Evaluation of the transmission risk of foot-and-mouth disease in Japan

The transmission risk of foot-and-mouth disease (FMD) in Japan was evaluated using a mathematical FMD transmission model. The distance-based transmission rate between farms, which was parameterized using the FMD epidemic data in 2010 in Japan, was used to calculate the local-level reproduction numbers—expected numbers of secondary infections caused by one infected farm—for all cattle and pig farms in the country, which were then visualized as a risk map. The risk map demonstrated the spatial heterogeneity of transmission risk in the country and identified risk areas with higher possibility of disease spread. This result suggests that, particularly in high-risk areas, it is important to prepare for the smooth and efficient implementation of control measures against FMD outbreaks.     

Comparative study of dermal components and plasma TGF-β1 levels in Slc39a13/Zip13-KO mice

Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-β1 (TGF-β1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-β1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-β1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-β1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-β1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-β homeostasis.     

Preparation of calibration standards of N1-H paralytic shellfish toxin analogues by large-scale culture of cyanobacterium Anabaena circinalis (TA04)

Mouse bioassay is the official testing method to quantify paralytic shellfish toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported. Some methods have been evaluated by a single laboratory validation. Among the different types of methods, chemical analyses are capable of identifying and quantifying the toxins, however a shortage of the necessary calibration standards hampers implementation of the chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in 10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration of 1.3 ± 0.1 μmol/L. The intracellular and extracellular toxins occupied 80% and 20%, respectively. Over 220 μmol of the toxins was obtained from approximately 200 L of the culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues. The toxins were chemically converted to six N1-H analogues. Preparation of the analogues was carried out at relatively high yields (50-90%). The results indicate that our preparation method is useful to produce N1-H toxins. In our present study, detailed conditions for preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated.     

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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.     

Clarification of water movement properties in a multi-soil-layering system

Maize (Zea mays L.) cv. T42 was grown in refined sand at low (0.1 μM) and normal (30 μu) concentrations of boron each under low (1 mM), normal (4 mM), and excess (8 mM) supply of calcium. Visible symptoms of boron deficiency which appeared first, were accentuated by calcium deficiency and were least evident when calcium was added in excess. The yield was maximum at normal levels of boron and calcium and was the lowest under boron and calcium deficiency.

In maize leaves when both calcium and boron were deficient together the activity of starch phosphorylase increased markedly and that of ribonuclease and polyphenol oxidase also increased. The increase in the calcium content inhibited the starch phosphorylase activity when boron was deficient. The activity of peroxidase was stimulated under boron deficiency at all levels of calcium and that of ATPase was depressed significantly when calcium was deficient alone.

A decrease in the tissue boron (except in old leaves) and tissue calcium content as well as sugar and starch contents was observed under the combined deficiency of calcium and boron.

Excess calcium at both levels of boron increased the tissue boron and calcium contents and decreased the concentration of starch, sugars, nucleic acids, total and inorganic phosphorus, and the activities of starch phosphorylase and ATPase.

The activity of ATPase increased upon the addition of calcium to plants deficient in calcium and boron respectively. The tissue concentration of the element added increased when the element was applied to calcium or boron deficient maize plants.     

Comparison of PCR assays for detection and quantification of Phomopsis sclerotioides in plant and soil

We developed a real-time PCR assay using a TaqMan probe (TM-qPCR) for specific detection and quantification of Phomopsis sclerotioides, causal agent of black root rot of cucurbit crops. The design of the primer sets and hybridization probe was based on the internal transcribed spacer region of the ribosomal DNA. The TM-qPCR assay was compared with a conventional, standard PCR (sPCR) assay and on a quantitative real-time PCR (SG-qPCR) assay based on SYBR Green I. The TM-qPCR assay had a detection limit of ca. 0.4 fg of P. sclerotioides DNA, which was approximately 100 times more sensitive than the sPCR assay and almost equivalent to the SG-qPCR assay. The TM-qPCR and SG-qPCR assays both were able to detect various quantities of P. sclerotioides DNA from diseased plants and infested soils, including DNA levels that were not detectable by the sPCR assay. However, the TM-qPCR was advantageous for samples containing PCR-inhibiting substances because its multiplex real-time PCR function allows the adjustment of cycle threshold values with an internal control. Based on the high specificity and sensitivity required for analyzing DNA in natural samples, the newly developed TM-qPCR assay was the most reliable tool for rapidly detecting and quantifying P. sclerotioides in plant and soil samples.     

Domain composition of rhamnose-binding lectin from shishamo smelt eggs and its carbohydrate-binding profiles

Osmerus (Spirinchus) lanceolatus egg lectin (OLL) is a member of the rhamnose-binding lectin (RBL) family which is mainly found in aqueous beings. cDNA of OLL was cloned, and its genomic architecture was revealed. The deduced amino acid (aa) sequence indicated that OLL was composed of 213 aa including 95 aa of domain N and 97 aa of domain C. N and C showed 73 % sequence identity and contained both -ANYGR- and -DPC-KYL-peptide motifs which are conserved in most of the RBL carbohydrate recognition domains. The calculated molecular mass of mature OLL was 20,852, consistent with the result, and 20,677.716, from mass spectrometry. OLL was encoded by eight exons: exons 1 and 2 for a signal peptide; exons 3–5 and 6–8 for N- and C-domains, respectively. Surface plasmon resonance spectrometric analyses revealed that OLL showed comparable affinity for Galα- and β-linkages, whereas Silurus asotus lectin (SAL), a catfish RBL, bound preferentially to α-linkages of neoglycoproteins. The Kd values of OLL and SAL against globotriaosylceramide (Gb3) were 1.69 × 10^?5 M for and 2.81 × 10^?6 M, respectively. Thus, the carbohydrate recognition property of OLL is slightly different from that of SAL. On the other hand, frontal affinity chromatography revealed that both OLL and SAL interacted with only glycolipid-type oligosaccharides such as Gb3 trisaccharides, not with N-linked oligosaccharides. The domain composition of these RBLs and an analytical environment such as the “cluster effect” of a ligand might influence the binding between RBL and sugar chains.     

Self-EcoTILLING to identify single-nucleotide mutations in multigene family

TILLING (Targeting Induced Local Lesions IN Genomes) is a low-cost, high-throughput reverse genetic technique that employs a mismatch-specific endonuclease CEL-1 to discover induced point mutations in the genes of interest. The use of the TILLING technique to survey natural variation in genes is called EcoTILLING. Here, we report a modified EcoTILLING method for the discovery of mutations in multigene family, which we coin “Self-EcoTILLING” by using an allotetraploid Monochoria vaginalis ALS multigene family as an example. The mutations could be detected by TILLING of PCR products resulting from the primers specific to both Als1 and Als3 without involving the experimental step of mixture of reference and query DNA. Either of the two co-amplified loci could serve as reference DNA to the other. We demonstrate with this example that Self-EcoTILLING is a fast, reliable and economical technique of detecting single-nucleotide mutations in polyploid plants containing multigene family.     

Electrophoretic karyotyping and mapping of pathotype-specific DNA sequences in Japanese isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG)₅, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate’s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.