Cloning of porcine interleukin (IL)-12 receptor beta2 (IL-12Rbeta2) gene and its application to a rapid biological assay for human/porcine IL-12

Porcine IL-12Rbeta2 gene was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs), and its complete nucleotide sequence was determined. To confirm the biological function, the entire open reading frame (ORF) was re-cloned into a mammalian expression vector, pcDNA3.1/Zeo(+), at the downstream of CMV promoter, and introduced to a Th1-like human lymphoma cell line, Jurkat E6-1. Antibiotic-resistant cells retaining the expression construct were selected then, isolated by the limiting dilution method. An established clone (10B10) constitutively expressed chimeric IL-12Rs composed of intrinsic (human) beta1 and extrinsic (porcine) beta2 subunits, and produced interferon (IFN)-gamma in response to IL-12 of both species with optimal PHA/PMA stimulation. The production of IFN-gamma was observed as early as 42 h after culture and appeared to be dose-dependent within the range between 20 and 2000 pg/ml. Thus, this clone not only reacts with IL-12 of both species but also provides a useful tool for quick and sensitive detection of IL-12 bioactivity.     

Inhibin secretion in the golden hamster (Mesocricetus auratus) testis during active and inactive states of spermatogenesis induced by the restriction of photoperiod

Gonadal function in the male golden hamster (Mesocricetus auratus) was investigated during exposure to a short photoperiod condition. Within 3 weeks of exposure to the short photoperiod condition, FSH and testosterone in the plasma significantly decreased, and subsequently immunoreactive (ir)-inhibin significantly decreased. Testicular contents of ir-inhibin and testosterone, and pituitary contents of LH and FSH also significantly decreased by 3 weeks with regression of weight of testes, epididymis and seminal vesicles and sperm head count. Circulating LH varied but not significantly. Thereafter, all reproductive parameters and secretion of LH, FSH, ir-inhibin and testosterone gradually recovered after 17 weeks of exposure even though animals continued to be subjected to the short photoperiod condition. Plasma concentrations of inhibin B and inhibin pro-alphaC were detectable and were significantly decreased after 15 weeks of exposure to the short photoperiod, but their levels were still detectable. Immunopositive reaction of inhibin alpha and betaB subunits was found in Sertoli cells and Leydig cells in the regressed testes of animals subjected to short photoperiod as was also seen in animals before exposure to the short photoperiod. Although the spermatogenic cycle was suppressed like prepubertal animals, the present study showed that the testicular recovery, so-called refractoriness, is functionally different from the developing stage of immature animals, especially with regard to inhibin secretion. The present results showed that changes in FSH preceded changes in inhibin during the regression and recovery phases, indicating that FSH is a major regulatory factor of inhibin secretion in male golden hamsters. The present study also demonstrated that regressed testes still secrete a small amount of bioactive inhibin during exposure to a short-photoperiod condition.     

An epidemic of caprine arthritis encephalitis in Japan: isolation of the virus

A disease characterized by arthritis of carpal joints and occasionally by pneumonia was seen among goats on a farm in Nagano prefecture of Japan in the summer of 2002. Serological investigation was done on 30 goats, that is one group on the farm by agar gel immunodiffusion tests using maedi-visna virus as the antigen, and 19 goats were positive. The caprine arthritis encephalitis virus genome was detected in peripheral blood leukocytes from several antibody-positive goats. Two goats among them were necropsied and used for pathological tests and virus isolation. The isolated virus was serially propagated in fetal lamb lung cell cultures. Goats inoculated with the culture fluid became antibody positive. These results suggested that a CAEV-infected goat had been introduced accidentally to the farm and that subclinical infection occurred among the flock.     

Low pathogenicity H5N2 avian influenza outbreak in Japan during the 2005-2006

At the end of May 2005, a low-pathogenicity avian influenza (LPAI) virus of subtype H5N2 was isolated for the first time from chickens in Japan. Through active and epidemiological surveillance, 5.78 million chickens on 41 farms were found to be affected and 16 H5N2 viruses were isolated. Antigenic analysis revealed antigenic similarity of these isolates. Phylogenetic analysis showed that they originated from a common ancestor and clustered with the H5N2 strains prevalent in Central America that have been circulating since 1994. Experimental infection of chickens with the index isolate (A/chicken/Ibaraki/1/05) demonstrated that this virus replicated efficiently in the respiratory tract without clinical signs, and dust-borne and/or droplet-borne transmission was considered as a possible mode of transmission. These results suggested that the H5N2 LPAI viruses isolated in Japan were highly adapted to chickens.     

Calf production from vitrified bovine sexed embryos following in‐straw dilution

The objective of this study was to develop an in‐straw dilution method suitable for direct transfer of vitrified bovine sexed embryos. Embryo sexing was performed by molecular diagnosis. Several sexed and vitrified‐warmed embryos were transferred after evaluation of morphologically embryonic survival at warming and in‐straw dilution (Evaluation group). The other embryos were immediately directly transferred to recipients without first being expelled from the straws after in‐straw dilution (Non‐evaluation group). The pregnancy rates of vitrified sexed embryos were 38.7% and 34.8% in the Evaluation group and Non‐evaluation group, respectively, which were not significantly different. The viability of lower quality embryos before vitrification tended to be lower (P = 0.087) than that of the higher quality embryos regardless of evaluating embryos after warming and in‐straw dilution. The abortion rates were similar, and there was no difference between the two groups (13.9% and 12.5%, respectively). These results demonstrate that vitrified bovine sexed embryos can be vitrified and diluted by the in‐straw method and that the vitrified and warmed sexed embryos can develop to term.     

Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes

Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non‐stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2‐bis(2‐aminophenoxy) ethane N,N,N’,N’‐tetraacetic acid tetrakis‐acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence.     

Establishment rate of direct-seeded rice in the relay-intercropping system in Kanto region of Japan

The relay intercropping system for cultivating direct-seeding rice and winter cereal is a low-cost method particularly for double cropping, because it eliminates the steps of raising seedlings, paddling, and transplanting. However, in this system, the seedling establishment rate (SER) of rice is low and unstable. The objective of this study was to identify the factors affecting SER to highlight the ways to improve SER. Experiments were conducted in experimental fields in Tsukuba City, Ibaraki Prefecture in 2015 and 2016. To determine the time of rice seed death and to calculate seed survival rates, ‘Akidawara’ seeds were embedded in soil (depth: 3 cm) and dug up after defined periods. We then analyzed the correlation between SER and meteorological factors at two sites (Ibaraki Prefecture and Gunma Prefecture) over the same two years. Based on mean air temperature (MT), we divided the period from seeding to June 20 (when irrigation had been initiated in almost all the fields) into four phases. In Phase 1, the number of days with rainfall (≥5 mm) and soil-wetting days (water potential ≥ ?100 kPa) were significantly and negatively correlated with SER. We found that most of the seed deaths occurred after germination, and the germination rates were presumably affected by water absorption during the low temperature phase. Further investigations are needed to understand the occurrences during the seedling emergence period. This study contributes to a better understanding of the factors affecting variations in the SER of direct-seeded rice grown in the relay-intercropping system.

List of Abbreviations: DSR: direct-seeded rice; SER: seedling establishment rate; TT: thiuram treatment; SSR: seed survival rate; DS: dead seeds; AGS: already germinated seeds; DSRI: direct-seeding of rice in the inter-row spaces of winter cereals; VWC: volume water content; WP: water potential     

Genetic and molecular analysis of the restoration of fertility ( Rf ) genes for Ogura male-sterility from a Japanese wild radish ( Raphanus sativus var. hortensis f. raphanistroides Makino)

Ogura male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. In this study, in order to gain better understanding of the variation and evolution of the restoration of the fertility (Rf) gene for Ogura male-sterile cytoplasm, the nucleotide sequence of the orf687 homologue in the Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides Makino) was analyzed using an F₂ population made with a cross between a Japanese wild radish plant containing the Rf gene and ‘Uchiki-Gensuke’ (a maintainer of Ogura-male sterility). Segregation of male-fertile/-sterile plants in the F₂ generation suggested that another unidentified Rf gene unlinked to orf687 exists in the Japanese wild radish. The genotype of orf687 was determined for each F₂ plant by Southern hybridization with an orf687 gene probe, mismatch-specific endonuclease digestion of PCR products, and direct sequencing of a PCR product. Genotyping revealed that some fertility-restored plants are homozygotic for the ‘Uchiki-Gensuke’ type orf687 allele, supporting the idea that another gene different from orf687 also functions as an Rf gene for Ogura male-sterility. Protein analysis using an antibody raised against the Ogura-specific ORF138 protein suggests a mechanism of fertility restoration by the unidentified Rf similar to that by orf687. Sequence analysis of orf687 from a Japanese wild radish plant and ‘Uchiki-Gensuke’ revealed that both orf687 regions encode a mitochondrially-targeted protein consisting of 687 amino acids with 16 PPR motifs. Comparison of the deduced amino acid sequences with those of the known orf687 sequences from ‘Yuan hong’ and ‘Kosena’ containing Rf and recessive one (rf), respectively, showed that three unique amino acid replacements are present in ORF687 of the Japanese wild radish. Two of the three replacements, that from lysine to isoleucine at position 232 and from asparagine to asparate at position 240, confer negative charges to the protein. Since the Rf of ‘Yuan hong’ was reported to have a unique replacement that confers a negative charge to ORF687 (from asparagine to aspartate at position 170), it is proposed that the amino acid replacements conferring a negative charge to ORF687 are important for determining the status of the Rf/rf gene.