In vitro development and postvitrification survival of cloned feline embryos derived from preadipocytes

The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.     

Amaranthus genetic resources in Indonesia: morphological and protein content assessment in comparison with worldwide amaranths

The morphological variations (growth habit, leaf shape, leaf color, height, stem diameter, number of branches and internodes, leaf area, blade ratio, leaf thickness, number of leaves, and total leaf area) and protein content of 53 Indonesian amaranths (Amaranthus spp.), consisting of weedy-, vegetable-, and ornamental-types, were assessed. The extent of variation in the Indonesian Amaranthus accessions were compared with the worldwide collection (26 accessions from United States Department of Agriculture (USDA) and 5 ornamentals from Nepal) in the experimental field of the University of Tsukuba, Japan. The variation in average values of most morphological traits and protein content in the Indonesian accessions were similar to those of the worldwide germplasm, but the important parameters that influence vegetable yield (e.g., number of leaves and stem diameter) were superior in the Indonesian accessions. Protein content showed a positive correlation with the number of leaves, whereas a negative correlation was observed with leaf thickness. The Indonesian accession of A. viridis and A. dubius showed a great potential to be further selected as parental lines for high protein content and number of leaves. The relatively high protein content of the leaves of Celosia may also potentially serve as an alternative protein source in the tropics.     

Squamous cell carcinoma in a digit of the hind limb with systemic metastasis in a 17-year-old female koala

We encountered a case of cutaneous squamous cell carcinoma (SCC) in a 17-year-old female koala at a zoo. A fragile, papillary, elevated mass was found on the third digit of the right hind limb. SCC was identified histopathologically: squamous cell-like polygonal tumor cells showed a nest-like growth pattern with epidermal down growth, central keratinization and necrotic foci, and invaded dermal connective tissues. Metastatic lesions were observed in various organs, including the lung and axillary lymph node: in the lung, multiple metastatic foci similar to the primary lesion, and in the axillary lymph node, individual polygonal tumor cells infiltrated the sinusoids. Immunohistochemistry revealed that the tumor cells were positive for proliferating cell nuclear antigen, which exhibited 32–33% of labeling indices in the tumor cells. To our knowledge, this is the first report of a case of SCC in a digit of a koala.     

Induction of Soil Suppressiveness Against Rhizoctonia solani by Incorporation of Dried Plant Residues into Soil

ABSTRACT Suppressive effects of soil amendment with residues of 12 cultivars of Brassica rapa on damping-off of sugar beet were evaluated in soils infested with Rhizoctonia solani. Residues of clover and peanut were tested as noncruciferous controls. The incidence of damping-off was significantly and consistently suppressed in the soils amended with residues of clover, peanut, and B. rapa subsp. rapifera 'Saori', but only the volatile substance produced from water-imbibed residue of cv. Saori exhibited a distinct inhibitory effect on mycelial growth of R. solani. Nonetheless, disease suppression in such residue-amended soils was diminished or nullified when antibacterial antibiotics were applied to the soils, suggesting that proliferation of antagonistic bacteria resident to the soils were responsible for disease suppression. When the seed (pericarps) colonized by R. solani in the infested soil without residues were replanted into the soils amended with such residues, damping-off was suppressed in all cases. In contrast, when seed that had been colonized by microorganisms in the soils containing the residues were replanted into the infested soil, damping-off was not suppressed. The evidence indicates that the laimosphere, but not the spermosphere, is the site for the antagonistic microbial interaction, which is the chief principle of soil suppressiveness against Rhizoctonia damping-off.     

Influence of weaning age on the villous height and disaccharidase activities in the porcine small intestine

Body weight gain after weaning is correlated with villous height and disaccharidase activity. This evidence suggests that the maintenance of the small intestinal structure and function after weaning is important for the growth of piglets. We demonstrated that the influence of weaning age was obtained by disaccharidase activities and villous height in eight sections of the porcine small intestine. Therefore, we designed three weaning ages (weaned at the ages of 14, 21 or 28 days) and the piglets were slaughtered after 7 or 14 days post‐weaning. The remaining suckling piglets were slaughtered at the age of 1, 7, 14, 21 and 28 days. Four piglets were slaughtered at each event; therefore, 44 piglets were used in this study. Villous height and disaccharidase activities were measured in each section of the small intestine. Early weaning such as that at 14 days had severe influence on villous and disaccharidase activities. In particular, weaning of 14‐day‐olds did not result in maltase activity at least 2 weeks post‐weaning. Accordingly, the weaning age of crossbred piglets is recommended to be at least 21 days or more on the basis of villous height and disaccharidase activity analyses.     

Immunohistochemical localization of enzymes related to lignin biosynthesis in the primary xylem of hybrid aspen

We have investigated the spatial regulation of the accumulation of enzymes involved in the biosynthesis of shikimate and lignin during differentiation of primary xylem from the apical meristem via procambium in hybrid aspen (Populus sieboldii x Populus grandidentata). Immuohistochemical staining revealed that, in the top part of shoots, lignification began in a single or just a few adjacent vessel elements and subsequently spread to neighboring cells. The spatial localization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is one of the key enzymes in the shikimate pathway, was tightly correlated with the cell-specific deposition of lignin in the primary xylem. We also found that the spatial localization of enzymes in the general phenylpropanoid pathway and in the lignin-specific pathway was closely associated with the cell-specific deposition of lignin and the accumulation of DAHPS. Our data suggest that enzymes that act in the shikimate, general phenylpropanoid, and lignin-specific pathways are initially produced and function coordinately in a single or a few adjacent elements at the start of primary xylem development.     

Influence of osmotic pressure on somatic embryo maturation in Pinus densiflora

The effect of an osmoticum, polyethylene glycol (PEG), on somatic embryo production was examined using embryogenic cells of Pinus densiflora. In the basal medium containing 30 μM abscisic acid and 6% maltose, the quality of the embryos formed was poor even though somatic embryos were produced. The addition of PEG with molecular weight of 4000 or 8000 significantly enhanced the development of both the quality and quantity of somatic embryos. Furthermore, higher levels of a constant osmotic pressure with PEG 8000 in a range from about 300 to 450 mmol/kg could remarkably enhance the morphogenesis of somatic embryos and their number of embryos produced. A higher stable osmotic pressure with an appropriate molecular weight of PEG is a key factor for the production of good quality somatic embryos in P. densiflora.     

Perceptions of “close-to-nature forestry” by German and Japanese groups: inquiry using visual materials of “cut” and “dead” wood

It is generally believed that forests are perceived differently according to the cultural background of the visitor. Different cultural groups tend to have diverse perceptions of tree species, landscapes, or gardens. In this research project, we conducted interviews of German and Japanese groups (each group consisting of three or more people), to identify their perspectives in exploring the theme of “cut” and “dead” wood. For this purpose, we interviewed 20 groups from each cultural background (40 data sets in total). Each group was asked to categorize 36 pictures chosen from photographic contests. The results were examined with PCA and cluster analysis. The quantified results of the two national groups highlighted the dichotomy of the cut and dead wood relationship. The method and materials used gave robust results. Differences between the two groups were identified by their perceptions of cut and dead wood. The results are insightful from a methodological and political rationale. First, new insights were gained by adopting group-based interviews using pictures from photographic contests. Research about public perceptions in forest science is often dominated by a focus on verbal inquiry. This study explored similar questions, using visual materials. The interviews consisted of visual materials to overcome bias in languages or the question order. Second, the results indicated the influence of “close-to-nature forestry” policy on public perceptions. While German society has a wide practice of leaving dead wood in forests based on their close-to-nature forestry policy, there is no equivalent Japanese concept. Although Japan and Germany both face pressures for economic restructuring and public participation, the difference in public perception of forestry can lead to a difference in acceptance for the same policy. If such differences are to exist, we can observe that the legitimacy of forestry in the two countries is reconstructed in different ways.     

Formation of the thiocyanate conjugate of chlorogenic acid in coffee under acidic conditions in the presence of thiocyanate and nitrite: possible occurrence in the stomach

The objective of the present study was to elucidate how chlorogenic acid in coffee was transformed under acidic conditions simulating the mixture of saliva and gastric juice. When coffee was incubated in acidified saliva that contained nitrite and SCN-, in addition to nitric oxide (NO), four major components were detected. Two of the four components (components 3 and 4) were generated when chlorogenic acid was incubated in acidified saliva and when incubated in an acidic buffer solution in the presence of both nitrite and SCN-. By the incubation of chlorogenic acid in acidic nitrite in the absence of SCN-, components 3 and 4 were not formed but the quinone of chlorogenic acid and nitrated chlorogenic acid were formed. The result indicates that SCN- was indispensable for nitrous acid induced formation of components 3 and 4. Component 4 was isolated and its structure was determined to be (E)-5'-(3-(7-hydroxy-2-oxobenzo[d] [1,3]oxathiol-4-yl)acryloyloxy)quinic acid. Component 3, which was suggested to be 2-thiocyanatochlorogenic acid, seemed to be formed by the reaction between SCN- and the quinone of chlorogenic acid. As it has been reported that the quinone of chlorogenic acid can react with thiols and can decompose producing H2O2, the formation of component 4 can reduce the toxic effects of the quinone of chlorogenic acid.