Identification of bovine serum albumin as an IgE-reactive beef component in a dog with food hypersensitivity against beef

IgE-reactive beef components were examined by an immunoblot analysis using a serum from a dog with food hypersensitivity against beef. The immunoblot analysis revealed a distinct band at approximately 66 kDa and a faint band at approximately 50 kDa. The immunoblot analysis for serum IgE reactivity to bovine serum albumin (BSA) also revealed a positive band at 66 kDa. Serum IgE reactivity to the 66-kDa protein of beef was diminished by pre-incubating the serum sample with BSA. Furthermore, a positive reaction to BSA was detected in intradermal testing in the dog. These results clearly indicated that BSA was an IgE-reactive beef component in the dog with food hypersensitivity against beef.     

Preliminary experiments on mechanical stretch-induced activation of skeletal muscle satellite cells in vivo

We have shown in vitro that mechanical stretch triggers activation of quiescent satellite cells of skeletal muscle to enter the cell cycle through an intracellular cascade of events including nitric oxide (NO) synthesis that results in the release of hepatocyte growth factor (HGF) from its extracellular association and its subsequent presentation to signaling receptors. In order to explore the activation mechanism in vivo, stretch experiments were conducted in the living animal using our suspension model developed. This system used the weight of the hind portion of rats to stretch the inside muscles of the left hind limb suspended for a period of 0.5–2.0 h. At the end of the stretch period, the rats received an intraperitoneal injection of bromodeoxyuridine followed by immunocytochemistry for its incorporation as an index of satellite cell activation in vivo. Depending on the period of stretch, bromodeoxyuridine labeling was increased significantly over the contralateral unstretched leg or control muscle from untreated rats. A stretched muscle extract prepared from the 2 h stretched tissue by incubating it in PBS, showed the active form of HGF as revealed by immunoblotting and it could stimulate the activation of unstretched satellite cells. Also, administering NO synthase inhibitor L‐NAME prior to muscle stretch abolished the stretch activation of satellite cells. Therefore, the results from these experiments demonstrate that stretching muscle triggers NO synthesis and HGF release, which could activate satellite cells in vivo.     

Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcepsilonRI on the cell surface. HRMC cells contained histamine and released beta-hexosaminidase through FcepsilonRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.     

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.     

H2 15O translocation in rice was enhanced by 10 μm 5-aminolevulinic acid as monitored by positron emitting tracer imaging system (PETIS)

5-Aminolevulinic acid (ALA) acts to increase chlorophyll biosynthesis, photosynthesis, cold stress tolerance, and salt tolerance at low concentrations. We studied the effects of ALA on H₂ ¹⁵O translocation from the roots to the shoots of rice plants (Oryza sativa L. cv. Nipponbare) in real time by a positron-emitting tracer imaging system (PETIS). When the plant was treated with 10 μm ALA, the velocity of the H₂ ¹⁵O translocation from 2 to 12 min after absorption increased to 126, 137, 140% that of the control at 1.5, 2.5, and 3.5 h after ALA treatment, respectively. However, ALA did not affect the H₂ ¹⁵O translocation within 0.5 h of treatment. When the plant was treated with 0.1 mM ABA at 4 h after 10 μm ALA treatment, the velocity of the H₂ ¹⁵O translocation decreased at 0.5 h after ABA treatment. Those observations suggested ALA might be absorbed and transported to the guard cells within 1.5 h and functioned to expand the stomatal aperture.     

Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.