Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV‐bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum‐free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum‐free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S‐200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin‐Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.     

Ghrelin and Leptin Did Not Improve Meiotic Maturation of Porcine Oocytes Cultured In Vitro

To improve culture system for in vitro maturation (IVM) of porcine oocytes, ghrelin, leptin or growth hormone (GH), at concentration of 0, 0.5, 5, 50 and 500 ng/ml were added to the porcine follicular fluid (pFF)‐supplemented medium NCSU23, and their effects on the maturation and cytoskeletal distribution of the oocytes with or without cumulus cells were compared. In the cumulus‐denuded oocytes, no significant changes were noted in the maturation rate by different hormone treatments due to a marked decline in the controls. Maturation of the cumulus intact oocytes was moderately interfered by ghrelin (0.5–50 ng/ml, p < 0.01), but not significantly affected by leptin and GH. Distribution density of the cytoplasmic microtubules was decreased significantly by addition of ghrelin (by approximately 30% in 50–500 ng/ml, p < 0.01), whereas no remarkable effect was noted by leptin supplementation. High concentration (500 ng/ml) of ghrelin or leptin decreased significantly the cytoplasmic microfilaments in density (by 43% and 38%, p < 0.01, respectively). GH did not affect cytoskeletal distribution. The results suggest, in the culture system using pFF‐supplemented medium that (i) ghrelin may have some inhibitory effect on the organization of microtubules and microfilaments, probably being a factor in lowered maturation rate and (ii) the addition of higher concentration of leptin may decrease microfilaments in density with no effect on meiotic maturation of the porcine oocytes.     

Interactions between canine RAD51 and full length or truncated BRCA2 BRC repeats

In humans, mutations in the gene for the breast cancer susceptibility protein BRCA2 affect its interactions with the recombinase RAD51 and are associated with an increased risk of cancer. This interaction occurs through a series of eight BRC repeat sequences in BRCA2. A mammalian two-hybrid assay using individual BRC repeats demonstrated that BRC6 did not bind to RAD51, whereas there was strong (BRC1, 2 and 4), intermediate (BRC8), or weak (BRC3, 5 and 7) binding of other BRC repeats to RAD51. In serial deletion mutation experiments, binding strengths were increased when the C-terminal BRC repeat was removed from BRC1-8, BRC1-5 and BRC1-3. These results may provide an insight into the effects of missense or truncation mutations in BRCA2 in canine tumours.     

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP), prostaglandin E2 (PGE2) and growth hormone releasing factor (GRF) on the release of growth hormone from cultured bovine anterior pituitary cells in vitro

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on growth hormone (GH) release was compared with that of prostaglandin E₂ (PGE₂) and growth hormone releasing factor (GRF) from cultured bovine anterior pituitary cells in vitro. Both PACAP and PGE₂ stimulated GH release at concentrations as low as 10⁻⁹ and 10⁻⁸ M, respectively, (P<0.01). However, GRF released GH at a concentration as low as 10⁻¹³ M (P<0.01). Percent increases of GH compared with controls were not significantly different among GRF, PACAP, and PGE₂ at 10⁻⁷ M; however, the increases of GH by the 10⁻⁸ M GRF, PACAP and PGE₂ were 196, 118, and 27%, respectively, (P<0.01), and 124, 65, and 1% in the 10⁻⁹ M media, respectively, (P<0.01). When GRF and somatostatin (SS) were added together, the GH releasing effect of GRF was blunted (P<0.01). Similar bluntness were observed in PACAP and PGE₂, when SS was added. The stimulatory effects of GRF and PGE₂ together were similar to that by either GRF or PGE₂ alone. When GRF and PACAP were added together, the GH released by both secretagogues was greater than that by PACAP alone (P<0.01); however, a synergistic effect was not clear when compared with GRF alone.

These findings suggest that PACAP and PGE₂ may modulate the release of GH in cattle.     

Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut

Rhipicephalus (Boophilus) microplus is an ectoparasite responsible for an important decrease in meat, milk and leather production, caused both by cattle blood loss and by the transmission of anaplasmosis and babesiosis. R. microplus is a rich source of serine protease inhibitors, including the trypsin inhibitors BmTI-A and BmTI-6, the subtilisin inhibitor BmSI, and the recently described thrombin inhibitor, boophilin. Boophilin is a double Kunitz-type thrombin inhibitor, with the unusual ability to form a ternary complex with a second (non-thrombin) serine proteinase molecule. The large-scale expression and purification of boophilin and of its isolated N-terminal (D1) domain in Pichia pastoris, its expression profile, and the effect of RNAi-mediated gene silencing in tick egg production are reported. Full-length boophilin and D1 were expressed at 21 and 37.5mg/L of culture, respectively. Purified boophilin inhibited trypsin (K(i) 0.65 nM), neutrophil elastase (K(i) 21 nM) and bovine thrombin (K(i) 57 pM), while D1 inhibited trypsin and neutrophil elastase (K(i) of 2.0 and 129 nM, respectively), but not thrombin. Boophilin gene silencing using RNAi resulted in 20% reduction in egg weight production, suggesting that the expression of boophilin in this life stage would be important but not vital, probably due to functional overlap with other serine proteinase inhibitors in the midgut of R. microplus. Considering our data, Boophilin could be combining with other antigen in a vaccine production for tick control.     

The influence of altitude on endophytic mycobiota in Quercus acuta leaves collected in two areas 1000 km apart

Endophytic fungi were isolated from healthy leaves of the evergreen oak Quercus acuta on Mt Takao in eastern Honshu and on Mt Osuzu in Kyushu, Japan, to study the effect of altitude on endophyte assemblages. Phomopsis sp. 1, Discula sp. and an unidentified species (QA-b) were isolated most frequently. Comparisons of the nucleotide sequence of the rDNA ITS regions of QA-b revealed a close match with species of Tubakia. On Mt Takao, Phomopsis sp. 1 and QA-b were isolated frequently, whereas on Mt Osuzu, Discula sp. and QA-b were dominant. The isolation frequency of QA-b decreased as altitude increased, whereas the isolation frequencies of Phomopsis sp. 1 and Discula sp. increased with altitude. In vitro, QA-b did not grow at temperatures <15°C whereas Phomopsis sp. 1 and Discula sp. still grew at 5°C. These results suggest that Phomopsis sp. 1 and Discula sp. are better adapted to lower temperatures and consequently to higher altitude than QA-b.     

Radiation use efficiency,N accumulation and biomass production of high-yielding rice in aerobic culture

The concept of aerobic culture is to save water resource while maintaining high productivity in irrigated rice ecosystem. This study compared nitrogen (N) accumulation and radiation use efficiency (RUE) in the biomass production of rice crops in aerobic and flooded cultures. The total water input was 800–1300 mm and 1500–3500 mm in aerobic culture and flooded culture, respectively, and four high-yielding rice cultivars were grown with a high rate of N application (180 kg N ha⁻¹) at two sites (Tokyo and Osaka) in Japan in 2007 and 2008. The aboveground biomass and N accumulation at maturity were significantly higher in aerobic culture (17.2–18.5 t ha⁻¹ and 194–233  kg N ha⁻¹, respectively) than in flooded culture (14.7–15.8 t ha⁻¹ and 142–173 kg N ha⁻¹) except in Tokyo in 2007, where the surface soil moisture content frequently declined. The crop maintained higher N uptake in aerobic culture than in flooded culture, because in aerobic culture there was a higher N accumulation rate in the reproductive stage. RUE in aerobic culture was comparable to, or higher than, that in flooded culture (1.27–1.50 g MJ⁻¹ vs. 1.20–1.37 g MJ⁻¹), except in Tokyo in 2007 (1.30 g MJ⁻¹ vs. 1.37 g MJ⁻¹). These results suggest that higher biomass production in aerobic culture was attributable to greater N accumulation, leading to higher N concentration (N%) than in flooded culture. Cultivar differences in response to water regimes were thought to reflect differences in mainly (1) early vigor and RUE under temporary declines in soil moisture in aerobic culture and (2) the ability to maintain high N% in flooded culture.