Structural basis of biological N2O generation by bacterial nitric oxide reductase

Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N(2)O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (Fe(B)) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.     

Sapling architecture and growth in the co-occurring species Castanopsis cuspidata and Quercus glauca in a secondary forest in western Japan

This study quantitatively compared the sapling (height 62–289cm) architecture and growth of Castanopsis cuspidata and Quercus glauca, both of which are major components in the temperate zone of western Japan, under shaded light conditions in secondary forest. When the sapling architectures were compared at the same support mass (trunk + branch mass), C. cuspidata had a larger crown area but a smaller height gain than did Q. glauca owing to the allocation of more biomass to lateral branches in C. cuspidata. The above-ground relative growth rate (RGR) of C. cuspidata (0.442gg^–1 year^–1) was nearly twice that of Q. glauca (0.256gg^–1year^–1), primarily as a result of a greater total leaf area per above-ground biomass (LAR) in C. cuspidata (56cm²g^–1) as compared to Q. glauca (33cm²g^–1). Because it has a disadvantage in height gain, related to its allocation pattern of biomass, C. cuspidata realized the same height growth (RGR_H) as Q. glauca, despite the large biomass production. The great potential for photosynthesis in C. cuspidata, which results from its vigorous lateral spreading, is presumed to give it a long-term advantage over Q. glauca in the shaded forest understory. Q. glauca invests preferentially in trunk biomass, possibly giving it an advantage in sunny sites as opposed to a shaded forest understory.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

Effects of Plant Species on CH4 and N2O Fluxes from a Volcanic Grassland Soil in Nasu, Japan

To investigate the effects of plant species in grassland on methane (CH₄) and nitrous oxide (N₂O) fluxes from soil, fluxes from an orchardgrass ( Dactylis glomerata L.) grassland, white clover ( Trifolium repens L.) grassland and orchardgrass/white clover mixed grassland were measured weekly from April 2001 to March 2002 using a vented closed chamber method. Related environmental parameters (soil inorganic N content, soil pH (H₂O) value, soil moisture content, soil temperature, grass yield, and the number of soil microorganisms) were also regularly monitored. On an annual basis, CH₄ consumption in the soil of the orchardgrass grassland, white clover grassland and orchardgrass/white clover mixed grassland was 1.8, 2.4, and 1.8 kg C ha⁻¹ year⁻¹, respectively. The soil bulk density of the white clover grassland was lower than that of the other grasslands. Fluxes of CH₄ were positively correlated with the soil moisture content. White clover increased the CH₄ consumption by improving soil aeration. Nitrogen supply to the soil by white clover did not decrease the CH₄ consumption in the soil of our grasslands. On the other hand, annual N₂O emissions from the orchardgrass grassland, white clover grassland, and orchardgrass/white clover mixed grassland were 0.39, 1.59, and 0.67 kg N ha⁻¹ year⁻¹, respectively. Fluxes of N₂O were correlated with the NO₃⁻ content in soil and soil temperature. White clover increased the N₂O emissions by increasing the inorganic N content derived from degrading white clover in soil in summer.     

Effects of Various Surfactants on Rheological Properties of Maize Starch Granules

In this study, the formation of complexes between surfactants and the helical chains of amylopectins was confirmed. Nonionic surfactants with hydrophobic and hydrophilic groups of appropriate size and chemical structure enhanced the swelling and gelatinization processes of starch granules. Hydrophobic groups form complexes with the amylose and linear chains of amylopectin by becoming inserted into the hydrophobic inner area of the helical structures. The hydrophilic groups help the approach of the hydrophobic groups into the hydrated molecular chains and thus aid the formation of the complex. Among the anionic surfactants tested, SDS and sodium n‐decyl benzenesulfate caused maximum swelling and gelatinization peaks. The average length of the amylopectin exterior chains is almost the same as that of the hydrophobic chains of SDS (16.9 Å) and of sodium decyl benzenesulfate (18.2 Å). This suggests that these anionic surfactants form rigid complexes with the exterior of the amylopectin by fitting their hydrophobic chains to the hydrophobic inside of the helical structures of these short exterior chains. This process was clarified by NMR analysis and by a decrease in the complex with the addition of iodine. The hydrophobic alkyl chains of anionic and cationic surfactants fix to the edge of the starch molecular chains by forming inclusion complexes with the helical chains of the amylopectin. Cationic ions interact with the starch molecular chains, causing a negative charge that results in a more rapid and efficient swelling of the starch granules. A decrease in setback value occurs due to the inhibition of rearrangement among the starch molecular chains. With SDS, the complex molecular chains become more extensively developed through the repulsion effects of the anionic ions resulting in a larger swelling power and gelatinization peak.     

The applicability of a color acetate film for estimating photosynthetic photon flux density in a forest understory

The suitability of a color acetate film for estimating photosynthetic photon flux density (PPFD) in a forest understory was examined. The fading ratio of the film (F), the total PPFD (PPFD_total) to which the film was exposed, and the average daily maximum temperature during exposure (T) were obtained from measurements at multiple sampling points throughout an entire year within a natural secondary forest (n = 42). The ranges of the recorded values were as follows: F 35%–99%, PPFD_total 1.4–28.3molm^–2, and T 6°–32°C. PPFD_total was regressed by F and T with a high r ² (=0.94; P < 0.0001): PPFD_total = (100 – F)/(1.085 + 0.051T). The absolute error (|estimated PPFD_total – measured PPFD_total|) averaged 1.3molm^–2 with a maximum of 5.7molm^–2, indicating a good fit. These results indicated broad applicability of the film, both spatially and temporally, for estimating forest understory PPFD.     

Addition of Wakame seaweed (Undaria pinnatifida) stalk to animal feed enhances immune response and improves intestinal microflora in pigs

This study was conducted to evaluate the effects of supplementation of Wakame seaweed stalks on the immunity and intestinal microflora of pigs. Three separate experiments were performed: Relatively young (start at 20–30 kg; Experiments 1 and 2) and fattening period (70 kg; Experiment 3). All pigs (including the control group) were fed the same commercial feed, free from antibiotic additives, but in the feed for the treatment groups, 1% seaweed powder was added. There were no group differences observed in daily weight gain and feed intake in Experiments 1 and 2 between groups; however, daily weight gain was significantly higher in the treatment group compared to the control group in Experiment 3. The percentage of peripheral blood natural killer cells of the treatment group was significantly higher than that of the control group in all experiments. Although addition of seaweed changed the gene expression of cytokine and toll‐like receptors of the small intestinal Peyer's patches slightly, seaweed seems to alter intestinal microflora preferentially, for instance, there was an increase in Lactobacillus and a decrease of Escherichia coli observed. These results suggest that Wakame seaweed can be used as supplement for pig feed to improve the gut health and immunity of pigs.     

Multiple copies of <Emphasis Type="Italic">AMT2</Emphasis> are prerequisite for the apple pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis> to produce enough AM-toxin for expressing pathogenicity

The apple pathotype of Alternaria alternata produces the cyclic depsipeptide AM-toxin and causes Alternaria blotch of apple. Previously, we cloned AMT2 from the apple pathotype as an orthologue of AFTS1, which is required for biosynthesis of the decatrienoic acid ester AF-toxin I of the strawberry pathotype. These genes were predicted to encode aldo-keto reductases involved in biosynthesis of a common precursor, 2-hydroxy-isovaleric acid, of AF-toxin I and AM-toxin. In this study, we analyzed the function of AMT2 in AM-toxin biosynthesis in the apple pathotype. DNA gel blot analysis of the apple pathotype strain IFO8984 with five restriction enzymes suggested that this strain has a single copy of AMT2 in the genome. However, gene disruption experiments showed that IFO8984 probably has three copies of AMT2. We made mutants having one or two copies of AMT2 disrupted. The single-copy mutants produced less AM-toxin than did the wild type and were still as pathogenic as the wild type. The two-copy mutants produced trace or undetectable amounts of AM-toxin and were markedly reduced in pathogenicity. Thus, AMT2 was verified to be required for AM-toxin biosynthesis and hence pathogenicity. The fact that the two-copy mutants have a remaining copy of AMT2 suggests that multiple copies of AMT2 are prerequisite for the pathogen to produce enough AM-toxin for full pathogenicity.     

Visualization of musculature of the larval greater amberjack Seriola dumerili (Risso) using whole‐mount immunostaining with special reference to cranial muscles

Muscles play important roles in feeding, respiration and swimming during not only adult stage but also larval stage in fish. However, there is no information of the muscle development in larval greater amberjack, Seriola dumerili (Risso). Here, we investigated muscle development in the greater amberjack, focusing primarily on the cranial muscles from 0 days post hatch (dph) to 12 dph using a modified whole‐mount immunohistochemical staining method. We found that the muscles required for feeding develop by 3 dph, when the larvae begin to feed. Subsequently, muscle composition in the dorsal branchial arches changes to the adult form between 5 and 8 dph. At 8 dph, all the muscles required for feeding and respiration appear, whereas the dorsal, pelvic and caudal fin muscles required for swimming develop later. This report provides fundamental information on larval greater amberjack muscle development, which will enable the detection of abnormal larval muscles and improve larval rearing techniques by modifying environmental conditions.     

First capture of post-spawning female of the Japanese eel Anguilla japonica at the southern West Mariana Ridge

Two adult male freshwater eels, Anguilla japonica, were captured in June 2008 in the West Mariana Ridge (13°N, 142°E) in the North Pacific, but collections of females have yet to be reported. In September 2008, we successfully caught two adult female A. japonica, 55.5 and 66.2 cm in total length, in the adjacent but northern area (14°N, 143°E). Six newly hatched eel larvae (pre-leptocephali) were also collected by subsequent plankton sampling conducted near the female catch area. Female adults appeared to be in the post-spawning state, probably a considerable time after spawning, since a small number of remarkably regressed oocytes (50–250 μm in diameter) were observed in the ovaries. Capture of post-spawning female eels and newly hatched larvae near the Suruga Seamount (14°N) together with the previous collection of mature males in the southern area (13°N) corroborates that the area along the West Mariana Ridge is the spawning area of this species, but suggests that the eel spawning may occur over a wider area than previously expected.