Comparisons of Ophiostomatoid fungi associated withTomicus piniperda andT. minor in Japanese red pine

Five species ofOphiostoma, twoLeptographium species and aGraphium species were isolated from two morphologically and ecologically similar bark beetle species,Tomicus piniperda andT. minor, and their infested Japanese red pine (Pinus densiflora) in Yamanashi Prefecture, central Honshu, Japan. An underscribedOphiostoma species andO. minus were isolated mainly fromT. piniperda and its galleries.Ophiostoma canum which was found for the first time in Japan was mainly fromT. minor and its galleries. Specific relationships between the beetles and fungal species are suggested. Contribution No.140, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba. Part of this study was presented at 108th Annual meeting of Japanese Forestry Society, April 2, 1997, Fukuoka, Japan.     

Development of a system for measuring calcitonin in the stingray Dasyatis akajei (a cartilaginous fish): the possible involvement of stingray calcitonin in gonadal development

To elucidate the physiological role of calcitonin (CT) in stingrays (cartilaginous fish), an enzyme-linked immunosorbent assay (ELISA) system using a specific antibody against stingray CT has been developed. Synthetic stingray CT was subcutaneously injected into mice four times—once every 2 weeks—together with an adjuvant. We purified the IgG antibody fraction using the protein A affinity chromatography from collected antiserum. Evaluating the antibody titer, we found the antibody’s optimum dilution ratio to be 600 times. Competitive ELISA has been developed using the antibody diluted 600 times. Our antibody did not cross-react with teleost CTs and muscle extraction, but cross-reacted with stingray plasma and the extract of the ultimobranchial gland, the secretary organ of stingray CT. Using this ELISA, we measured the plasma CT level in stingrays and examined its correlation with several mineral concentrations. Plasma CT did not show significant correlation to calcium, magnesium, inorganic phosphorus, sodium, chlorine, or urea, although there was a correlation among the factors involved in osmoregulation, such as sodium, chlorine, and urea. On the other hand, plasma CT was significantly correlated to body weight and length. Furthermore, there was a significant correlation between plasma CT and gonad weight. Since plasma CT was correlated with the weight of liver, which is involved in the synthesis of egg yolk protein, we examined the influence of 17β-estradiol (E2) on CT secretion. After E2 injection, the plasma CT level increased significantly. This is the first study to demonstrate that E2 induced plasma CT secretion in cartilaginous fish.     

Purification and characteristics of trypsin from masu salmon (<Emphasis Type="Italic">Oncorhynchus masou</Emphasis>) cultured in fresh-water

Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS–PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N ^α -p-tosyl-l-lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60°C, and the trypsin was stable below 50°C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.     

Assessment of the measurement of canine and feline serum fibroblast growth factor-23 concentrations by automated chemiluminescence immunoassay

This study compared canine and feline fibroblast growth factor (FGF)-23 concentration measurements between automated chemiluminescence assay (CLEIA) and enzyme-linked immunosorbent assay (ELISA). Seventy serum samples each from dogs and cats were evaluated. FGF-23 measurements by CLEIA significantly correlated with those of ELISA in both dogs and cats. The Bland–Altman test showed that FGF-23 between CLEIA and ELISA had fixed and proportional biases, respectively, in both dogs and cats. Measurements by CLEIA were lower than those of ELISA, especially in higher serum FGF-23 concentrations. This study showed that FGF-23 concentrations in dogs and cats can be evaluated by automated CLEIA. However, FGF-23 cannot be directly compared between CLEIA and ELISA.     

Establishment of a canine lens epithelial cell line

Some immortalized lens epithelial cell lines have been established and are useful for molecular analysis. The establishment of additional cell lines must, however, enable a variety of in-vitro examinations. The objective of this study was to establish a new canine lens epithelial cell line by isolating CLC-1 cells from the lens tissue of a dog with cataracts. In CLC-1 cells, transforming growth factor beta (TGF-β) treatment significantly decreased gene expression of an epithelial marker and elevated that of mesenchymal markers; these characteristics are similar to those of a human lens epithelial cell line. Interestingly, CLC-1 cells exhibited lower expression of an epithelial marker and higher expression of mesenchymal markers than an anterior lens capsule. These results suggest that CLC-1 cells were derived from a cell population that was committed to epithelial-mesenchymal transition in cataract lens tissue. In conclusion, CLC-1 cells could be useful for analyzing molecular pathogenesis in canine cataracts.     

Role of sorbitol in manufacturing dried seafood from heated squid meat

SUMMARY: Japanese common squid meat was heat-treated at 80°C for 1 min, cured with 1.0 M sorbitol solution (pH 7.0) at 4°C for 18 h, and dried further at 30°C (60% relative humidity) for 16 h. Osmotic dehydration during the sorbitol curing process and slow moisture vaporization at the initial drying period were observed regardless of the heat denaturation of muscle protein. Simultaneously, lowering the amount of moisture vaporized in the falling rate of the drying period caused a shortening of the total drying time. Furthermore, the shear force of the dried product from heat-treated meat was kept at a lower value by sorbitol curing, although the suppression effect of sorbitol on the hardening of dried meat was lost by protein denaturation. These results are useful for understanding the role of sorbitol in reducing drying time and in eliminating excess hardening of dried squid products.     

Temperature and metal ions-dependent activity of the family I inorganic pyrophosphatase from the swine roundworm Ascaris suum

Temperature dependence, heat stability and metal ions-dependent activity were examined on the Family I inorganic pyrophosphatase (PPase) recently identified from Ascaris suum. Recombinant A. suum PPase (rAsPPase) showed an optimal activity at 55 degrees C. The rAsPPase was heat stable at 40 degrees C in the absence of added Mg(2+) and at 50 degrees C in its presence. The enzyme required divalent metal ions for its activity. The preferences for the metal ions (5 mM concentration) were in the order: Mg(2+)> Co(2+)> Cu(2+)> Fe(2+)> Zn(2+)> Mn(2+). On the contrary, enzyme activity was inhibited by Ca(2+). These findings suggest that catalytic features of AsPPase are consistent with the Family I PPases reported from a wide range of organisms.     

No effect of bovine interferon-tau for control of calf diarrhea and immunomodulation in calves

Newborn calves received a low dose of bovine interferon-tau (boIFN-tau) orally for 4 weeks and calves that had developed diarrhea received a low dose of boIFN-tau orally for 5 days. No effects of boIFN-tau were seen in the duration of the diarrhea, or in daily weight gain. Calves received a high dose of boIFN-tau subcutaneously 3 times and they were then stimulated with bovine herpesvirus type 1 vaccine. No adverse effects were observed after the administration of boIFN-tau and lymphocyte subsets from calves did not change after the stimulation. Our results suggest that boIFN-tau does not seem protecting for preventing calves from diarrhea, recovering the health of calves with diarrhea or immunomodulation, although the treatment itself is not toxic.     

Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein

Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzyme-linked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP(Sc)) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP(Sc) detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP(Sc) was observed in WB, even when performed after 4 days of incubation at 37 degrees C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP(Sc) due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses.     

Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity

Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34(cdc2) kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started.