Characterization of bullet tuna myoglobin with reference to the thermostability-structure relationship

Myoglobin (Mb) was isolated from bullet tuna (Auxis rochei) skeletal muscle and characterized from the viewpoint of the thermostability-structure relationship. Differential scanning calorimetry (DSC) measurement showed that the thermostability of bullet tuna Mb was the lowest among all the scombridae fish Mbs so far examined. The highest value (72.8 degrees C) of melting temperature (Tm) was obtained at pH 6.52. alpha-Helical content at 10 degrees C was 34.5%, clearly lower than that of horse Mb (55.3%). The amino acid sequence was then deduced by cloning cDNA which encodes bullet tuna Mb. Bullet tuna Mb consisted of 147 amino acids, and the sequence identity was very close to that of skipjack (Katsuwonus pelamis) Mb (91.8%). A few amino acid substitutions, which could be involved in stability difference of Mb, were recognized. By mass spectrometry of lysyl endoproteinase digest of Mb, the N-terminus was found to be acetylated like that of other fish Mbs.     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002     

Development of two immunochromatographic tests for the serodiagnosis of bovine babesiosis

In this study, we developed two immunochromatographic tests (ICTs), which are nitrocellulose membrane-based immunoassays for the convenient and rapid serodiagnosis of bovine babesiosis caused by Babesia bovis (BoICT) and Babesia bigemina (BiICT). The efficacy of two ICTs was evaluated using 13 positive sera from experimentally infected cattle with B. bovis or B. bigemina. Clear results showed that the BoICT and ELISA detected antibodies in sera collected from 14 to 93 days post-infection, while BiICT and ELISA detected from 13 to 274 days post-infection. In additon, non-infected cattle, Neospora caninum, and Cryptosporidium parvum were negative in two ICTs. To evaluate the field utility of the ICTs, we tested 186 field bovine sera collected from cattle living in Yanbian (China) and Mato Grosso do Sul (Brazil). The results of ICTs were compared to those of classical serodiagnostic methods, enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence assay (IFAT). The overall concordances of BoICT were determined as 92.5 and 90.3% when the results of ELISA and IFAT were set as the reference standards, respectively. In contrast, those of BiICT showed 96.8 and 92.5% relative to the results of standard ELISA and IFAT, respectively. Conventional and rapid diagnosic devices for bovine babesiosis may provide a valuable tool in clinical and field applications.     

Effect of replacing fishmeal with canola meal on growth and nutrient utilization in kuruma shrimp Marsupenaeus japonicus (Bate)

A feeding trial was conducted to assess the nutritional values of canola meal as a substitute for fishmeal in diets for kuruma shrimp using five isocaloric diets (190 kJ kg^?1) prepared by replacing 0%, 10%, 20%, 30% and 40% of fishmeal protein with canola meal protein in CM0, CM10, CM20, CM30 and CM40 diets respectively. Triplicate groups of juveniles (0.19 g) were fed the respective diets for 60 days in tanks. At the end of trial, weight gain (%) and specific growth rate (% day^?1) were not significantly (P > 0.05) different among shrimp fed CM0, CM10 and CM20 diets. However, growth was significantly (P < 0.05) decreased in shrimp fed CM30 and CM40 diets. As with growth performance, feed intake, feed conversion ratio and protein efficiency ratio were also decreased (P < 0.05) in CM30 and CM40 groups. Whole body dry matter, methionine and proline were significantly decreased with canola meal substitution levels. In general, retention efficiency of protein and indispensible amino acids were decreased as canola meal increased in diets. It may be concluded that 20% fishmeal protein can be successfully replaced with canola meal, while supplementation of amino acids or blending with complementary proteins could facilitate higher fishmeal replacements in kuruma shrimp diets.     

Assessment of the decay risk of airborne wood-decay fungi III: decay risks at different sampling sites

The decay risk of airborne wood-decay fungi in the same volume of air was investigated by using an air sampler over the course of a year at three different sampling sites. Japanese cedar disks measuring 7.8 cm in diameter and about 3 mm in thickness, and with a moisture content of about 100 % were placed in a “BIOSAMP” air sampler and then exposed to 1000 l of air in the northern, central, and southwest parts of Japan. The exposed disks were incubated for 20 weeks in a damp container maintained at 26 ± 2 °C and degraded by fungi trapped on the disks. The decay risk was calculated from the mass loss during incubation, and the factors affecting the said risk were explored. The results showed that sampling sites apparently do not affect decay risk, even though the Scheffer’s climate indexes of the sites were quite different. The relation between the sampling month and decay risk reveals that decay risk remains virtually the same year-round. Relative humidity on a sampling day is one of the key factors affecting decay risk in sampling conducted at the central or southwest site. In contrast, no weather factors influenced decay risk at the northern sampling site.     

Influence of truck-transportation on the function of bronchoalveolar lavage fluid cells in cattle

The study sought to evaluate whether truck-transportation had an impact on the respiratory immune system of cattle. Six castrated 6-10-month-old Holstein calves were shipped approximately 100 km by road for 4 h. Plasma and bronchoalveolar lavage (BAL) fluid samples, collected immediately before transportation, at 4 h (soon after transportation), and on days 3 and 7 after transportation, were examined. A marked elevation of plasma cortisol concentration was observed at 4 h, but this level was unchanged in controls. The chemiluminescence (CL) response of phagocytes in BAL fluid cells, composed mainly of alveolar macrophage, decreased significantly after transportation (P<0.05). Transportation increased the CD3+ T cell population significantly (P<0.05), and a significant increase (P<0.05) in the ratio of CD4+ to CD8+ cells in BAL fluid was evident. We conclude that short-term road transportation alters pulmonary cells and their function, which may engender bovine respiratory disorders.     

Nucleocapsid-like structures of Ebola virus reconstructed using electron tomography

Electron tomography (ET) is a new technique for high resolution, three-dimensional (3D) reconstruction of pleiomorphic macromolecular complexes, such as virus components. By employing this technique, we resolved the 3D structure of Ebola virus nucleocapsid-like (NC-like) structures in the cytoplasm of cells expressing NP, VP24, and VP35: the minimum components required to form these NC-like structures. Reconstruction of these tubular NC-like structures of Ebola virus showed them to be composed of left-handed helices spaced at short intervals, which is structurally consistent with other non-segmented negative-strand RNA viruses.     

Molecular cloning, chromosomal location, and biological activity of porcine interleukin-21

A pig interleukin-21 (IL-21) cDNA was successfully cloned and sequenced from porcine peripheral blood lymphocytes (PBL) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin P (PHA), 50 ng/ml phorbol 12-myristate 13-acetate (PMA), and 0.5 microg/ml anti-porcine CD3 antibody for 48 hr. The open reading frame of the porcine IL-21 cDNA is 459 base pairs in length and encodes 152 amino acids. The predicted amino acid sequence of the porcine IL-21 shows 86.2%, 77.7%, and 58.4% identity to the bovine, human, and murine IL-21, respectively. The porcine IL-21 gene was mapped to porcine chromosome 8 (8q22-->q23) by means of fluorescence in situ hybridization and radiation hybrid mapping, where the porcine IL-2 gene had been mapped nearby. The recombinant porcine mature IL-21 expressed by E. coli induced dose-dependent proliferation and IFN-gamma production from a human NK cell line, NK0. The porcine IL-21 identified in this study will be helpful for the enhancement of innate immune responses of pigs.     

Evaluation of fetal growth and estimation of fetal age based on skeletal growth in Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884)

We investigated fetal development and the estimation of fetal age of 127 Hokkaido sika deer fetuses, categorizing them into three groups according to the nutritional condition of populations. The order and time of the appearance of ossification centers were clarified, and fetal age was determined based on bone length and the appearance of ossification centers. Then we observed the differences in fetal growth among three populations, and discussed the effect of poor nutrition on the fetal growth. The results suggest that fetal diaphysial length of the femur was affected very little by nutritional conditions, whereas conception dates were delayed and fetal weight was restricted as the nutritional condition became poorer. Although it is impossible to know the exact accurate fetal age in wild populations, it was possible to create a standard to estimate fetal age more precisely by the method described in this study. Both the bone length and the appearance of ossification centers are reliable indices to estimate fetal age precisely in measurements available from fetuses of unknown age, and can be applied to estimate the fetal age of other populations of sika deer, whereas estimation of fetal age based on weight is prone to great errors.