Evaluating antigen-specific cytotoxicity of CD8+ T cells in fish by granzyme B-like activity

Granzyme B plays an important role in granule-mediated apoptosis by CTL. It is a well characterized component of the cytolytic machinery in mammals and a candidate for the evaluation of cytotoxic activity of CTL as an alternative to conventional cytotoxicity assay. In this study, we examined the effects of granzyme inhibitors to assess the characteristics of fish granzymes in terms of substrate specificity and the involvement of granzyme B-like in the cytotoxic response. 3,4-dichloroisocoumarin (DCI), which inhibit the activity of serine protease including all members of the granzyme family, markedly suppressed the cytotoxic activity of CTL. However, CTL-mediated cytotoxicity was significantly but not completely suppressed by the addition of carbobenzyloxy-Ile-Glu-Thr-Asp-fluoromethyl ketone (Z-IETD-FMK) that specifically blocks granzyme B activity. These results suggest that additional serine proteases as well as granzyme B-like are involved in cytotoxicity of CTL in fish. We further compared cytotoxicity with the granzyme B-like hydrolytic activity against fluorogenic substrate acetyl-Ile-Glu-Thr-Asp-4-methylcoumaryl-7-amide (Ac-IETD-MCA) and found that granzyme B-like activity correlated well with the cytotoxicity of CTL in ginbuna crucian carp. Present results suggest that the granzyme activity assays is useful to assess cytotoxic activity of CTL in fish in which genetic information on granzymes and specific tools for cytotoxicity assay are not available because of well conserved catalytic triad residues and substrate binding sites in granzyme B throughout vertebrates.     

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.     

Successive deaths of a captive snow leopard (Uncia uncia) and a serval (Leptailurus serval) by infection with feline panleukopenia virus at Sapporo Maruyama Zoo

Feline parvoviruses were isolated from frozen samples of intestines taken from a snow leopard (Uncia uncia) and a serval (Leptailurus serval) that died successively at Sapporo Maruyama Zoo in Hokkaido, Japan. Isolates possessed an antigenic epitope for both the feline panleukopenia virus (FPLV) and mink enteritis virus, identified with a hemagglutination inhibition test. Sequencing analyses of the VP2 region of the isolates revealed that the two isolates were identical and of the FPLV-type. These results suggested that FPLV was introduced from a feral cat which entered the zoo and transmitted the virus inside the zoo.     

Experimental transplacental transmission of canine herpesvirus in pregnant bitches during the second trimester of gestation

The effects of canine herpesvirus (CHV) on fetuses were studied after IV inoculation of pregnant bitches in the 2nd trimester of gestation. Cesarean sections were performed on 2 bitches that were inoculated with CHV on the estimated 30th day of gestation. Bitch M-1 had 2 mummified fetuses and bitch M-2 had 4 mummified and 2 dead fetuses and 3 live-born pups. Infection by CHV was confirmed histopathologically by the presence of focal areas of necrosis associated with intranuclear inclusion bodies in heart muscle sections of 1 dead fetus; CHV was not recovered from other organs. Abortion occurred between the 2nd and 3rd week after inoculation of another pregnant bitch inoculated with CHV on the estimated 30th day of gestation. Two bitches inoculated with CHV on the estimated 40th day of gestation gave birth prematurely to 10 pups. The detection of characteristic herpesviral lesions in various organs and the reisolation of CHV from the liver, spleen, kidneys, and lungs of premature pups indicated CHV infection. Transplacental infection of fetal pups by CHV resulted in their death and subsequent mummification. It appears that abortion and premature birth also may occur in pregnant bitches infected during the 2nd trimester of gestation.     

Developmental competence of cat oocytes from ovaries stored at various temperature for 24 h

We conducted two experiments to investigate the effects of storage temperature and period for cat ovaries on the meiotic and developmental competence of oocytes collected from the ovaries. In Experiment 1, ovaries were stored in physiological saline for 24 h at 4 C, 23-25 C, or 38 C (cold, room, and incubator temperature groups, respectively), and then oocytes were collected from the ovaries in each group. Morphologically intact oocytes were then selected and cultured in maturation medium for 24 h. Significantly more oocytes reached metaphase II (MII) in the cold temperature group (53.4%) than in the room and incubator temperature groups (20.0 and 2.4%, respectively). In Experiment 2, ovaries were stored in physiological saline at room temperature for 0, 6, 12 or 18 h, and then they were stored at 4 C (cold storage) until reaching a total storage period of 24 h. After storage of the ovaries, morphologically intact oocytes were matured, fertilized with frozen-thawed spermatozoa, and cultured in vitro. The rates of morphologically intact oocytes obtained from the ovaries stored at room temperature for 0, 6, 12 or 18 h were 35.3, 30.0, 26.4 and 14.7%, respectively, and the rates of intact oocytes that reached MII were 63.2, 36.4, 26.5 and 11.9%, respectively. The results suggested that the numbers of morphologically intact oocytes and intact oocytes that reached MII after in vitro maturation decrease gradually as the period of storage at room temperature before cold storage increases. Only oocytes from ovaries stored for 6 h developed to the blastocyst stage after in vitro maturation and fertilization when ovaries were stored at room temperature before cold storage. These results indicate that 24 h storage of ovaries at high temperatures (>23 C) decreases the meiotic competence of oocytes. The quality and developmental competence of oocytes are influenced by the length of storage at room temperature before cold storage.     

Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi Nuclear Power Plant

The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium (¹³⁷Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a ¹³⁷Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood ¹³⁷Cs and muscle ¹³⁷Cs (Y = 28.0X, R² = 0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high‐risk cattle before they enter the market, and will contribute to food safety.     

Structural and physical properties ofAntheraea pernyi silk fibroin fiber treated with I2-KI aqueous solution

Silk fibroin (SF) fiber from theAntheraea pernyi silkworm was treated with a 1.23 N iodine-potassium iodide (I₂-KI) aqueous solution, and the structure and physical properties were investigated to clarify the effects of the iodine treatment. The noticeably high weight gain value of SF fiber, about 25 wt% was attributed to the absorption of polyiodide ions in the form of I₃ ^? and I₅ ^?. Fourier transform infrared spectroscopy and X-ray diffraction measurements suggested that polyiodide ions mainly entered the amorphous region. In addition, a new sharp reflection on the meridional direction, corresponding to a period of 7.0 Å, was observed and indicated the possibility of the formation of mesophase structure ofβ-conformation chains. Dynamic viscoelastic measurements showed that the damping tanδ peak at 270°C gradually shifted to lower temperature in the iodinated SF fibers, suggesting an enhancement of the molecular motion of the fibroin chains induced by the presence of polyiodide ions. With heating above 254 °C, the iodine component introduced intermolecular cross-linking of SF, and the melt flow of the sample was inhibited. The thermal decomposition stability of fibroin molecules was greatly enhanced by iodine treatment.     

Effect of extraction method on the concentrations of selected bioactive compounds in mandarin juice

A mandarin-type citrus fruit, ponkan (Citrus reticulata), was processed by in-line, chopper pulper, and hand-press extractions to investigate the effect of extraction method on the concentrations of bioactive compounds in processed juice. Concentrations of polymethoxylated flavones (tangeretin, nobiletin, and sinensetin) and beta-cryptoxanthin in juice, and inhibitory activities against arachidonate cyclooxygenase and lipoxygenases of the juice extract were analyzed. The juice processed by hand-press extraction contained the largest amounts of nobiletin (3.56 mg/100 mL), tangeretin (4.10 mg/100 mL), and sinensetin (0.13 mg/100 mL). Concentrations of beta-cryptoxanthin were 0.66, 0.59, 0.55, and 0.50 mg/100 mL in chopper pulper, in-line (5/64 in.), in-line (8/64 in.) and hand-press juices, respectively. Both extracts of in-line juices showed greater inhibitory activity toward platelet 12-lipoxygenase than the others. The inhibitory effect of hand-press juice extract on platelet cyclooxygenase activity was remarkable among juice extracts. All juice extracts effectively inhibited polymorphonuclear 5-lipoxygenase activity at nearly the same rate.