Determination of polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins/dibenzofurans in marine products

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in plastics and textile coatings, and these compounds have been recognized as ubiquitous environmental contaminants. Furthermore, it is considered a serious problem that polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs), having toxicities similar to those of chlorinated dioxins, are generated by the manufacture of brominated flame retardants (BFRs) such as PBDEs, and formed by the combustion of substances containing BFRs. Several congeners of PBDD/DFs and PBDEs have been detected in the adipose tissue of the Japanese. Although food is suspected as an exposure source, little information is available regarding the levels of these brominated compounds in food, as compared with information regarding dioxin or polychlorinated biphenyls. It is necessary to investigate the levels of these brominated organic compounds in various foods and to estimate their influence in the case of human exposure. We developed an efficient method of analyzing PBDEs and PBDD/DFs contents in food samples using accelerated solvent extraction and determined the concentrations in several marine products such as raw fish, processed foods, and seaweed purchased in Japan. A recovery test (n = 5) using the method and involving dried fish showed acceptable recoveries of 57.7-78.5% (RSD 5.4-15.9%) for PBDEs and 50.0-56.4% (RSD 1.5-7.9%) for PBDD/DFs. In the analysis of marine product samples, several congeners of PBDEs were detected in raw fish, processed fish, and seaweed; the highest concentration of sigmaPBDEs was detected in yellowtail (1161 pg/g whole basis), followed by mackerel (553.5 pg/g whole basis). The most dominant congener present in these marine samples was 2,2',4,4'-tetraBDE (#47).     

Antioxidant activities of black and yellow soybeans against low density lipoprotein oxidation

Several studies have demonstrated that the daily intakes of soy foods were associated with a reduced cardiovascular risk. The aim of our study was to investigate the inhibitory effect of black soybeans on low density lipoprotein (LDL) oxidation in comparison to yellow soybeans. The extract from black soybean had a longer LDL oxidation lag time than that from yellow soybean (205 +/- 16 and 65 +/- 3 min, respectively). When both soybeans were divided into the seed coat and the mixture of the germ and cotyledon, the diluted extract solution from the black soybean seed coat prolonged the lag time significantly more than the original extract of the yellow soybean seed coat. On the other hand, antioxidant effects of the extract from the mixture of germs and cotyledons were similar in both soybeans. Regarding total polyphenol contents, the seed coat of black soybean had a higher polyphenol content than that of yellow soybean (29.0 +/- 0.56 and 0.45 +/- 0.02 mg/g, respectively). Interestingly, the mixture of the germ and cotyledon hydrolyzed by beta-glucosidase in both soybeans showed a stronger inhibitory effect on LDL oxidation than that before being hydrolyzed by beta-glucosidase. These results suggest that black soybeans may be more effective in inhibiting LDL oxidation than yellow soybeans because of total polyphenols contents in its seed coat. In addition, aglycones, which are rich in soybeans fermented or hydrolyzed by beta-glucosidase, may play a crucial role in the prevention of oxidation-related diseases.     

A model to estimate available timber and forest biomass and reforestation expenses in a mountainous region in Japan

We developed a model to estimate supply potentials and available amounts of timber and forest biomass resources from profitable sub-compartments of thinning and final felling operations. Economic balances were estimated while considering not only harvesting expenses but also reforestation expenses after final felling, which should be considered for sustainable forest management. Harvesting expenses were estimated based on two types of timber harvesting systems and three types of forest biomass harvesting systems in each sub-compartment. Then, the model was applied to Nasushiobara city of Tochigi prefecture, Japan. Reforestation expenses had large negative impacts on the financial balances of final felling operations. Few sub-compartments were profitable after considering reforestation expenses. Most profitable sub-compartments were those with mechanized operation systems and landing sales. These accounted for 17.19% of all sub-compartments, while only 5.75% of the sub-compartments were profitable based on their current operation systems and landing sales. Although the overall supply potentials of timber and forest biomass resources were 380,000 m3 and 210,000 Mg, respectively, and 15 times the planned harvest of coniferous tree volume of 25,000 m3year-1 and 50 times the annual demand for the woody gasification power generation of 4,000 Mg year-1 in Nasushiobara, available amounts of timber and forest biomass resources were only49,429 m3 and 33,333 Mg, which were 13.0% and 15.7% of supply potentials for landing sales with mechanized operation systems.     

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

Variation in herbivory-induced responses within successively flushing Quercus serrata seedlings under different nutrient conditions

Herbivore damage can induce the host plant to alter the chemical and physical qualities of its leaves, which is thought to be a plant strategy—termed “induced response”—for avoiding further herbivory. In woody plants, many studies have considered variation in induced response with resource availability, but few studies have examined this variation in relation to growth patterns of woody plants. We studied the phenotypic variability of induced response within successively flushing Quercus serrata seedlings. Q. serrata seedlings were grown under controlled conditions. The controlled factors were herbivore damage (herbivore-damaged and -undamaged) and soil fertility (low and high). At each flush stage, the concentrations of condensed tannin (CT), total phenolics (TP), and nitrogen (N) in leaves were analyzed, and the leaf mass per area (LMA) was measured. CT and TP concentrations in leaves and LMA were higher in herbivore-damaged seedlings. Leaves of the first flushes showed greater sensitivity to herbivore damage and had a higher CT concentration than leaves of the later flushes. Furthermore, seedlings growing in low-fertility soil showed a greater induced response. The results suggest that the induced response of Q. serrata seedlings was related to the contributions of the tissue to current productivity. Leaves of the first flush showed a greater induced response, possibly because they play an important role in subsequent growth. The potential of Q. serrata seedlings to adjust the properties of leaves depending on herbivory and soil fertility in relation to growth patterns may be advantageous on the forest floor, where seedlings grow in soil of heterogeneous fertility and are constantly exposed to herbivory.     

Cloning and sequence analysis of llama cytokines related to cell-mediated immunity

In order to characterize the T helper 1 (Th1) cytokines of llama, we have cloned several llama cytokine genes and compared them to those of other mammalian species. The cDNAs encoding for interleukin (IL)-2, interferon (IFN)gamma, IL-12p35 and IL-12p40 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-2, IFN-gamma, IL-12 p35 and IL-12p40 were found to be 465, 501, 669 or 993 bp in length, with open reading frames encoding 154, 166, 222 or 330 amino acids, respectively. Homology analyses of nucleotide and deduced amino sequences of llama IL-2, IFN-gamma, IL-12p35 and IL-12p40 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla, which includes pig and cattle.     

Generation of multinucleated giant cells in vitro from bovine monocytes and macrophages

The generation of multinucleated giant cells (MGC) from cells of the bovine monocyte-macrophage lineage was investigated. Freshly isolated monocytes were incubated with the conditioned medium (CM) of peripheral blood mononuclear cell cultures treated with Concanavalin A for 1-4 days (CM1 to CM4). Only CM1 generated MGC despite similar concentrations of IFNgamma in all CMs. Nevertheless, MGC formation from monocytes was enhanced by adding either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), MGC formations from macrophages were observed only when macrophages were cultured with GM-CSF plus CM. These results indicate that several mechanisms to generate MGC from bovine monocytes-macrophage lineage cells exist, and that GM-CSF is a major mediator of MGC formation in cattle.     

An outbreak of chlamydiosis in captive blue-fronted Amazon parrots (Amazona aestiva) in Brazil.

Fifty-eight blue-fronted Amazon parrot (Amazona aestiva) nestlings, recovered from the illegal trade, became ill at a wildlife rehabilitation center in S?o Paulo State, Brazil. Clinical signs observed were nonspecific, and the mortality rate was 96.5% despite initial treatment with norfloxacin. Postmortem examinations were performed on 10 birds. Liver and spleen smears showed structures suggestive of Chlamydophila psittaci in four cases. Diagnosis was confirmed by seminested polymerase chain reaction on tissue samples. Other birds from the same location showed no clinical signs of the disease, although high complement fixation titers to C. psittaci were found in 10 adult psittacines. All birds in the facility were treated with doxycycline. The two surviving nestlings did not recover after two doxycycline treatments and were euthanatized. The high mortality rate observed in this outbreak was attributed to poor conditions of husbandry and delays in the diagnosis and treatment of the disease. After diagnosis, improved control measures for chlamydiosis were instituted.