Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor

Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3×10⁶ U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.     

The fate and tissue disposition of deoxynivalenol in broiler chickens

To evaluate the fate of deoxynivalenol (DON) in broilers, DON was administered either intravenously or orally to broilers at a dose of 1 mg/kg BW. Concentrations of DON in plasma were measurable up to 4 hr and 2 hr after intravenous and oral administration, respectively. Following intravenous administration, the values for the elimination half-life, the volume of distribution and the clearance were 1.25 ± 0.25 hr, 7.55 ± 2.03 l/kg and 4.16 ± 0.42 l/hr/kg, respectively. The oral bioavailability was 15.46 ± 4.02%. DON was detectable in all tissues examined after oral administration. These results suggest that DON is able to penetrate into the various tissues in broilers, though poorly absorbed from their gastrointestinal tract.     

Methane production potential and methanogenic archaeal community structure in tropical irrigated Indian paddy soils

Soil characteristics regulate various belowground microbial processes including methanogenesis and, consequently, affect the structure and function of methanogenic archaeal communities due to change in soil type which in turn influences the CH₄ production potential of soils. Thus, five different soil orders (Alfisol, Entisol, Inceptisol, Podzol and Vertisol) were studied to assess their CH₄ production potential and also the methanogenic archaeal community structure in dryland irrigated Indian paddy soils. Soil incubation experiments revealed CH₄ production to range from 178.4 to 431.2 μg CH₄ g^-1 dws in all soil orders as: Vertisol<Inceptisol<Entisol<Podzol<Alfisol. The numbers of methanogens as quantified using real-time quantitative polymerase chain reaction (qPCR) targeting mcrA genes varied between 0.06 and 72.97 (×10⁶ copies g^-1 dws) and were the highest in Vertisol soil and the least in Alfisol soil. PCR-denaturing gradient gel electrophoresis (DGGE)-based approach targeting 16S rRNA genes revealed diverse methanogenic archaeal communities across all soils. A total of 43 DGGE bands sequenced showed the closely related groups to Methanomicrobiaceae, Methanobacteriaceae, Methanocellales, Methanosarcinaceae, Methanosaetaceae and Crenarchaeota. The composition of methanogenic groups differed among all soils and only the Methanocellales group was common and dominant in all types of soils. The highest diversity of methanogens was found in Inceptisol and Vertisol soils. Methane production potential varied significantly in different soil orders with a positive relationship (p?<?0.05) with methanogens population size, permanganate oxidizable C (POXC) and CO₂ production. The present study suggested that CH₄ production potential of different soils depends on physicochemical properties, methanogenic archaeal community composition and the population size.     

Bacterial community in plant residues in a Japanese paddy field estimated by RFLP and DGGE analyses

Plant residues (PRs) are “hot spots” of microbial activities in soil. PRs with the size more than 0.5 mm were collected from a Japanese paddy field during rice cultivation period (from May to September) and fractionated into four categories by size (>4, 2-4, 1-2, and 0.5-1 mm) using sieves. Restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) patterns were compared among the fractions after DNA extraction from the PRs and PCR amplification. The total amount of PRs with the size over 0.5 mm decreased in the field with the first-order kinetics (r²=0.810, p<0.01) with time from rice transplanting to harvest. RFLP analysis showed that the bacterial community structure in PRs with the 0.5-2 mm fraction was different from that in PRs with the >2 mm fraction and the latter community structure changed after the midseason drainage. In contrast, the DGGE patterns of the bacterial community in the PRs indicated the succession from June to September during rice cultivation forming three major groups irrespective of the fraction size. Sequence analysis of DGGE bands showed that Firmicutes (clostridia), α-, γ-, δ-Proteobacteria (myxobacteria), Nitrospira, Acidobacteria, Bacteroidetes, Verrucomicrobia and Spirochaetes were predominant members in the PRs irrespective of fraction size.     

Community structure of bacteria and fungi responsible for rice straw decomposition in a paddy field estimated by PCR-RFLP analysis

Rice straw including leaf sheaths and blades put in nylon mesh bags was placed in the plow layer of a Japanese paddy field after harvest under upland conditions and after transplanting of rice seedlings under flooded conditions. In addition, rice straw that was decomposed under the upland conditions during the off-crop season in winter was placed again in soil at the time of transplanting. The materials were collected periodically to analyze the community structure of the bacteria and fungi responsible for rice straw decomposition by PCR-RFLP analysis. The PCR products with 27f and 1492r primers designed for bacterial 16S rDNA and with EF3 and EF4 primers designed for fungal 18S rDNA were digested with four restriction endonucleases (Hinf I, Sau3A I, Hae III, EeoR I). Bacterial communities in the decomposing rice straw were different from each other between upland and flooded conditions, between leaf sheaths and blades, and between straw samples with and without decomposition under upland conditions during the off-crop season. Fungal communities in the decomposing rice straw were also different between the leaf sheaths and blades under upland soil conditions. Score plots of bacterial and fungal communities in the principal component analysis were separated from the plot of the straw materials along with the duration of the placement, indicating the succession of bacterial and fungal communities in decomposing rice straw with time.     

Bacterial communities associated with nodal roots of rice plants along with the growth stages: Estimation by PCR-DGGE and sequence analyses

Bacterial communities in rice roots that developed from different nodes and at different growth stages were compared by using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA. Rice root samples were collected at three stages, namely tillering (July 2), maximum tillering (July 21), and ripening (September 12). The bacterial diversity in rice roots was found to increase along with the growth stages of the rice plants as well as the root age from the numbers of DGGE bands. The community structure of the bacteria was also found to change with the growth stages and root age from cluster analysis. Sequence analysis of the DGGE bands indicated that the dominant bacteria associated with rice roots were Gram-negative bacteria, especially β-Proteobacteria irrespective of the growth stages and root age. DGGE bands related to Janthinobacterium agaricidamnosum W1r3T and Clostridium sp. FCB90-3 were ubiquitous in many roots irrespective to the sampling date. Principal component analysis enabled to characterize the DGGE bands related to nitrogen-fixing Azoarcus spp., and Azovibrio sp. BS20-3 in the samples collected on July 2 and on July 21, and the myxobacteria collected on September 12, respectively, as representative bacteria in the bacterial communities. The habitat around older rice roots at every sampling date was more reductive than that around younger rice roots, and the DGGE bands related to Spirochaeta spp. were specific in older roots at every sampling date. Some specific bacteria that were most closely related to the DGGE bands were found from principal component analysis to characterize young and old. roots at each growth stage as follows: aerobes Flavobacterium sp. 90 clone 2 and Janthinobacterium agaricidamnosus W1r3T in young roots and facultative anaerobes Dechloromonas sp. MissR and Anaeromyxobacter dehalogenans 2CP-3 in old nodal roots on July 2, strict anaerobe Geobacter pelophilus Dfr2 and aerobes Nitrosospira sp. Nsp17 and uncultured Nitrospira sp. clone 4-1 in old roots on July 21, and different Clostridium spp. in both young and old roots and Desulfovibrio magneticus RS-1 in old roots on September 12, respectively. A larger number of the closest relatives of anaerobic bacteria grew at the late stage than at the early stages, and in old roots than in younger roots. Thus, the environment of paddy roots was remarkably heterogeneous as a bacterial habitat, where not only the whole root system but also a root may create oxic and anoxic environments.     

On the semi-micro determination of total nitrogen (part 4) the decision of number of samples in rice experiment fields

To obtain a value of soil total nitrogen, which represents nitrogen content of soils of rice field under experiment, statistical consideration will be required, because the value of total nitrogen of soil may differ from sample to sample, owing to heterogeneity of the soil in the field.     

Microbial communities responsible for the decomposition of rice straw compost in a Japanese rice paddy field determined by phospholipid fatty acid (PLFA) analysis

To identify the microbial communities responsible for the decomposition of rice straw compost in soil during the rice cultivation period, phospholipid fatty acid (PLFA) composition of rice straw compost was determined by periodically sampling the compost from a Japanese rice field under flooded conditions. About 21% of the compost was decomposed within a period of 3 months. The total amount of PLFAs, as an indicator of microbial biomass, was significantly lower under drained conditions than under flooded conditions and was relatively constant during the flooding period. This indicates that the microbial biomass in the compost samples did not increase during the gradual decomposition of rice straw compost under flooded conditions. The proportion of branched-chain PLFAs (biomarker of Grampositive and anaerobic Gram-negative bacteria) slightly decreased during the early period after placement, and increased gradually afterwards. Among the branched-chain PLFAs, i15:0, ail5:0, i16:0 and i17:0 PLFAs predominated and their proportions increased gradually except for i16:0. The proportion of straight mono-unsaturated PLFAs (biomarker of Gramnegative bacteria) was almost constant throughout the period, and 18:1ω9 and 18:1ω7 PLFAs predominated. The proportion of straight poly-unsaturated PLFAs as a biomarker of eukaryotes including fungi was also constant throughout the period, except for a decrease under drained conditions. Straight poly-unsaturated PLFAs consisted mainly of 18:2ω6c PLFA. Therefore, these results suggest that the proportions of Gram-positive and anaerobic Gram-negative bacteria increased during the decomposition of rice straw compost in flooded paddy field. Statistical analyses enabled to divide PLFA patterns of microbiota in the rice straw compost into two groups, one group consisting of rice straw compost samples collected before mid-season drainage and the other of samples collected after mid-season drainage. Small squared distances among samples in cluster analysis indicated that the community structure of microbiota was similar to each other as a whole. These results suggest that the microbial communities changed gradually during the period of placement, and that mid-season drainage may have affected the community structure of microbiota. Principal component analysis of the PLFA composition suggested that the succession of microbiota along with the decomposition in flooded soil was similar between rice straw compost and rice straw and that the changes in the community structure during the decomposition in flooded soil were more conspicuous for rice straw than for rice straw compost.     

Nutrient storage in termite (Macrotermes bellicosus) mounds and the implications for nutrient dynamics in a tropical savanna Ultisol

The role of mounds of the fungus-growing termite Macrotermes bellicosus (Smeathman) in nutrient recycling in a highly weathered and nutrient-depleted tropical red earth (Ultisol) of the Nigerian savanna was examined by measuring stored amounts of selected nutrients and estimating their rates of turnover via the mounds. A study plot (4?ha) with a representative termite population density (1.5?mounds?ha^?1) and size (3.7?±?0.4?m in height, 2.4?±?0.2?m in basal diameter) of M. bellicosus mounds was selected. The mounds were found to contain soil mass of 9249?±?2371?kg?ha^?1, composed of 7502?±?1934?kg?ha^?1 of mound wall and 1747?±?440?kg?ha^?1 of nest body. Significant nutrient enrichment, compared to the neighboring topmost soil (Ap1 horizon: 0–16?cm), was observed in the nest body for total nitrogen (N) and exchangeable calcium (Ca), magnesium (Mg) and potassium (K), and in the mound wall for exchangeable K only. In contrast, available (Bray-1) phosphorus (P) content was found to be lower in both the mound wall and the nest body than in the adjacent topmost soil horizon. Consequently, the mounds formed by M. bellicosus contained 1.71?±?0.62?kg?ha^?1 of total N, 0.004?±?0.003?kg?ha^?1 of available P, 3.23?±?0.81?kg?ha^?1 of exchangeable Ca, 1.11?±?0.22?kg?ha^?1 of exchangeable Mg and 0.79?±?0.21?kg?ha^?1 of exchangeable K. However, with the exception of exchangeable K (1.2%), these nutrients amounted to less than 0.5% of those found in the topmost soil horizon. The soil nutrient turnover rate via M. bellicosus mounds was indeed limited, being estimated at 1.72?kg?ha^?1 for organic carbon (C), 0.15?kg?ha^?1 for total N, 0.0004?kg?ha^?1 for available P, 0.15?kg?ha^?1 for exchangeable Ca, 0.05?kg?ha^?1 for exchangeable Mg, and 0.06?kg?ha^?1 for exchangeable K per annum. These findings suggest that the mounds of M. bellicosus, while being enriched with some nutrients to create hot spots of soil nutrients in the vicinity of the mounds, are not a significant reservoir of soil nutrients and are therefore of minor importance for nutrient cycling at the ecosystem scale in the tropical savanna.