Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.     

Vibrational property changes of spruce wood by impregnation with watersoluble extractives of pernambuco (Guilandina echinata Spreng.) II: structural analysis of extractive components

Heartwood of pernambuco (Guilandina echinata Spreng, synCaesalpinia echinata Lam.), which has been used as material for violin bows, was extracted by soaking in water, and the obtained extractives were analyzed. The main components of the extractives were identified to be protosappanin B and brazilin. In particular, protosappanin B occupied about 40% of the pernambuco extractives. The loss tangent (tan) of spruce wood impregnated with protosappanin B decreased markedly, the same as that of specimens impregnated with extractives before being purified. It is expected that protosappanin B can make a contribution to the decrease in tan due to impregnation with extractives.Part of this work was presented at the 48th annual meeting of the Japan Wood Research Society, Shizuoka, April 1998; and the 49th annual meeting of the Japan Wood Research Society, Tokyo, April 1999     

Structure, allometry, and biomass of plantation Metasequoia glyptostroboides in Japan

We quantified structural features and the aboveground biomass of the deciduous conifer, Metasequoia glyptostroboides (Hu and Cheng) in six plantations in central Japan. In order to derive biomass estimates we dissected 14 M. glyptostroboides trees into three structural components (stem wood, branch wood and foliage) to develop allometric equations relating the mass of these components and of the whole tree to diameter at breast height (DBH). We found robust relationships at the branch and whole tree level that allow accurate prediction of component and whole tree biomass. Dominant tree height was similar within five older (>40 years) plantations (27–33 m) and shorter in a 20-year-old plantation (18 m). Average stem diameter varied from 12.8 cm in the youngest stand to greater than 35 cm in the oldest stand.

Metasequoia have relatively compact crowns distributed over the top 30% of the tree although the youngest stand had the deepest crown relative to tree height (up to 38%). At the individual tree level in older stands, 87% of the aboveground biomass was allocated to the stem, 9% to branch wood and 4% to foliage. We found little difference in the relative distribution of above ground biomass among the stands with the exception of lower foliage biomass in larger diameter trees. Total aboveground biomass of the older stands varied twofold, ranging from a maximum of 450 Mg ha⁻¹ in a 42-year-old stand to a minimum of 196 Mg ha⁻¹ in a 48-year-old stand. Total above ground biomass of the 20-year-old stand was 176 Mg ha⁻¹.     

Mapping of QTLs for leaf developmental behavior in rice (Oryza sativa L.)

Leaf developmental behavior in rice (Oryza sativa L.) is one of the important agronomic characteristics, which not only determines vegetative growth but also influences grain yield. This study was conducted to identify quantitative trait loci (QTLs) for total number of leaves (TNL), days to the emergence of flag-leaf (DEF) and the leaf emergence rates (LER) on main stem, which mainly represent leaf developmental behavior, using recombinant inbred lines (RILs) derived from a cross between a japonica variety, Asominori and an indica variety, IR24, cultivated in 2001 and 2002. The transgressive segregations in both parental directions and continuous variations of all three tested traits were observed. Significant correlations among these traits were detected. A total of fourteen QTLs for leaf development behavior were detected with 289 RFLP markers. Six QTLs controlling TNL were mapped to chromosomes 3, 5, 6, 8, 9, 12, and accounted for 5.615.7% of the total phenotypic variations, and three QTLs for DEF were mapped to chromosome 3, 6, 8 and accounted for 10.735.4% of total phenotype variation and five QTLs for LER were mapped to chromosome 1(two QTLs), 2, 4, 9 and explained 6.217.5% of phenotype variation. The identification of QTLs for leaf developmental behavior in rice may be useful for selection of fast growing genotype before heading using maker-assisted selection.     

Trisomic genetic analysis of aroma in three Japanese native rice varieties (Oryza sativa L.)

Information on the genetics of aroma in rice facilitates breeding and selection of new aromatic varieties with high yield and good quality. Objective of the present study was to make clear the number of genes controlling aroma, and the allelism of aroma genes and the location of aroma gene(s) on the chromosome in three Japanese native aromatic rice varieties (Kabashiko, Shiroikichi and Henroyori). Lack of leaf aroma in all F₁ plants of non-aromatic/aromatic crosses indicated the recessive nature of aroma, and the segregation ratios (3:1) of non-aromatic to aromatic plants in its F₂ populations from Nipponbare/aromatic varieties crosses revealed that each of the three aromatic varieties contains a single recessive gene for aroma. Through trisomic analysis, the segregation of non-aromatic and aromatic plants in all F₂ populations from the crosses between trisomics lines NT8, with an extra chromosome 8, and aromatic varieties deviated significantly from disomic segregation of 3:1 ratios, and fitted to trisomic segregation, however, in other F₂ populations derived from other 7 types of trisomic F₁ plants, the segregation ratios of non-aromatic to aromatic were 3:1, indicating that the single recessive aroma gene was located on chromosome 8 in three aromatic varieties. This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of GHG and ammonia emissions from stored dairy cattle slurry by using a floating dynamic chamber

We developed a system for measuring emissions from stored slurry by using a floating dynamic chamber. CH₄, CO₂, N₂O and NH₃ emitted from the storage tank of a dairy cattle farm in eastern Hokkaido were measured during summer 2008 (7/16–8/6), fall 2008 (10/2–10/26), spring 2009 (6/2–6/21) and winter 2009 (3/11). Average daily gas emission rates in summer, fall and spring were, respectively, 54.8, 54.2 and 34.3 g/m² for CH₄; 602, 274 and 254 g/m² for CO₂; 55.4, 68.2 and trace mg/m² for N₂O; and 0.55, 0.73 and 0.46 g/m² for NH₃. CH₄, CO₂ and NH₃ emission rates during the brief measurement period in winter were reduced to 1/4, 1/23 and 1/2, respectively, of summer emission rate levels. All gas emissions showed diurnal fluctuation and were greatest during the daytime, when the ambient temperature rose. CH₄, NH₃ and CO₂ emissions increased significantly during the daytime, and the daily emission (in grams) of each gas was positively correlated with maximum daily temperature. According to the combined spring, summer and fall measurements, the CH₄, N₂O and NH₃ annual emission factors were 1.42% (g CH₄/g volatile solids), 0.02% (g N₂O‐N/g total N) and 0.43% (g NH₃‐N/g total N), respectively.     

Influence of the nitrogen form on in vitro organogenesis in Equisetum arvense

The physiological and biochemical mechanisms of organogenesis in Equisetum arvense have not been clarified yet. However, high concentrations of nitrogen have been shown to exert an inhibitory effect on in vitro tuber formation in E. arvense. The aim of this study was to clarify the influence of the form of nitrogen in a medium on in vitro organogenesis in E. arvense. Single‐node segments of E. arvense rhizomes were cultured in the test medium. The NH₄‐N and NO₃‐N concentrations of the test medium, respectively, were adjusted by adding NH₄H₂PO₄ and KNO₃ to the basal medium. The basal medium was a nitrogen‐free, modified form of White's medium. Vegetative shoots were newly formed in the test tubes for concentrations of NO₃‐N and NH₄‐N that exceeded 56 mg L^?1. However, no rhizome was formed at NH₄‐N concentrations exceeding 28 mg L^?1. The number of newly formed tubers decreased at an NH₄‐N concentration of 28 mg L^?1 and no tuber was formed at NH₄‐N concentrations exceeding 56 mg L^?1. In summary, although the presence of NO₃‐N in the medium did not inhibit in vitro rhizome or tuber formation in E. arvense, the presence of NH₄‐N in the medium exerted a strong inhibitory effect on the in vitro formation of both of these organs.     

Microstructure and glycosaminoglycan ratio of canine cornea after reconstructive transplantation with glycerin-preserved porcine amniotic membranes

Objective  Although amniotic membranes of canine, feline, and equine species have some advantages as corneal transplantation material in many canine ocular diseases, their softness, thinness, and low availability can pose problems. As an alternative, the more abundant porcine amniotic membranes may be used. This paper describes the use of glycerin-preserved porcine amniotic membranes in corneal transplantation in eight normal dogs.
Method  A 0.4-mm deep recipient bed in the axial cornea of the OS of all dogs was created using an 8-mm Barron radial vacuum trephine. The recipient bed was then filled with amnion, and the entire cornea was covered with another piece of the glycerin-preserved membrane. The ocular signs evaluated were corneal opacity and corneal vascularization. The dogs were euthanized on days 5, 10, 20, or 40 after surgery, and samples were collected to evaluate corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content and the glycosaminoglycan (GAG) ratio.
Results  Corneal opacity was observed immediately after surgery. Restoration of corneal transparency, regression of corneal vascularization, and visualization of the pupil and iris were noted on day 40.
Conclusions  The clinical observations were supported histologically by regained corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content, and GAG ratio, suggesting that this technique may be a novel method for the treatment of ocular surface disorders.     

The tyrosine kinase inhibitor imatinib [STI571] induces regression of xenografted canine mast cell tumors in SCID mice

Canine mast cell tumors (MCTs) are the most common cutaneous tumors in the dog. They have a wide range of behaviour, which can make these tumors challenging to treat. Recently, mutations in c-kit proto-oncogene have been identified in several canine MCTs. Imatinib is the first member of a new class of agents that act by inhibiting particular tyrosin kinase enzymes, including KIT which is a product of the c-kit. In this study the efficacy of imatinib to reduce or abolish canine MCT [CMC-1] using xenografted MCT in severe combined immunodeficient [SCID] mice was evaluated. Imatinib was administered at doses of 200 mg/kg and 100 mg/kg once a day for one week. The antitumor responses in SCID mice with CMC-1 xenografts following treatment with imatinib were observed. Significant tumor regression occurred with 100 mg/kg on days 7, 10, 14 and 21, and 200 mg/kg on all days. Our results indicate that imatinib is effective against canine mast cell tumor in mouse xenograft models. Canine MCTs might be a potential target for imatinib therapy.