Rapid and simple mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.     

Numerical experiments of watershed-scale soil water movement and bedrock infiltration using a physical three-dimensional simulation model

Numerical experiments of soil water movement and bedrock infiltration based on a simplified simulation method were conducted to analyze watershed-scale rainfall-runoff processes. To verify the model accuracy, it was applied to a Minamitani watershed (0.45 ha). The simulation was performed with 2.5-m space grids horizontally and five cells vertically. Results of long-term calculation of this model proved that this simulation model is robust and demonstrated good computational water mass conservation. Calculation results showed the best agreement with observed hydrographs and the number of groundwater levels simultaneously when laboratory-tested soil hydraulic characteristics for topsoil were used and infiltration into bedrock was included in numerical calculations. Numerical experiments show that bedrock infiltration generated a stable base flow and suppressed the secondary discharge peak. The reproducibility achieved by observed soil hydraulic characteristics with the assumption of bedrock flow demonstrates the effectiveness of the simulation model used in this article for analyses of watershed-scale soil water movements.     

Effects of light and microsite conditions on tree size of 6-year-old Cryptomeria japonica planted in a group selection opening

We examined the extent to which direct and indirect measures of light and microsite conditions could explain variation in tree height and diameter at the base of 6-year-old Cryptomeria japonica trees planted in a group selection opening of about 0.32 ha on a steep slope at Shiiba, Miyazaki Prefecture, southern Japan. We first used the gap light index (GLI) and soil thickness (ST) as directly measured indices. For an indirect measure of light, we used a between-cohort competition index (BCI) estimated from the position and total height of residual trees. For indirect measures of microsite, we examined topographic indices (slope, plan and profile curvature, average slope gradient, and relative elevation) derived from digital elevation models (DEMs) with different resolutions ranging from 2 to 10 m. The multiple linear regression using GLI and ST explained about 45% of variation in tree size, while simple regression using only GLI explained about 35%. The contribution of ST was about half of GLI. The multiple regressions using BCI and the topographic indices did not explain any more variation than using BCI alone (R ² of about 0.26). We conclude that microsite conditions with shallower soil and steeper slope have negative effects on tree growth in group selection openings, although the relative importance is smaller than light conditions. More comprehensive studies considering several openings with more heterogeneous topography including different species are needed to generalize our growth prediction using the indirect measures, which are useful for practical forest management.     

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.     

Methylation of free-floating deoxyribonucleic acid fragments in the bronchoalveolar lavage fluid of dogs with chronic bronchitis exposed to environmental tobacco smoke

Background

The etiology of canine chronic bronchitis (CB) is not completely understood, although exposure to environmental tobacco smoke (ETS) affects the airway inflammatory responses in some dogs with CB. The mechanism by which this occurs is unknown.

Findings

We investigated the concentrations and methylation rates of free-floating DNA fragments in bronchoalveolar lavage fluid (BALF) from dogs with chronic bronchitis. Based on serum cotinine levels, dogs with CB were divided into 2 groups: dogs that either had or had not been exposed to ETS. Our results demonstrated that the total nucleated cell and macrophage numbers increased in BALF of ETS-exposed dogs with CB. There were no significant differences in DNA concentrations and methylation rates in BALF between the 2 groups. However, 3 out of 8 dogs exposed to ETS had high DNA methylation rates in their BALF samples.

Conclusion

Our results suggest that ETS exposure leads to epigenetic modifications of cellular components in BALF in dogs diagnosed with CB.     

Increased proportions of CCR4+ cells among peripheral blood CD4+ cells and serum levels of allergen-specific IgE antibody in canine chronic rhinitis and bronchitis

Canine chronic rhinitis (CR) and bronchitis (CB) are suspected to be allergic diseases. The present study tested whether dogs diagnosed with CR or CB present an atopic predisposition based on the ratio of CC chemokine receptor 4 (CCR4)-positive cells among peripheral blood CD4-positive cells (CCR4/CD4) and the serum levels of allergen-specific IgE antibodies. We found that most dogs with CR and CB have a possibility of atopic predisposition, and macrolide therapy constitutes an alternative to corticosteroid therapy in controlling the clinical signs.     

Pharmacokinetics of metoclopramide in calves with renal dysfunction

To clarify the effect of renal dysfunction on pharmacokinetics of the prokinetic agent metoclopramide (MCP), we administered intravenously 0.4 mg/kg MCP to healthy calves and calves subjected to right kidney vessel ligation (ligation) without or with a subsequent left nephrectomy (ligation plus removal). Plasma MCP concentration, glomerular filtration rate (GFR) and plasma prolactin level were measured by liquid chromatography-tandem mass spectrometry, simplified equation using iodixanol and enzyme-linked immunosorbent assay, respectively. Only in calves with ligation plus removal, plasma MCP concentrations were increased significantly 6, 8 and 12 hr after injection, showing that a negative correlation was observed between the plasma MCP concentrations and GFR value. A tendency to increase in plasma PRL concentration was noted also in these calves. In conclusions, plasma MCP concentrations depend on the GFR mode in calves, and its critical GFR value was estimated.     

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.