Japan

ABSTRACT

Emission of methane (CH₄), a major greenhouse gas, from submerged paddy soils is generally reduced by introducing intermittent drainage in summer, which is a common water management in Japan. However, such a practice is not widely conducted in Hokkaido, a northern region in Japan, to prevent a possible reduction in rice grain yield caused by cold weather. Therefore, the effects of intermittent drainage on CH₄ emission and rice grain yield have not been investigated comprehensively in Hokkaido. In this study, we conducted a three-year field experiment in Hokkaido and measured CH₄ and nitrous oxide (N₂O) fluxes and rice grain yield to elucidate whether the reduction in CH₄ emission can be achieved in Hokkaido as well as other regions in Japan. Four experimental treatments, namely, two soil types [soils of light clay (LiC) and heavy clay (HC) textures] and two water management [continuous flood irrigation (CF), and intermittent drainage (ID)], were used, and CH₄ and N₂O fluxes were measured throughout the rice cultivation periods from 2016 to 2018. Cumulative CH₄ emissions in 2016 were markedly low, suggesting an initially low population of methanogens in the soils presumably due to no soil submergence or crop cultivation in the preceding years, which indicates a possible reduction in CH₄ emission by introducing paddy-upland crop rotation. Cumulative CH₄ emissions in the ID-LiC and ID-HC plots were 21–91% lower than those in the CF-LiC and CF-HC plots, respectively, whereas the cumulative N₂O emissions did not significantly differ between the different water managements. The amount of CH₄ emission reduction by the intermittent drainage was largest in 2018, with a comparatively long period of the first drainage for 12 days in summer. Rice grain yields did not significantly differ between the different water managements for the entire 3 years, although the percentage of well-formed rice grains was reduced by the intermittent drainage in 2018. These results suggest that CH₄ emission from paddy fields can be reduced with no decrease in rice grain yield by the intermittent drainage in Hokkaido. In particular, the first drainage for a long period in summer is expected to reduce CH₄ emission markedly.     

Preventive effects of CIDR-based protocols on premature ovulation before timed-AI in Ovsynch in cycling beef cows

Ovsynch is a program developed to synchronize ovulation for timed breeding. In this paper, the authors investigate whether controlled internal drug release (CIDR)-based protocols prevent premature ovulation before timed-artificial insemination (AI) when Ovsynch is started a few days before luteolysis in cycling beef cows. Nine beef cows at 16 days after oestrus were treated with (1) Ovsynch, i.e. gonadotropin releasing hormone (GnRH) analogue on day 0, prostaglandin (PG) F(2alpha) analogue on day 7 and GnRH analogue on day 9 with timed-AI on day 10, (n=3); (2) Ovsynch+CIDR (Ovsynch protocol plus a CIDR for 7 days from day 0, n=3), or (3) oestradiol benzoate (OB)+CIDR+GnRH (OB on day 0 in lieu of the first GnRH treatment, followed by the Ovsynch+CIDR protocol, n=3). In the Ovsynch group (1) plasma progesterone concentrations fell below 0.5 ng/mL earlier (day 5) than in both CIDR-treated groups (2) and (3), where this occurred on day 8. Plasma oestradiol-17beta concentrations peaked on day 8 in the Ovsynch group and on day 9 in both CIDR-treated groups. The dominant follicle ovulated on day 10 in the Ovsynch group and on day 11 in both CIDR-treated groups. Thus, both CIDR-based protocols prevented premature ovulation before timed-AI in Ovsynch when the protocol was started a few days before luteolysis. This reflects the fact that progesterone levels remained high until the beef cattle were treated with PGF(2alpha).     

Quantitative structure—activity relationships of light-dependent herbicidal 4-pyridone-3-carboxanilide derivatives II. Substituent effects of anilide and pyridone moieties

The variations in the light-dependent herbicidal activity against Echinochloa oryzicola of a number of 4-pyridone-3-carboxanilides with various substituents on the anilide and pyridone rings have been examined by use of physicochemical substituent parameters and regression analysis. The effects of substituents of the anilide ring were such that the activity was related parabolically not only with the substituent hydrophobicity (π) but also with the steric bulk parameter (E_s) of the bulkier ortho substituent. The effect of substituents at the 1-position of the pyridone ring was analysed with steric(STERIMOL L and B₅) and electronic(γ₁) parameters. That of substituents at the 5-position of the pyridone ring was explained by a parabolic function of a steric (MR) parameter. Analyses of the activity of 105 analogs confirmed that our previous selection of a candidate compound in this series for field trials was indeed appropriate in terms of physicochemical substituent effects.     

Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.     

Novel plasmodesmata association of dehydrin-like proteins in cold- acclimated Red-osier dogwood (Cornus sericea)

Dehydrins are proteins associated with conditions affecting the water status of plant cells, such as drought, salinity, freezing and seed maturation. Although the function of dehydrins remains unknown, it is hypothesized that they stabilize membranes and macromolecules during cellular dehydration. Red-osier dogwood (Cornus sericea L.), an extremely freeze-tolerant woody plant, accumulates dehydrin-like proteins during cold acclimation and the presence of these proteins is correlated with increased freeze tolerance (Karlson 2001, Sarnighausen et al. 2002, Karlson et al. 2003). Our objective was to determine the location of dehydrins in cold-acclimated C. sericea stems in an effort to provide insight into their potential role in the freeze tolerance of this extremely cold hardy species. Abundant labeling was observed in the nucleus and cytoplasm of cold-acclimated C. sericea stem cells. In addition, labeling was observed in association with plasmodesmata of cold-acclimated vascular cambium cells. The unique association of dehydrin-like proteins with plasmodesmata has not been reported previously.     

Development of a quantification assay for the cysts of the toxic dinoflagellate <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> using real-time polymerase chain reaction

ABSTRACT:   The cysts of toxic dinoflagellate Alexandrium tamarense are the seed population for the bloom responsible for paralytic shellfish poisoning (PSP). However, it is impossible to identify the Alexandrium spp. cyst on the basis of morphological features. In this study, we prepared A. tamarense cysts by sexual conjugation in laboratory conditions and developed an efficient DNA extraction method for polymerase chain reaction (PCR) assay. Using the A. tamarense cysts, we established the identification and quantification method showing the species specificity and the high sensistivity for A. tamarense cysts using real-time PCR. This assay was also able to detect and quantify the A. tamarense cysts accurately when mixed with excess cysts of A. catenella (Whedon and Kofoid) Balech prepared by conjugation experiment.