Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.     

Effects of nutrient loadings from catchments on Asajino mire, a small coastal ombrotrophic mire in northernmost Japan

The relationship between water chemistry and vegetation was studied in a coastal ombrotrophic mire in northern Hokkaido, Japan. The distributions of Sphagnum and Phragmites communities were separated clearly by the pH and ion concentration of the peat surface-pore water. The drainage ditches along the road across the center of the mire had a high pH and ion concentration, as did the peat water in the western part of the mire. It was found that fields used for livestock farming on a hill to the west of the mire leached materials into the mire through drainage ditches, surface runoff, and probably also through ground water, and thus influenced the water chemistry of the mire. Management of the water, including that in the catchment of the mire, should be introduced before biological buffering capacity against excess nutrient loading caused by human activity is exceeded and the mire loses its ombrotrophic status.     

Production of M-/GM-group aflatoxins catalyzed by the OrdA enzyme in aflatoxin biosynthesis

Aspergillus parasiticus produces the minor aflatoxins M(1) (AFM(1)), M(2) (AFM(2)), GM(1) (AFGM(1)), and GM(2) (AFGM(2)), as well as the major aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), and G(2) (AFG(2)). Feeding of A. parasiticus with aspertoxin (12c-hydroxyOMST) caused AFM(1) and AFGM(1), and cell-free experiments using the microsomal fraction of A. parasiticus and aspertoxin caused production of AFM(1), indicating that aspertoxin is a precursor of AFM(1) and AFGM(1). Feeding of the same fungus with O-methylsterigmatocystin (OMST) caused AFM(1) and AFGM(1) together with AFB(1) and AFG(1); feeding with dihydroOMST (DHOMST) caused AFM(2) and AFGM(2) together with AFB(2) and AFG(2). Incubation of either the microsomal fraction or OrdA enzyme-expressing yeast with OMST caused production of aspertoxin together with AFM(1) and AFB(1). These results demonstrated that the OrdA enzyme catalyzes both 12c-hydroxylation reaction from OMST to aspertoxin and the successive reaction from aspertoxin to AFM(1). In contrast, feeding of the fungus with AFB(1) did not produce any AFM(1), demonstrating that M-/GM-aflatoxins are not produced from B-/G-aflatoxins. Furthermore, AFM(1) together with AFB(1) and AFG(1) was also produced from 11-hydroxyOMST (HOMST) in feeding experiment of A. parasiticus, whereas no aflatoxins were produced when used the ordA deletion mutant. These results demonstrated that OrdA enzyme can also catalyze 12c-hydroxylation of HOMST to produce 11-hydroxyaspertoxin, which serves as a precursor for the production of AFM(1) and AFGM(1). The same pathway may work for the production of AFM(2) and AFGM(2) from DHOMST and dihydroHOMST through the formation of dihydroaspertoxin and dihydro-11-hydroxyaspertoxin, respectively.     

Directed evolution of lectins with sugar-binding specificity for 6-sulfo-galactose

6-sulfo-galactose (6S-Gal) is a prevalent motif observed in highly sulfated keratan sulfate, which is closely associated with the glioblastoma malignancy while acting as a critical determinant for endogenous lectins. However, facile detection of this unique glycoepitope is greatly hampered because of a lack of appropriate probes. We have previously reported tailoring an α2-6-linked sialic acid-binding lectin from a ricin-B chain-like galactose-binding protein, EW29Ch, by a reinforced ribosome display system following an error-prone PCR. In this study, we challenged the creation of novel lectins to recognize 6S-Gal-terminated glycans by incorporating a high-throughput screening system with a glycoconjugate microarray. After two rounds of selection procedures, 20 mutants were obtained and 12 were then successfully expressed in Escherichia coli, 8 of which showed a significant affinity for 6'-Sulfo-LN (6-O-sulfo-Galβ1-4GlcNAc), which the parental EW29Ch lacked. Analysis of two representative mutants by frontal affinity chromatography revealed a substantial affinity (K(d) ～3 μm) for a 6S-Gal-terminated glycan. On the basis of the observation that all eight mutants have a common mutation at Glu-20 to Lys, site-directed mutagenesis experiments were performed focusing on this aspect. The results clearly indicated that the E20K mutation is necessary and sufficient to acquire the specificity for 6S-Gal. We also confirmed a difference in binding between E20K and EW29Ch to CHO cells, in which enzymes to catalyze the synthesis of 6S-Gal were overexpressed. The results clearly demonstrate that these mutants have potential to distinguish between cells containing different amounts of 6S-Gal-terminated glycans. This new technology will be used to provide novel tools essential for sulfoglycomics.     

Characterization of a tobacco TPK-type K+ channel as a novel tonoplast K+ channel using yeast tonoplasts

The tonoplast K(+) membrane transport system plays a crucial role in maintaining K(+) homeostasis in plant cells. Here, we isolated cDNAs encoding a two-pore K(+) channel (NtTPK1) from Nicotiana tabacum cv. SR1 and cultured BY-2 tobacco cells. Two of the four variants of NtTPK1 contained VHG and GHG instead of the GYG signature sequence in the second pore region. All four products were functional when expressed in the Escherichia coli cell membrane, and NtTPK1 was targeted to the tonoplast in tobacco cells. Two of the three promoter sequences isolated from N. tabacum cv. SR1 were active, and expression from these was increased approximately 2-fold by salt stress or high osmotic shock. To determine the properties of NtTPK1, we enlarged mutant yeast cells with inactivated endogenous tonoplast channels and prepared tonoplasts suitable for patch clamp recording allowing the NtTPK1-related channel conductance to be distinguished from the small endogenous currents. NtTPK1 exhibited strong selectivity for K(+) over Na(+). NtTPK1 activity was sensitive to spermidine and spermine, which were shown to be present in tobacco cells. NtTPK1 was active in the absence of Ca(2+), but a cytosolic concentration of 45 microM Ca(2+) resulted in a 2-fold increase in the amplitude of the K(+) current. Acidification of the cytosol to pH 5.5 also markedly increased NtTPK1-mediated K(+) currents. These results show that NtTPK1 is a novel tonoplast K(+) channel belonging to a different group from the previously characterized vacuolar channels SV, FV, and VK.     

Factors controlling changes in the aquatic macrophyte communities from 1984 to 2009 in a pond in the cool-temperate zone of Japan

We investigated changes in aquatic macrophyte communities over 25 years in Utonai-ko (42°42′N, 141°42′E) in northern Japan and determined the major change-producing factors using canonical correspondence analysis (CCA) of 21 measured hydrochemical variables with potential to influence the occurrence of communities. We then examined the corresponding changes in the 25-year fluctuation trends in the communities and measured variables. The most prominent changes were a decline in the Hippuris community and an increase in the Vallisneria and Myriophyllum communities. CCA revealed that the leading variable was significant wave height (SWH), followed by water depth (WD), total nitrogen (T-N), dissolved oxygen (DO), pH, chlorine ion (Cl^?), transparency, mud thickness, and suspended solids (SS). The Hippuris community was positively correlated with T-N and Cl^? and negatively with SWH, WD, DO, and pH. All of these variables were likewise correlated with the Vallisneria and Myriophyllum communities but in the reverse direction. SS and transparency exhibited no correlations. During the 25 years, WD and T-N increased, but annual maximum wind velocity and Cl^? decreased. Fluctuation of DO was <2 mg ml^?1 and pH was consistent. Considering the direction of correlations and 25-year trends, vital factors for the change in aquatic macrophyte communities were WD and Cl^?. Because the concentration of C1^? was low, the change in aquatic macrophyte communities likely resulted from the increase in WD.     

Induction of NGF synthesis in astrocytes by onjisaponins of Polygala tenuifolia, constituents of kampo (Japanese herbal) medicine, Ninjin-yoei-to.

The effect of a kampo medicine, Ninjin-yoei-to (NYT; Ren-shen-yang-rong-tang in Chinese) on nerve growth factor (NGF) secretion from the cultured rat astrocytes was examined in vitro. When rat embryo astrocytes were cultured in the presence of NYT for 24 h, the amount of NGF in the medium was significantly increased in a dose dependent manner. Among 14 kinds of component herbs in NYT, the roots of Polygala tenuifolia and roots of Panax ginseng extracts increased NGF levels from the astrocytes. Saponin fraction from the roots of P. tenuifolia enhanced the production of NGF, however phenolic glycoside fraction showed no effect. Onjisaponins A, B, E, F and G as major saponins of the root of P. tenuifolia strongly increased the NGF level, whereas ginsenosides Rb1 and Rg1 did not affect the NGF level. Onjisaponin F also induced ChAT mRNA level in rat basal forebrain cells. These results indicate the possibility that NYT and/or onjisaponins in P. tenuifolia may have potential therapeutic effects for the treatment of Alzheimer disease patients.     

Evaluation of a field experiment for the conservation of a <Emphasis Type="Italic">Magnolia stellata</Emphasis> stand using clear-cutting

Magnolia stellata is a rare subcanopy tree species that grows in secondary forests in warm temperate zones. It is now endangered due to habitat degradation by vegetation succession. In an attempt to improve the habitat, a 30 m?×?10 m plot (0.03 ha) was set up with all vegetation including M. stellata being clear-cut in January 2012. The number of sprouts increased for 1–2 years after clear-cutting and then gradually decreased or remained constant. Five years after clear-cutting, the numbers of individuals and stems, and the total basal area (BA), were 87.0, 165.5 and 3.2%, respectively, of the values before clear-cutting. BA was highest for Ilex pedunculosa, followed by M. stellata and Hydrangea paniculata. Some sprouted individuals of M. stellata produced flower buds in the second year after clear-cutting, and flowered and fruited in the spring and summer of the third year, respectively. The densities of potential canopy species were 18,533 ha^?1 (height >?0.5 m) and 7,267 ha^?1 (height >?1.2 m), vastly exceeding the value of the criterion for successful natural regeneration after clear-cutting of warm temperate forests in the region (3,000 ha^?1). Based on this criterion, it is thus considered that the natural regeneration has reached completion. However, 45.1% (height >?0.5 m) and 95.5% (height >?1.2 m) of M. stellata individuals were regenerated by sprouting. Further research is needed into how individuals, regenerated from seedlings, develop and reach sexual maturity, and how successive generations change.