Development of an in vitro culture system for producing eel larvae from immature ovarian follicles in Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To standardize conditions during the final maturation and ovulation of ovarian follicles from Japanese eel, we have developed a culture system for the production of fertilizable eggs from post-vitellogenic ovarian follicles in vitro. Post-vitellogenic ovarian follicles were incubated in culture medium supplemented with 17α,20β-dihydroxy-4-pregnen-3-one (DHP) with or without bovine serum albumin (BSA) to assess the effects of protein concentration. Eggs that ovulated during incubation were fertilized, and the remaining follicles were incubated in prostaglandin F_2α (PGF_2α) for a further 3 or 6 h before fertilization. Male eels were injected repeatedly with human chorionic gonadotropin. The quality of eggs obtained under the different culture conditions was evaluated after artificial fertilization in terms of hatching success. Hatching rates tended to decrease with increasing concentrations of BSA in the incubation medium in a dose-dependent manner. The addition of PGF_2α drastically increased the number of eggs that ovulated, but the rate of hatching was greatly decreased compared with eggs obtained earlier by DHP incubation alone. The larvae obtained from artificially fertilized eggs produced in vitro survived for 14 days without feeding. We conclude that in vitro culture systems thus have a great potential for the acquisition of good quality eggs under tightly controlled artificial conditions, culminating in the production of eel larvae.     

A putative RNA silencing component protein FoQde-2 is involved in virulence of the tomato wilt fungus <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

RNA silencing pathways in filamentous fungi are composed of multiple component proteins and known to be involved in vegetative growth, virulence or sexual reproduction. We found that the tomato wilt fungus, Fusarium oxysporum f. sp. lycopersici (Fol), carries four homologues genes of Qde-2, an argonaute protein gene and one of the main component protein genes in Neurospora crassa. Gene targeting revealed that FoQde-2, one of the Qde-2 homologues in Fol, is involved in virulence to tomato but not in vegetative growth.     

A bronchopulmonary artery fistula in a horse

A 24-year-old, Thoroughbred gelding presented with difficulty breathing for a few days and intermittent nose bleeding before dying. At necropsy, the bronchoesophageal artery and the bronchial artery that flowed into the left anterior lobe were tortuous and dilated, and it was found that dilated tortuous branches of the bronchial artery ran over the dorsal and ventral surfaces of the left anterior lobe. Histopathologically, an anastomosis between a muscular artery and an elastic artery were demonstrated, which were identified as bronchial and pulmonary arteries, respectively. Based on the gross and histopathological findings, a bronchopulmonary artery fistula was diagnosed. To the best of our knowledge, this is the first case report of a pulmonary vascular anastomosis in a horse.     

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Reasons for performing study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n = 100) and sera (n = 100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n = 95) and horses (n = 50). Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K⁶³ and K²³⁸ of the EGF‐like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 × biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Conclusions and potential relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.     

Estimation of stiffness and strength in timber knee joints with adhesive and verification by experiment

The use of adhesive joints is gradually increasing, especially those with glued-in steel rods (GIRs). There are, however, some problems with the design methods when used for moment-transmitting applications. In this article, design methods for GIRs and cross-lapped glued joints (CLJs) are proposed. For CLJs, we made a hypothesis that both rotational deformation of CLJ and stress of the glue line occurred with bending and shearing deformation of the timber. Using this hypothesis and Kelvin’s theorem, a mechanical model of CLJ is proposed. For GIRs, the axial stress component of the rod and the lateral stress component of the rod were taken into account using the theory of a beam on an elastic foundation. From the comparisons between calculations and experimental results, it was recognized that the stiffness and strength of CLJs and GIRs could be predicted precisely using our proposed models.     

Development of a joint system using a compressed wooden fastener I: evaluation of pull-out and rotation performance for a column—sill joint

Compressed wooden plates and dowels were used to connect members in post-and-beam structures as a substitute for a steel fastener. In order to take advantage of the characteristic properties of compressed wood and to achieve optimum joint performance, two compressed wooden plates were used in each joint to give multiple shear planes for each compressed wooden dowel. Consequently, this type of joint showed very good properties in pull-out and momen-trotation performance, and its engineering design could be further optimized. This joint is expected to be introduced to many kinds of structural systems, including long-span frame structures made of domestic timber found in Japanese residential houses.     

Ceramide aminoethylphosphonate from jumbo flying squid Dosidicus gigas attenuates the toxicity of cyanotoxin microcystin-LR

We have previously established the method for isolation of ceramide aminoethylphosphonate (CAEP) from jumbo flying squid Dosidicus gigas. In this study, we performed a MTT assay to evaluate the safety of CAEP to the cell lines for the application to health food and supplements. The CAEP did not show any cytotoxicity to various HEK293-transfectant cells. Next, we elucidated the positive function of CAEP to the somatic cells. Recently, we have reported that hepatotoxin microcystin-LR was taken up into the hepatocytes mediated by hepatocellular uptake transporters OATP1B1 and OATP1B3, and the cells were induced cytotoxicity subsequently. Cytotoxicity of microcystin-LR to permanently OATP1B3-expressing HEK293-OATP1B3 cells rather than to HEK293-OATP1B1 cells was preferentially attenuated by CAEP in a concentration-dependent manner. In addition, the enzyme activity of serine/threonine phosphatase, which was inhibited by microcystin-LR, was recuperated by co-exposure to CAEP. Furthermore, microcystin-LR-induced cellular protein phosphorylation were disrupted by CAEP exposure. These results suggested that CAEP is a promising remedy and/or preventive medicine for liver damage with microcystin-LR.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.