Manipulated Mouse Embryos as Bioassay System for Water Quality Control

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K⁺ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D‐O) water and Milli‐Q system (M‐Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA‐supplemented protein‐free culture media, whereas in EDTA‐ and protein‐free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M‐Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein‐free culture media were very low (0–7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M‐Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.     

Accuracy of imputation of single nucleotide polymorphism marker genotypes from low‐density panels in Japanese Black cattle

Using target and reference fattened steer populations, the performance of genotype imputation using lower‐density marker panels in Japanese Black cattle was evaluated. Population imputation was performed using BEAGLE software. Genotype information for approximately 40 000 single nucleotide polymorphism (SNP) markers by Illumina BovineSNP50 BeadChip was available, and imputation accuracy was assessed based on the average concordance rates of the genotypes, varying equally spaced SNP densities, and the number of individuals in the reference population. Two additional statistics were also calculated as indicators of imputation performance. The concordance rates tended to be lower for SNPs with greater minor allele frequencies, or those located near the ends of the chromosomes. Longer autosomes yielded greater imputation accuracies than shorter ones. When SNPs were selected based on linkage disequilibrium information, relative imputation accuracy was slightly improved. When 3000 and 10 000 equally spaced SNPs were used, the imputation accuracies were greater than 90% and approximately 97%, respectively. These results indicate that combining genotyping using a lower‐density SNP chip with genotype imputation based on a population of individuals genotyped using a higher‐density SNP chip is a cost‐effective and valid approach for genomic prediction.     

Androgen induces production of male effect pheromone in female goats

Previously we showed that the primer pheromone responsible for the "male effect" was produced in specific skin regions of castrated male goats by androgen treatments. In the present study, we examined whether androgen can also induce production of the male effect pheromone in female goats. Capsules containing dihydrotestosterone (DHT) or testosterone (T) were subcutaneously implanted into six ovariectomized (OVX) goats for 28 days. Small skin samples were collected from the head and rump regions, and the pheromone activity of their ether extracts was examined using a bioassay that monitors the electrophysiological manifestation of the hypothalamic gonadotropin-releasing hormone pulse generator as multiple-unit activity. Behaviors of OVX goats towards ovary-intact estrous goats were also examined before and at the end of DHT or T treatment. Before androgen treatment, neither the head nor rump skin samples in OVX goats showed pheromone activity. DHT treatment induced pheromone activity in the head skin sample of six OVX goats and in the rump skin sample of two OVX goats. Similar results were obtained by T treatment. In addition, OVX goats treated with T showed masculine-type sexual behaviors such as courtship and mounting behaviors towards the estrous goats. These results demonstrate that androgen is capable of inducing primer pheromone activity in the female and suggest that the synthesis pathway of the male effect pheromone exists in both sexes in the goat.     

Expression and functional analysis of chemokine receptor 7 in canine lymphoma cell lines

C-C chemokine receptor 7 (CCR7) contributes to cell homing to lymph nodes (LNs). Recent studies reported that CCR7 is also expressed in tumor cells, which correlates with LN metastasis in various cancers. However, the expression of CCR7 in tumor cells is unknown in dogs due to the lack of appropriate antibodies. In the present study, a fusion protein of C-C chemokine ligand 19 (CCL19) was employed as an alternative method to CCR7 antibodies. The fusion CCL19 protein specifically detected CCR7 expressed in canine lymphoma cell lines, which showed active chemotaxis to both canine and mouse ligands. The present study will help further research on the involvement of canine CCR7 in LN metastasis.     

Ceramide aminoethylphosphonate from jumbo flying squid Dosidicus gigas attenuates the toxicity of cyanotoxin microcystin-LR

We have previously established the method for isolation of ceramide aminoethylphosphonate (CAEP) from jumbo flying squid Dosidicus gigas. In this study, we performed a MTT assay to evaluate the safety of CAEP to the cell lines for the application to health food and supplements. The CAEP did not show any cytotoxicity to various HEK293-transfectant cells. Next, we elucidated the positive function of CAEP to the somatic cells. Recently, we have reported that hepatotoxin microcystin-LR was taken up into the hepatocytes mediated by hepatocellular uptake transporters OATP1B1 and OATP1B3, and the cells were induced cytotoxicity subsequently. Cytotoxicity of microcystin-LR to permanently OATP1B3-expressing HEK293-OATP1B3 cells rather than to HEK293-OATP1B1 cells was preferentially attenuated by CAEP in a concentration-dependent manner. In addition, the enzyme activity of serine/threonine phosphatase, which was inhibited by microcystin-LR, was recuperated by co-exposure to CAEP. Furthermore, microcystin-LR-induced cellular protein phosphorylation were disrupted by CAEP exposure. These results suggested that CAEP is a promising remedy and/or preventive medicine for liver damage with microcystin-LR.     

Effects of a macrolide antibiotic on enamel formation in rat incisors--primary lesion of ameloblast at the transition stage

A novel macrolide antibiotic was administered orally to 5-week-old Jcl:Wistar rats at a dose of 5,000 mg/kg/day for 5 weeks, and then a half of animals were maintained without any treatment for 10 weeks. A white discolored lesion with horizontal stripes developed on the surface of the upper and lower incisors after dosing for 4 weeks, and these macroscopical incisal lesions disappeared with the eruption in 4 weeks after stop of administration. Histopathologically, increase in number of karyopycnosis of ameloblast at the transitional stage, vacuolar degeneration of ameloblast and cystic change in the maturation stage, and impaired iron pigment secretion at the pigmentation stage were observed. Microradiography, calcio-traumatic zones, which means hypocalcification, were observed on the superficial layer of enamel. These results suggest that the primary lesion induced by a novel macrolide antibiotic is the increased karyopycnosis of ameloblast at the transitional stage, and followed by later stage.     

Down regulation of a novel protein,PSP, in rat hepatoma cdRLh 84-beared tumor

Perchloric acid-soluble protein (PSP) is a members of a new hypothetical family (YER057c/YJGF family) of small proteins with presently unknown function. The high degree of evolutionary conservation of these proteins reflect an involvement in basic cellular regulation. The expression of PSP was examined in rat hepatoma cell dRLh 84-beared rats. The tumor weight increased to 4.24 g at 3 weeks after the transplantation of hepatoma cells and hepatoma which has less differentiated characteristics were observed in rat liver. The expression of PSP in rat hepatoma was down regulated as compared with intact tissue. Thus the expression of PSP seems to be associated with the differentiation process in these transformed cells. On the other hand, some positive cells against the PSP-antibody were observed in the central region of tumor tissue by immunohistochemistry. These cells were shown to be the lymphocytes and the macrophages. The involvement of PSP to cellular growth and differentiation is discussed.     

Genetic and biological comparison of tick-borne encephalitis viruses from Hokkaido and far-eastern Russia

We compared the biological properties of Oshima 5-10 (tick-borne encephalitis [TBE] virus isolated in Hokkaido, Japan) and Sofjin-HO (Far-Eastern subtype TBE virus) including plaque formation, virus replication and virus protein synthesis in BHK-21 cell cultures to reveal strain differences. We also determined the complete nucleotide sequences of both strains and compared the deduced amino acid sequences. Plaques of Oshima 5-10 were smaller than those of Sofjin-HO. Virus titers in culture fluid of Oshima 5-10 were 1/100 of those of Sofjin-HO at 9 and 12 hr after infection. Less viral protein and RNA syntheses of strain Oshima 5-10 was observed than with Sofjin-HO. Genetic analysis revealed 1.4% of amino acids to differ with Sofjin-HO. No difference between the two strains was detected in the motif sequence of the viral enzyme, cleavage sites of viral protein or glycosylation sites of NS1.     

Nucleotide sequence homology to bovine viral diarrhea virus 2 (BVDV 2) in the 5′ untranslated region of BVDVs from cattle with mucosal disease or persistent infection in Japan

Cytopathogenic and non-cytopathogenic bovine viral diarrhea viruses (BVDVs) were isolated from cattle with mucosal disease or persistent infection in Japan. These isolates were compared for antigenic properties by cross-neutralization tests with Japanese reference strains of BVDV belonging to classical type 1. Significantly low cross-reactivity to reference strains was noted, indicating the viruses to possibly represent a new serotype in Japan. Thus, to determine the genotype of the isolates, nucleotide sequences of the 5′ untranslated region were determined and compared with those of previously reported BVDV 1 and 2. The isolates were clearly shown to belong to BVDV 2, not to BVDV 1.