A single gene transfer of gibberellin biosynthesis gene cluster increases gibberellin production in a Fusarium fujikuroi strain with gibberellin low producibility

Fusarium fujikuroi, the causative agent of bakanae disease in rice, produces many kinds of secondary metabolites. Recently, two phylogenetic subgroups (F and G groups) of Japanese F. fujikuroi have been identified and found to have differences in their gibberellin (GA) and fumonisin production. G-group F. fujikuroi produces large amounts of GA, but is a fumonisin nonproducer. F-group produces large amounts of fumonisin, but is a GA low or nonproducer. We investigated the cause of low GA production in the F-group. Genetic mapping suggests that low GA production in the F-group strain Gfc0825009 is due to a GA gene cluster for GA biosynthesis. Analysis of the nucleotide and amino acid sequences of the genes in the GA gene cluster showed >98.4% homology between the F-group strain Gfc0825009 and the G-group strain Gfc0801001. Following a 7-day culture under low nitrogen conditions, we found that expression of P450-1, P450-4, and P450-2 in the cluster increased in the G-group strain and not in the F-group strain. We hypothesized that complementation by GA genes in the G-group strain would be required to increase GA production in the F-group strain. However, we found that this occurred with a single gene complementation of DES, P450-1, P450-4, or P450-2. Simultaneous increase in the expression of P450-1, P450-4, and P450-2 were detected in the complementary transformants. Moreover, the same phenomenon was observed by reintegration of its own P450-1. Our results suggest the presence of unknown regulatory mechanisms of the GA gene cluster in F. fujikuroi.     

Enhancement of interleukin-2 production by bovine peripheral blood mononuclear cells treated with the combination of anti-programmed death-ligand 1 and cytotoxic T lymphocyte antigen 4 chimeric monoclonal antibodies

Our previous studies demonstrate the therapeutic efficacy against bovine diseases of an anti-bovine programmed death-ligand 1 (PD-L1) chimeric antibody. In humans, PD-1 and PD-L1 antibodies are more effective when combined with an antibody targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and these combination therapies are therefore clinically used. Here we generated an anti-bovine CTLA-4 chimeric antibody (chAb) to enhance the therapeutic efficacy of the PD-L1 antibody. We further analyzed the effects of dual blockade of CTLA-4 and PD-1 pathways on T-cell responses. The established anti-bovine CTLA-4 chAb showed comparable blocking activity on the binding of bovine CTLA-4 to CD80 and CD86 as the anti-bovine CTLA-4 mouse monoclonal antibody. Anti-bovine CTLA-4 chAb also significantly increased IL-2 production from bovine peripheral blood mononuclear cells (PBMCs). Further, the combination of anti-CTLA-4 chAb with anti-PD-L1 chAb significantly upregulated IL-2 production by PBMCs. These results suggest that the combination of antibodies have higher potential to enhance immune responses against pathogens compared with single administration.     

Nitrification, ammonia-oxidizing communities, and N2O and CH4 fluxes in an imperfectly drained agricultural field fertilized with coated urea with and without dicyandiamide

Agricultural soil is a major source of nitrous oxide (N₂O), and the application of nitrogen and soil drainage are important factors affecting N₂O emissions. This study tested the use of polymer-coated urea (PCU) and polymer-coated urea with the nitrification inhibitor dicyandiamide (PCUD) as potential mitigation options for N₂O emissions in an imperfectly drained, upland converted paddy field. Fluxes of N₂O and methane (CH₄), ammonia oxidation potential, and ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) abundances were monitored after the application of PCU, PCUD, and urea to upland soil. The results showed that urea application increased the ammonia oxidation potential and AOB and AOA abundances; however, the increase rate of AOB (4.6 times) was much greater than that of AOA (1.8 times). These results suggested that both AOB and AOA contributed to ammonia oxidation after fertilizer application, but the response of AOB was greater than AOA. Although PCU and PCUD had lower ammonia oxidation potential compared to urea treatment, they were not effective in reducing N₂O emissions. Large episodic N₂O emissions (up to 1.59 kg N ha^?1 day^?1) were observed following heavy rainfall 2 months after basal fertilizer application. The episodic N₂O emissions accounted for 55–80 % of total N₂O emissions over the entire monitoring period. The episodic N₂O emissions following heavy rainfall would be a major source of N₂O in poorly drained agricultural fields. Cumulative CH₄ emissions ranged from ?0.017 to ?0.07 kg CH₄ ha^?1, and fertilizer and nitrification inhibitor application did not affect CH₄ oxidation.     

Reproductive biology of male skipjack tuna <Emphasis Type="Italic">Katsuwonus pelamis</Emphasis> (Linnaeus) in the tropical western and central Pacific Ocean

The reproductive biology of male skipjack tuna Katsuwonus pelamis was examined from May 2005 to December 2007 in the tropical western and central Pacific Ocean. Testis maturity was classified into five stages (i.e., immature, early maturation, mid maturation, late maturation, and regression) on the basis of morphological changes in the germinal epithelium. The testis duct system (TS), which was characterized by lobules that lack spermatocysts, store only sperm, and form an anastomosing network, was located under the main sperm duct. The TS was observed at all stages of maturity except the immature stage, and first appeared in individuals with a fork length (FL) of 38–40 cm. The estimated minimum size at first maturity was 35.5 cm FL and the size at 0.5 maturity was 40.7 cm in FL. It was difficult to clearly differentiate the testis maturity stage based solely on the gonad index (GI) class because each GI class contained mature fish. Monthly changes in testis height index and GI did not follow a seasonal cycle. Mature individuals dominated throughout the year. These results imply that male skipjack tuna in this area showed evidence of spawning activity throughout the year without a clear seasonal pattern.     

Relationships between ammonia oxidizers and N2O and CH4 fluxes in agricultural fields with different soil types

Nitrous oxide (N₂O) is a greenhouse gas that contributes to the destruction of stratospheric ozone, and agricultural soil is an important source of N₂O. Aerobic soils are sinks for atmospheric methane (CH₄), a greenhouse gas. Ammonia monooxygenase (AMO) can oxidize CH₄, but CH₄ is mostly oxidized by methane monooxygenase (MMO), and CH₄ oxidation by AMO is generally negligible in the soil. We monitored the N₂O and CH₄ fluxes after urea application in fields containing different soils using an automated sampling system to determine the effects of environmental and microbial factors on the N₂O and CH₄ fluxes. The soil types were Low-humic Andosol (Gleyic Haplic Andosol), yellow soil (Gleyic Haplic Alisol) and gray lowland soil (Entric Fluvisol). Cumulative N₂O emissions from the yellow soil were higher than those from other soil types, although the difference was not significant. The CH₄ uptake level by Andosol was one order of magnitude higher than that by other soils. There were significant relationships between the ammonia oxidation potential, AOB and AOA amoA copy numbers, and the CH₄ uptake. In contrast, the gene copy numbers of methane-oxidizing bacteria (MOB) pmoA were below the detection limit. Our results suggested that the AMOs of AOB and AOA may have more important roles than those previously considered during CH₄ oxidation in agricultural soils treated with N fertilizers.     

Production of bioactive bovine fibroblast growth factor 4 in E. coli based on the common nucleotide sequence of its structural gene in three breeds

Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the proper development of bovine embryos. We recently determined the FGF4 gene sequence in eight cattle derived from three breeds and revealed a common nucleotide sequence of the structural gene encoding FGF4, which leads to the deletion and mutation of amino acid sequences in the mature FGF4 (Pro³²‐Leu²⁰⁶) compared with the sequence previously reported. In the present study, HisbFGF4, a 6× histidine‐tagged bovine FGF4 (Pro³²‐Leu²⁰⁶), was produced in Escherichia coli based on the validated nucleotide sequence and purified by heparin column chromatography. In primary bovine fibroblasts, HisbFGF4 showed significant mitogenic activity, whereas, intriguingly, the activity of a commercially available recombinant human FGF4 (Gly²⁵‐Leu²⁰⁶) produced in E. coli was weaker than that of HisbFGF4. In conclusion, the present study provides a simple method for the production of a bioactive bovine FGF4 derivative in E. coli utilizing its structural gene elucidated by us.     

Effect of modified ultrafiltration on cytokines and hemoconcentration in dogs undergoing cardiopulmonary bypass

Cardiac surgery using cardiopulmonary bypass (CPB) generates severe inflammatory reactions secondary to hemodilution and surgical stress. This study was conducted to evaluate whether modified ultrafiltration (MUF) could be performed safely and to clarify its effects during mitral valve repair in dogs in terms of hemodilution and the status of inflammatory cytokines. We retrospectively studied 38 dogs with mitral valve disease who underwent MUF immediately after mitral valve repair under CPB. To determine the effect of MUF, we measured the pre- and post-MUF blood dilution and blood cytokine levels. The levels of red blood cells, hematocrit (HCT), and albumin were significantly increased after MUF, whereas interleukin (IL)-6 levels were significantly increased from 24.3 (range 9.6–54.6) to 32.3 (15.9–65.1) pg/ml. The levels of IL-8 and IL-10 declined significantly after MUF, from 368.2 (246.1–669.4) and 45.4 (28.6–76.1) to 272.2 (174.1–414.4) and 28.8 (18.8–44.5) pg/ml, respectively. Our results demonstrated that MUF can be applied in dogs undergoing CPB and is effective in achieving hemoconcentration. Moreover, MUF may be useful for the removal of cytokines. Further studies are needed to validate these findings and clarify the effects of inflammatory cytokines after CPB.