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.     

Cell-cycle-dependent Colonization of Mouse Spermatogonial Stem Cells After Transplantation into Seminiferous Tubules

Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5–10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.     

Evaluation of Live Feeds for Larval and Postlarval Mahimahi Coryphaena hippurus

Live hatchery feeds were assayed for fatty acids (FA), amino acids (AA), and their ability to support growth and survival of larval and postlarval mahimahi Coryphaena hippurus at two different hatchery stages. Euterpina acuritrons copepods (C), mahimahi yolk-sac larvae (YSL), Artemia parthenogenica brine shrimp nauplii (BSn), A. parthenogenica juveniles (BSj), and Brachionus plicatilis rotifers (R) were assayed, using several enrichment media. There was little difference in AAs among feeds.
Levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were about 10 times higher in YSL than in their feeds. This explains previous findings where first stage larval survival (0–9 days) was not affected by feed HUFA levels. Second stage survival was significantly higher when larvae were fed copepods. Enrichment with 100 ppm SuperSelco greatly improved the survival of larvae that were fed brine shrimp. The even higher omega-3 fatty acids found in copepods appear to be important for survival of larvae under more stressful conditions.
Brine shrimp juveniles enriched with SuperSelco are a good food for postlarval mahimahi. Yolksac larvae of mahimahi are an even better food, promoting faster growth at less cost, for large scale mahimahi aquaculturists. Different batches of yolksac mahimahi larvae varied by a factor of 10 in their concentration of DHA, but always had the highest level of DHA as much as 40% of total fatty acids (FAs). These "high HUFA" batches of YSL also had the highest levels of EPA and total fatty acids. The data suggest that climate and broodstock age may have considerable influence on larval nutrition.     

Long Term Trend of Chemical Constituents in Tokyo Metropolitan Area in Japan

In recent years, acid rain has been a social problem all over the world. In Japan, it is also a big problem especially in the metropolitan area. Then, we have measured major ions such as H⁺, Na⁺, NH₄ ⁺, K⁺, Mg²⁺, Ca²⁺, Cl^?, NO₃ ^?, and SO₄ ^2? in precipitation and dry deposition samples which had been collected at 9 sampling sites at Hiyoshi, Mita, Kashiwa, Shiki, Fujisawa, Yokosuka, Mitaka, Hachiouji, and Ashikaga in Tokyo Metropolitan area for 10 years since 1990. The average pH of precipitation in their sites was 4.56 (n=1906). As the results of multiple regression analysis showed that pH of precipitation was determined by 5 ions such as NH₄ ⁺, nssCa²⁺(non sea salt calcium), nssCl^?(non sea salt chloride), NO₃ ^?, nssSO₄ ^2?(non sea salt sulfate) in the most of the sampling sites. Therefore, it is very important to investigate the behavior of these ions to understand the acidification of rain in Tokyo Metropolitan area. In this study, a long term trend of each ion concentration in precipitation and wet deposition was also investigated the base on the data we had observed at 7 sites for 10 years by the statistical method.     

Nondestructive measurement of cross-sectional shape of a tree trunk

To evaluate windthrow resistance with respect to stem breakage, a nondestructive method for determining the shape of trunk cross sections was developed. In this method, the coordinates of multiple gauge points set on the perimeter of a trunk are calculated by measuring the distances between them. The shape between the gauge points is generated with the use of a profile gauge placed between them. Measurement tests were conducted using profile gauges with lengths of 300 and 900 mm on model specimens with four shape patterns and four different diameters. The accuracy of the estimation was verified by comparing the section modulus calculated for the generated image and for the photograph. The average ratio of section modulus (generated/photo) for all specimens was 0.994, which indicates that the proposed method is highly accurate. The section moduli of hollow trunks can be evaluated using the profile method together with the drill resistance technique on the condition that 26% of the trunk diameter could be drilled without skew.     

The structure of the first coordination shell in liquid water

X-ray absorption spectroscopy and x-ray Raman scattering were used to probe the molecular arrangement in the first coordination shell of liquid water. The local structure is characterized by comparison with bulk and surface of ordinary hexagonal ice Ih and with calculated spectra. Most molecules in liquid water are in two hydrogen-bonded configurations with one strong donor and one strong acceptor hydrogen bond in contrast to the four hydrogen-bonded tetrahedral structure in ice. Upon heating from 25 degrees C to 90 degrees C, 5 to 10% of the molecules change from tetrahedral environments to two hydrogen-bonded configurations. Our findings are consistent with neutron and x-ray diffraction data, and combining the results sets a strong limit for possible local structure distributions in liquid water. Serious discrepancies with structures based on current molecular dynamics simulations are observed.