Prediction of grain protein content in winter wheat through leaf color measurements using a chlorophyll meter

A field trial was conducted over a 3-year period at the Hokkaido Kitami Agricultural Experiment Station to examine whether the grain protein content (GPC) of a winter wheat cultivar (Triticum aestivum L. cv. Chihokukomugi) suitable for Japanese noodle-making could be predicted before harvest. The prediction of the GPC was accurate based on the color of the second leaf (just below the flag leaf) at the end of the emergence of the inflorescence, when nitrogen application was graded. In order to evaluate the reliability of this test, a survey of 95 wheat fields in the eastern part of Hokkaido was also carried out during a 3-year period. The prediction of the GPC for this cultivar based on the color of the second leaf was less accurate across many sites. The results of this survey, however, suggested that the leaf color could be used as an index for ranking the GPC as low or high in relation to processing requirements. When the leaf color value of the second leaf measured with a chlorophyll meter at the end of the emergence of the inflorescence was less than 40, it was predicted that the GPC would be lower than the processing requirement. This index could be applied to the cultivars grown in the eastern part of Hokkaido, except for those grown on peat soils.     

Changes in organosulfur compounds in garlic cloves during storage

We determined the changes in the contents of three gamma-glutamyl peptides and four sulfoxides in garlic cloves during storage at -3, 4, and 23 degrees C for 150 days using a validated high-performance liquid chromatography method that we reported recently. When garlic was stored at 4 degrees C for 150 days, marked conversion of the gamma-glutamyl peptides, gamma-L-glutamyl-S-allyl-L-cysteine and gamma-L-glutamyl-S-(trans-1-propenyl)-L-cysteine (GSPC), to sulfoxides, alliin and isoalliin, was observed. Interestingly, however, when garlic was stored at 23 degrees C, a decrease in GSPC and a marked increase in cycloalliin, rather than isoalliin, occurred. To elucidate in detail the mechanism involved, the conversion of isoalliin to cycloalliin in both buffer solutions (pH 4.6, 5.5, and 6.5) and garlic cloves at 25 and 35 degrees C was examined. Decreases in the concentration of isoalliin in both the solutions and the garlic cloves during storage followed first-order kinetics and coincided with the conversion of cycloalliin. Our data indicated that isoalliin produced enzymatically from GSPC is chemically converted to cycloalliin and that the cycloalliin content of garlic cloves increases during storage at higher temperature. These data may be useful for controlling the quality and biological activities of garlic and its preparations.     

Morphological analysis of viral communities in the floodwater of a Japanese paddy field

Capsid size distributions of viral communities in the floodwater of a Japanese paddy field under a long-term fertilizer trial were surveyed during the rice cultivation period by using transmission electron microscopy. The capsid size distributions were monophasic, and the median values fell within the range of 50-70 nm. The quartile intervals were narrow from either 40-50 nm or 50-60 nm to either 60-70 nm or 70-80 nm for most samples. There was no clear seasonal variation in the capsid size distribution of viral communities. The difference in size distributions among different fertilizer plots was also not found. Viral communities in the floodwater were predominated by isometric icosahedral forms. Viruses with octahedral capsids and elongated ones were rare and sporadic in the floodwater.     

Metabarcoding of feces and intestinal contents to determine carnivorous diets in red-crowned cranes in eastern Hokkaido,Japan

The red-crowned crane Grus japonensis in Hokkaido, Japan forms a closed population as a residence that is independent of the mainland population. Based on observations of a limited number of individuals as well as cranes in captivity, red-crowned cranes are omnivores and eat fish, worms, insects and plants in their own territories except in winter, when they are fed with dent corn that is supplied in eastern Hokkaido. DNA metabarcoding based on high throughput sequencing was carried out using universal primer sets for cytochrome oxidase subunit I gene. Feces from 27 chicks collected in June and July in the period from 2016 to 2018 and intestinal contents from 33 adult and subadult cranes that were found dead almost throughout year in 2006–2013 in the field in eastern Hokkaido were used. Although compositions varied considerably in the cranes, both insects and fish were found in adults and subadults to the same extents, while insects were predominant in chicks. Both insects and fish were detected in all seasons for adults and subadults. Horse flies, scarab beetles and weevils accounted for the most of the insects regardless of the life stage. Dace, stickleback, flatfish and sculpin were the major fish species in adults, while chicks ate almost only stickleback. The results provide the first comprehensive data on carnivorous diets in wild red-crowned cranes in eastern Hokkaido as basis for conservation of red-crowned cranes, for which the life style and area continue to change.     

Effects of tris(1,3-dichloro-2-propyl) phosphate on epididymal sperm parameters in adult male rats

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is widely used as a flame retardant and is known to exhibit anti-androgenic effects in vitro and in vivo. To assess the reproductive toxicity potency of TDCIPP, we investigated the effects of 7 days of TDCIPP oral administration on epididymal sperm motion and concentration in adult male Wistar–Imamichi rats. Thirty-five days after the final administration, sperm parameters were evaluated by computer-assisted sperm analysis. Results showed that sperm swimming progression and vigor and sperm concentration in TDCIPP-treated rats were unexpectedly higher than those in control rats. TDCIPP did not significantly affect the percentage of motile sperms or sperm swimming pattern. These results contribute to the understanding of the biological effects of TDCIPP.     

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.     

Determination of total dietary fiber in Japanese foods.

Total dietary fiber was determined in Japanese foods by the Prosky-AOAC method. To accomplish the analyses of unsuitable samples, we introduced a few minor modifications to the versions for (i) seaweed and fruits, (ii) cereals, and (iii) fish and meats. These modified methods were used together with the standard method to obtain results with reasonably good relative standard deviation for 231 foods and 21 groups of mixed foods. In this study, dietary fiber was defined so as not to exclude the nondigestible polysaccharide portions of animal foods. A method was proposed which could estimate more accurately the fiber components of animal foods by measuring the "nondigestible protein" of the fiber sample of the fiber sample by the Biuret colorimetric method, instead of the Kjeldahl method, to avoid deducting the values for aminopolysaccharides. In Japanese diets, the amount of fiber obtained from animals foods was less than 5% of the total intake of dietary fiber.