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     

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.     

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.     

Effects of delayed first feeding on the survival and growth of tiger grouper,Epinephelus fuscoguttatus (Forsskål, 1775), larvae

The effects of delayed first feeding on the survival and growth of tiger grouper, Epinephelus fuscoguttatus (Forsskål 1775), larvae were examined under controlled conditions. The total length, yolk sac volume, oil globule volume, yolk sac absorption time and nutritional transition period (NTP) of the larvae fed at different first times (0, 6, 12, 18 and 24 h after the mouth opening stage; h AMO) were compared. Larval first feed intake was observed at 54 h after hatching (h AH) at 27.5 ± 0.5 °C. The yolk sac was consumed more rapidly with an increase in delayed first feeding and was significantly different among treatments (P<0.05). Larvae first fed at 0 h AMO had the longest yolk sac absorption (72 h AH) and NTP (20 h) times and had the highest survival and growth rates at the end of the experimental period (360 h AH), being significantly higher (P<0.05) than the other treatments. First mortality was observed at 69 h AH, approximately 2 h after point of no return (PNR) occurred. This study suggests that first feeding of tiger grouper larvae should commence at 0 h AMO for enhancement of larval survival and growth.     

Organ-specific allocation pattern of acquired phosphorus and dry matter in two rice genotypes with contrasting tolerance to phosphorus deficiency

ABSTRACT

Our earlier study demonstrated that the landrace of Japonica rice, Akamai exhibits low-P (phosphorous) tolerance mechanisms compared to the conventional type cultivar, Koshihikari. The present study examined the genotypic difference of yield, plasticity of root growth, and internal utilization of acquired P (allocation pattern of biomass and P among different vegetative and reproductive organs) of two contrasting cultivars in response to P-deficiency. Each cultivar was grown until maturity with (+P) and without (–P) P supply in pots (two plants per pot) filled with 15 kg of Regosol soil. Grain yield and yield components were determined along with biomass and P accumulation in different vegetative and reproductive organs. To assess the plasticity of root growth, the soil column in the pot was divided into two equal portions (upper and lower soil layers) in which the root dry weight and length were measured separately. Among the investigated yield components, the number of filled grains per panicle was the key parameter determining genotypic differences of grain yield of two cultivars. P-deficiency had a marked influence on grain filling of Koshihikari where the filled grain percentage under –P condition was reduced by 29% compared to that under +P condition. However, the respective reduction for Akamai was only 11%. Low-P tolerance ability of Akamai imparts a yield advantage over Koshihikari under P-deficient conditions because of the production of the higher number of filled grains per panicle. Akamai explored both upper and lower soil layers of the pot more efficiently in search of P through greater root biomass and length. Akamai grown under P-deficient conditions had remarkably lower P concentrations in less active vegetative tissues (partly and fully senesced leaves) than those of Koshihikari; whereas, more active organs (green leaves and panicles) contained a greater amount of P. Akamai’s higher plasticity to external P availability can be a genetic resource for developing low-P tolerant, high-yielding rice genotypes suitable for predicted future P-limited environments.     

Metabolite profiling of shoot extract,root extract,and root exudate of rice under nitrogen and phosphorus deficiency

ABSTRACT

Root exudate is derived from plant metabolites and its composition is affected by plant nutrient status. A deficiency of mineral nutrients, such as nitrogen (N) and phosphorus (P), strongly affects the type and amount of plant metabolites. We applied a metabolite profiling technique to investigate root exudates of rice plants under N and P deficiency. Oryza sativa was grown in culture solution containing two N levels (0 and 60 mg N L^?1) or two P levels (0 and 8 mg P L^?1). Shoot extracts, root extracts, and root exudates were obtained from the rice plants 5 and 15 days after transplanting and their metabolites were determined by capillary electrophoresis/time-of-flight mass spectrometry. Shoot N concentration and dry weight of rice plants grown at ?N level were lower than those of plants grown at +N level. Shoot P concentration and dry weight of rice plants grown at ?P level were lower than those of plants grown at +P level. One hundred and thirty-two, 127, and 98 metabolites were identified in shoot extracts, root extracts, and root exudates, respectively, at the two N levels. One hundred and thirty-two, 128, and 99 metabolites were identified in shoot extracts, root extracts, and root exudates, respectively, at the two P levels. Seventy-seven percent of the metabolites were exuded to the rhizosphere. The concentrations of betaine, gamma-aminobutyric acid, and glutarate in root exudates were higher at both ?N and ?P levels than at their respective high levels. The concentration of spermidine in root exudates was lower at both ?N and ?P levels than at their respective high levels. The concentrations of the other metabolites in root exudates were affected differently by plant N or P status. These results suggest that rice roots actively release many metabolites in response to N and P deficiency.     

Changes in the pH,EC, available P,SOC and TN stocks in a single rice paddy after long-term application of inorganic fertilizers and organic matters in a cold temperate region of Japan

Purpose

The objective of this study was to determine the changes in the main soil chemical properties including pH, electrical conductivity (EC), available phosphorus (P), soil organic carbon (SOC) and total nitrogen (TN) stocks after long-term (31 years) additions of two types of organic matters—rice straw and rice straw compost, combined with NPK fertilizers in single rice paddy in a cold temperate region of Japan.

Materials and methods

A long-term experiment on combined inorganic fertilizers and organic matters in paddy rice cultivation began in May 1982 in Yamagata, northeastern Japan. After the 31st harvest, soil samples were collected from five treatments [(1) PK, (2) NPK, (3) NPK + 6 Mg ha^?1 rice straw (RS), (4) NPK + 10 Mg ha^?1 rice straw compost (CM1), and (5) NPK + 30 Mg ha^?1 rice straw compost (CM3)] at five soil depths (0–5, 5–10, 10–15, 15–20, and 20–25 cm). Soil chemical properties of pH, EC, available P, SOC, and TN were analyzed.

Results and discussion

The pH decreased significantly only at the higher compost rate of 30 Mg ha^?1, while EC increased in all the organic matter treatments. Available P significantly increased in the CM1 and CM3 treatments by 55.1 and 86.4 %. The amounts of SOC stock increased by 67.2, 21.4, and 8.6 %, and soil TN stock by 64.1, 20.2, and 8.5 % in CM3, RS, and CM1, respectively, compared to NPK treatment.

Conclusions

Significant changes in soil properties were observed after 31 years of organic matter applications with reference to PK- and NPK-fertilized rice paddy soils. A significant decrease in pH was observed with the application of a high rate (30 Mg ha^?1) of rice straw compost but not with the conventional rate of 10 Mg ha^?1. However, EC increased significantly relative to that of the PK- and NPK-fertilized plots in all the organic matter treatments. Available P significantly increased in the CM1 and CM3 treatments by 55.1 and 86.4 %. The amounts of SOC stock expressed as a percentage of total C applied to the soil were higher from 10 Mg ha^?1 compost (28.7 %) than that from 6 Mg ha^?1 rice straw (17.4 %), indicating a more effective soil organic C accumulation from rice straw compost than that from original rice straw.

     

Pilot evaluation of the efficacy of shampoo treatment with ultrapure soft water for canine pruritus

Ultrapure soft water (UPSW) is water in which calcium and magnesium ions have been replaced with sodium ions using a cation‐exchange resin. We recently demonstrated that washing with soap and UPSW reduced the clinical severity of dermatitis and improved the skin barrier function in NC/NgaTnd mice, a murine model for human atopic dermatitis. The purpose of this pilot study was to evaluate the efficacy of shampoo treatment with UPSW for dogs with pruritus. Eleven dogs with pruritus were randomly assigned to two groups depending on whether they received weekly shampoo treatment with UPSW or tap water for 4 weeks. After a washout period, the treatment protocol was switched such that each dog received both treatments. The pre‐treatment and post‐treatment values of the following were compared: pruritus scores assessed by the owners; dermatitis scores recorded by an investigator; and transepidermal water loss (TEWL). Shampoo treatment with UPSW significantly decreased pruritus and dermatitis scores in the dogs, whereas shampoo treatment with tap water did not. In addition, shampoo treatment with UPSW, but not with tap water, significantly reduced TEWL in the dogs. Adverse events due to the treatment were not observed in the dogs. Furthermore, we found that topical application of UPSW for barrier‐disrupted skin caused by tape stripping in healthy dogs decreased TEWL more rapidly than topical application of tap water. Our findings suggest that shampoo treatment with UPSW promotes skin barrier recovery and thus could be considered as a possible therapeutic option in the management of pruritus and dermatitis in dogs.