Basic characterization of avian β‐defensin genes in the Japanese quail,Coturnix japonica

In this study, we identified a cluster of 14 avian β‐defensins (AvBD; approximately 66 kbp) in the Japanese quail, Coturnix japonica. Except for AvBD12 (CjAvBD12) and ‐13, the CjAvBDs coding sequences exhibited greater than 78.0% similarity to the respective orthologous chicken AvBD genes (GgAvBD). The putative amino acid sequence encoded by each CjAvBD contained six cysteine residues and the GXC (X1‐2) motif considered essential for the β‐defensin family. Each CjAvBDs also formed a sub‐group with the respective orthologous genes of various bird species in a phylogenetic tree analysis. Synteny between the CjAvBD cluster and GgAvBD cluster was confirmed. The CjAvBD cluster was mapped on the long‐arm end of chromosome 3 by linkage analysis based on single nucleotide polymorphisms (SNPs) of CjAvBD1 and CjAvBD12 (approximately 46kbp), as well as GgAvBD cluster. We also confirmed that CjAvBD1, ‐4, ‐5, ‐9, and ‐10 are transcribed in 20 tissues, including immune and digestive tissues. However, our experimental data indicated that the CjAvBD cluster lacks the AvBD3 and ‐7 loci, whereas the CjAvBD101α, ‐101β, and ‐101θ loci arose from gene duplication of the AvBD6 orthologous locus in the CjAvBD cluster after differentiation between Coturnix ‐ Gallus.     

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Development of a shuttle polymerase chain reaction for the detection of bovine herpesvirus 2

Three different polymerase chain reaction (PCR) protocols were evaluated for their ability to detect bovine herpesvirus 2 (BoHV-2): single-step PCR with 3 reaction stages (denaturation, annealing and extension), 2 reaction stages (denaturation and annealing/extension; shuttle PCR), and semi-nested PCR with 3 reaction stages. All the PCR protocols showed the same sensitivity (detection limit of 0.4 TCID(50)). A non-specific band sometimes appeared in mock cell DNA at annealing temperatures below 64 degrees C. The shuttle PCR was found to be superior to the other protocols under consideration because of the speed of its application. Furthermore, no non-specific band was detected in DNAs of eight other DNA viruses. Thus, the shuttle PCR seems to be an excellent diagnostic tool for BoHV-2 infections.     

Prevention of progeny formation in Drosophila melanogaster by 1-arylimidazole-2(3H)-thiones

Effects of 1-arylimidazole-2(3H)-thiones (AITs) and 1-(substituted benzyl)imidazole-2(3H)-thiones (BITs) were tested on progeny formation in Drosophila melanogaster. Some AITs showed inhibitory activities at laying eggs and delayed eclosion by 1 day. The inhibitory activity was nullified by adding octopamine (OA) or noradrenaline (NA) to the medium for progeny formation in D. melanogaster. The effect of AIT on the contents of OA and NA was analyzed in adults of D. melanogaster by high-performance liquid chromatography with electrochemical detection. Flies fed with AIT decreased OA and NA levels and increased TA content. Taken together, the inhibitory activity of AIT could be due to inhibition of tyramine β-hydroxylase and dopamine β-hydroxylase.     

Root and stem rot of chrysanthemum caused by five <Emphasis Type="Italic">Pythium</Emphasis> species in Japan

Root and stem rot with wilt of above ground parts of cultivated chrysanthemums was first found in Ibaraki, Toyama and Kagawa prefectures, Japan in 2002 and 2003. Pythium species were isolated from the diseased tissues and identified as P. dissotocum, P. oedochilum, P. sylvaticum, P. ultimum var. ultimum and asexual strains of P. helicoides based on their morphologies and sequences of rDNA-ITS region. All the Pythium species were strongly pathogenic to chrysanthemums in pot conditions and were reisolated from the inoculated plants. Because Pythium root and stem rot of chrysanthemum has never been reported in Japan, we propose that this is a new disease that can be caused by the five Pythium species.     

Analysis of mesh breaking loads in cotton gill nets: Possible solution to ghost fishing

ABSTRACT:   A small number of fishers in Chiba Prefecture of eastern Japan use cotton gill nets to catch Japanese spiny lobster Panulirus japonicus. To examine the advantages of cotton gill nets, we analyzed changes in mesh breaking load of a new cotton gill net used in a fishing operation. A new cotton gill net was also soaked in a seawater tank to simulate ghost fishing conditions. The average mesh breaking load of new cotton mesh was 50.3 N. This value decreased to 19.0 N after 38 days (∼912 h), and after 82 days (∼1968 h) the mesh could be easily torn (breaking load 0.07 N). Under fishing conditions, the cumulative soak time was only 744.4 h over 19 months. The average breaking load at the end of this period was 43.1 N, a strength 86% that of the presoaked mesh. The mesh breaking load of a cotton gill net continuously soaked for 744.4 h was 26.1 N, as estimated from tank experiment data. Thus, a cotton gill net maintains reasonable strength under typical use conditions, but will degrade if lost at sea.     

Reactivity of a condensed–type lignin model compound in the Mannich reaction and preparation of cationic surfactant from sulfuric acid lignin

 The chemical conversion of phenolized sulfuric acid lignin (P-SAL), prepared from sulfuric acid lignin (SAL) by phenolation with sulfuric acid catalyst, to novel cationic surfactant was investigated. To elucidate the chemical reactivity of the P-SAL to a Mannich reaction, 1-guaiacyl-1-p-hydroxyphenylethane (I) as a simple phenolized sulfuric acid lignin model compound was reacted with dimethylamine and formaldehyde. Quantitative analysis of the products by gas-liquid chromatography suggested that the p-hydroxyphenyl nucleus was more reactive than the guaiacyl nucleus. The Mannich reaction of SAL with dimethylamine did not yield a soluble cationic surfactant, but P-SAL produced water-soluble cationic surfactant in a quantitative yield. The Mannich reaction products (MP-SAL) of P-SAL had 1,3-dimethylaminomethyl groups/C₉-C₆. The results of the surface tension measurements showed that the decrease in surface tension of MP-SAL was much larger than that of lignosulfonate as a commercial surfactant from lignin.     

Genotype distribution of Raffaelea quercivora in the oak galleries and its composition in the mycangia of Platypus quercivorus

Raffaelea quercivora is the pathogenic fungus that causes Japanese oak wilt. The female monogynous ambrosia beetle, Platypus quercivorus, carries this fungus in mycangia on the pronotum. These beetles bore galleries in oak trees with their partners to produce offspring, and they deposit fungus on the gallery walls from their mycangia. The offspring mature in the gallery, before loading the fungal pathogen and flying from the gallery to other healthy trees. To investigate the unloading and loading modes of the fungus within the gallery, we developed four polymorphic microsatellite markers for R. quercivora and identified the fungal genotypes in the galleries and mycangia of the beetles. Small wood chips were sampled at 5–10‐mm intervals from the walls of five galleries in a dead Quercus serrata tree. The pronota were also sampled from five female adult beetles. The genotypes of the R. quercivora isolates from the wood chips and pronota were identified using the microsatellite makers. The genotypic analysis showed that each gallery was inhabited patchily by 5–10 genotypes of R. quercivora, and the mycangia of each beetle contained 3–6 genotypes. These results indicate that diverse R. quercivora genotypes are unloaded repeatedly from the mycangia of female beetles onto the gallery wall, which results in their patchy distribution on the walls. When the offspring leave the host tree, the fungal clones that proliferate in the walls are also loaded repeatedly into the mycangia of the mature beetles.