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.     

Moisture distribution in rice grains used for sake brewing analyzed by magnetic resonance imaging

Water absorption in individual white-core and non-white-core grains of rice varieties used in sake brewing was observed during soaking by magnetic resonance imaging (MRI). Water was absorbed faster in white-core grains than in non-white-core grains and was pooled in the core. Moisture distributions were compared using the line profiles of nuclear magnetic resonance signal intensities (SI profiles) generated from magnetic resonance images of grains soaked for 2 h. SI profiles of white-core and non-white-core grains overlapped despite slight differences in the central and intermediate regions of the grains. A white-core-like structure with loosely packed starch granules was found to exist in the endosperm of non-white-core grains. SI profiles of grains polished to 70% of the yield weight of brown rice were similar in shape to those of grains polished to 90% of the yield, but their overall moisture content was higher after the removal of the hydrophobic grain periphery. A varietal difference in the thickness of the intermediate region, which may affect fissuring resistance in the polishing process, was detected in SI profiles. Differences in water absorbability between parents and their offspring were also apparent from their SI profiles. MRI enabled the detection of variety-specific water-absorption properties in grains.     

Interaction between the helicase domain of the tobacco mosaic virus replicase and a tobacco arginine decarboxylase

In a yeast two-hybrid screening test for tobacco proteins that interact with TMV replicase using the helicase (H) domain as bait, a cDNA clone was selected that encodes a polyamine biosynthetic enzyme, arginine decarboxylase (ADC). In yeast cells, the C-terminal internal region of ADC interacted with the H domain. This observation was confirmed in vitro by far-Western blotting. Inhibition of the binding between the H domain and the IRnHEL (I region and N-terminus of helicase domain) region by ADC using a yeast three-hybrid assay suggested possible interference of the heterodimerization of 126K and 183K by ADC.The nucleotide sequence data of pADCF reported in this study is available in the DDBJ/EMBL/GenBank databases under accession number AB110952     

Suppression of MyoD induces spontaneous adipogenesis in skeletal muscle progenitor cell culture

The degree of intramuscular adipose tissue accumulation is one of the factors affecting meat quality. Accumulation of adipocytes is also observed under the pathological condition of skeletal muscle such as muscular dystrophy and sarcopenia. The origin of adipocytes seen in skeletal muscle is mesenchymal progenitor cells that can give rise to both adipocytes and fibroblasts. In the present study, we demonstrated that siRNA-mediated suppression of MyoD expression in rat skeletal muscle progenitor cell culture, which comprises both myogenic satellite cells and mesenchymal progenitor cells, resulted in diminished myotube formation and an unexpected spontaneous appearance of white adipocytes. Suppressing myomaker expression also resulted in complete absence of myotube formation without reducing MyoD expression, but no adipogenesis was seen in this scenario, indicating that decline in MyoD expression rather than decreased myotube formation is necessary to induce adipogenesis. In addition, spontaneous adipogenesis induced by suppressing MyoD expression in culture was inhibited by the conditioned medium from control culture, indicating that anti-adipogenic factor(s) are secreted from MyoD-positive myogenic cells. These results indicate the presence of regulatory mechanism on adipogenesis by myogenic cells.     

Improvements in the qualities of gluten-free bread after using a protease obtained from Aspergillus oryzae

From a nutritional perspective, rice flour is one of the most valuable flours and it is suitable for preparing food for people suffering from wheat allergy. However, bread made from rice flour is very difficult to bake because it lacks gluten. We found that pre-fermenting of rice flour using Aspergillus oryzae facilitated a better formulation of gluten-free rice bread. Bread swelling was remarkably improved with a longer pre-fermenting period at 55 °C. The specific loaf volume (SLV) without polymeric thickeners after a 12 h fermentation was approximately 2.2-fold (2.98 ml/g) higher than that after 0 h (1.36 ml/g). An enzymatic assay of the batter indicated that protease activity during the pre-fermentation period increased from 0.38 to 1.44 U/ml and this activity correlated with bread swelling. Furthermore, a commercial protease from A. oryzae also produced similar results with an adequate SLV of 3.03 ml/g. Rheological analysis showed that batter treated with protease had an increased batter viscosity and decreased flour settling behavior because of the aggregation of flour particle after partial cleavage of storage proteins. These results indicated that the improved SLV was mainly because of an A. oryzae protease, which affected the batter rheology thereby improving gas retention before baking.     

Development of a Simple Method for Evaluation of Water Absorption Rate and Capacity of Rice Flour Samples

A simple method employing a commercially available canister was developed for the determination of mode of water absorption of rice flour samples. The samples prepared by four different grinding methods were used to analyze water absorption. The total amount of water in a flour sample was described by using an exponential model. Capacity and rate of water absorption of the samples were determined, and the relationship to baking quality of partially substituted rice bread was investigated. The water absorption was highly dependent on the method of grinding. Flours produced by wet jet‐milling of the grains, which absorbed a small amount of water at high speed, were most suitable for rice bread. The method was applicable to other food powders, provided that flour particles do not stick together or swell immediately upon contact with water.     

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.     

Simultaneous steroids measurement in dogs with hyperadrenocorticism using a column-switching liquid chromatography-tandem mass spectrometry method

We developed an analytical method using an on-line column-switching liquid chromatography with triple quadrupole mass spectrometry (LC/MS/MS) for quantifying multiple steroids in serum. Using the developed method, we evaluated the serum concentration of nine steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 21-deoxycortisol, deoxycorticosterone, progesterone, 17α-OH-progesterone and aldosterone) in dogs with hyperadrenocorticism (HAC). Serum was mixed with stable isotope internal standards and thereafter purified by the automated column-switching system. The limit of detection ranged 2–16 pg/ml for nine steroids. In the baseline samples, five steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, and 17α-OH-progesterone) were detected in all dogs. The concentrations of cortisone, 11-deoxycortisol, and 17α-OH-progesterone in dogs with HAC (n=19) were significantly higher those in dogs without HAC (n=15, P<0.02). After the adrenocorticotropic hormone stimulation test, six steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 17α-OH-progesterone, and deoxycorticosterone) were above the limit of quantification in all dogs. Cortisol, corticosterone, cortisone, and deoxycorticosterone concentrations of dogs with HAC were significantly higher than those of dogs without HAC (P<0.02). In addition, 11-deoxycortisol and 17α-OH-progesterone concentration was higher in dogs with HAC than in dogs without HAC (P=0.044 and P=0.048, respectively). The on-line column-switching LC/MS/MS would be feasible for measuring multiple steroids in dog serum. The results suggest that cortisone, 11-deoxycortisol, and 17α-OH-progesterone would be related to HAC. Further studies are warranted to assess the clinical feasibility of steroid profile in dogs with HAC.