Semi-micro chiral HPLC analysis of lignans

Chiral high-performance liquid chromatography (HPLC) separation of lignans with semi-micro columns of 1.0–2.0mm inner diameter (i.d.) was established for the first time. Practical sensitivity was increased 5 to 20-fold compared with that of conventional chiral HPLC using analytical columns with 4.6mm i.d. The semi-micro chiral HPLC system can be applied to high-sensitivity enantiomeric separation of many chiral organic compounds in addition to lignans.     

Vitelline cyst in the rat ileum

Congenital vitelline duct anomalies other than Meckel’s diverticulum are rare in animals. A cyst of approximately 8 mm in diameter was observed on the antimesenteric surface of the ileal serosa in a 10-week-old female Crl:CD(SD) rat. Microscopically, the cyst closely resembled the ileum, but it did not communicate with the ileal lumen. We diagnosed this case as a vitelline cyst derived from the vitelline duct based on the location where it developed and its histological behavior. In rats, only Meckel’s diverticulum has been reported with a congenital anomaly of the vitelline duct, and no other spontaneous anomalies including a vitelline cyst have been reported. This case may be the first report concerning a vitelline cyst in the rat ileum.     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.     

Severe oxidative stress is thought to be a principal cause of jaundice of yellowtail Seriola quinqueradiata

The jaundiced yellowtail Seriola quinqueradiata had yellow discolored skin and hyperbilirubinemia. Histology revealed that hemosiderin and ceroid occurred in the spleen. In the vascular system, there were vasculitis and sinusoidal dilatation. Hepatic cells showed degenerative figures with pyknotic or karyolytic nuclei, basophilic cytoplasm, cytoplasmic vacuoles in various sizes, and ceroidosis. In the bile duct system, however, there was no noticeable change. Hemoglobin contents of jaundiced fish were similar to those of control fish. Plasma taurine levels of jaundiced fish were significantly lower than those of control (p0.05). Judging from 2-thiobarbituric acid reactive substance values, in vivo lipid peroxidation progressed in the liver of jaundiced fish. In the plasma of jaundiced fish, contents of unconjugated bilirubin were significantly higher than those of conjugated bilirubin. A large amount of substance Xs, breakdown products of bilirubin scavenging active oxygen species, existed in the plasma of jaundiced fish. Therefore, jaundiced fish may suffer from severe oxidative stress, which might be a principal cause of jaundice. Liver superoxide dismutase and glutathione peroxidase activities of jaundiced yellowtail were significantly lower than those of controls.     

Persistence of cyprinid herpesvirus 2 in asymptomatic goldfish Carassius auratus (L.) that survived an experimental infection

Cyprinid herpesvirus 2 (CyHV‐2) is the causative agent of herpesviral haematopoietic necrosis (HVHN) in goldfish, Carassius auratus, and Prussian carp, C. auratus gibelio. In this study, we investigated virus persistence in goldfish experimentally infected with CyHV‐2. Virus DNA presence in organs was monitored in survivors reared at a virus permissive temperature and also in survivors treated with a non‐permissive temperature for 4 days, initiated at three different time points post‐infection in order to obtain fish with different virus loads. We detected virus DNA in all organs tested at 51 days post‐infection (dpi) and in the spleen, trunk kidney and gills of survivors at 81 dpi, although the virus load in fish influenced the subsequent number of organs that tested positive for virus DNA. In addition, some organs dissected from four out of five asymptomatic survivors tested positive by PCR following incubation in vitro in a medium for 5 days. Following inoculation with the homogenate of PCR‐positive kidney incubated in vitro, one of the three inoculated fish died, showing that the detected virus by PCR produced infectious particles. This study suggests that CyHV‐2 can establish a persistent infection in some organs, especially the spleen and trunk kidney, and that asymptomatic surviving fish can be a source of infection.     

Enzyme-linked immunosorbent assay with monoclonal and single-chain variable fragment antibodies selective to coplanar polychlorinated biphenyls

Coplanar polychlorinated biphenyls (Co-PCBs) consisting of non-ortho and mono-ortho-chlorinated PCBs are dioxin-like compounds and cause wide contamination in the environment. To monitor Co-PCB residues, it was attempted to establish an enzyme-linked immunosorbent assay (ELISA) with monoclonal and recombinant antibodies selective to Co-PCBs. When 3,3',5,5'-tetrachlorobiphenoxybutyric acid (PCBH)-keyhole limpet hemocyanin conjugate was immunized into mice, two monoclonal antibodies, Mab-0217 and Mab-4444, were obtained. 3,3',5,5'-Tetrachlorobiphenyl (PCB80) was determined with an IC(50) value of 2.6 and 0.46 ng mL(-1) in ELISA based on Mab-0217 and Mab-4444, respectively. Mab-4444 cross-reacted with Co-PCB congeners, except for PCB77 and PCB81. Mab-0217 reacted with PCB80 and cross-reacted with PCB111. A single-chain variable fragment (scFv) antibody derived from Mab-4444 was produced in recombinant Escherichia coli cells. The scFv antibody showed nearly the same sensitivity toward PCBH as the parent monoclonal antibody in ELISA. These results clearly suggested that Mab-4444 and its scFv antibodies were suitable for monitoring the representative congeners of Co-PCBs.     

Estimation of the root colonization of soybean by an arbuscular mycorrhizal fungus,Gigaspora rosea,based on specific fatty acid profiles

Root colonization by arbuscular mycorrhizal (AM) fungi has traditionally been analyzed by microscopy. However, this method is time consuming and it is often difficult to distinguish between AM and non-AM fungi. In this study, we analyzed the fatty acid profiles in soybean roots colonized by AM fungi to determine if specific fatty acids derived from AM fungi can be used as markers for the intensity of the AM fungal colonization. The wild-type Enrei and hypernodulating Kanto100 soybean cultivars were inoculated with an AM fungus (Gigaspora rosea) alone or with Bradyrhizobium diazoefficiens, which nodulates soybean roots. Fatty acids 20:1ω9, 20:4ω6, and 20:5ω3 were specifically detected in the lateral roots of AM fungus-inoculated and dual-inoculated soybean plants. In the second lateral roots, the percentage of AM-specific fatty acids (i.e., 20:1ω9, 20:4ω6, and 20:5ω3) derived from AM fungi was closely correlated with the intensity of the AM fungal colonization. We propose that the AM-specific fatty acids represent useful markers for estimating the degree of AM fungal colonization. The percentage of AM-specific fatty acids was more than twofold higher in the second lateral roots than in the first lateral roots. Thus, the degree of AM fungal colonization is probably twofold higher in the second lateral roots than in the first lateral roots.     

Involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization in soybean roots

Soybean plants autoregulate to suppress excessive nodulation. It has been revealed recently that the autoregulation of various legumes controls both nodulation and arbuscular mycorrhizal (AM) fungal colonization. We investigated the involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization. We used a wild-type soybean cv. Enrei and its hypernodulating mutant Kanto100, defective in the autoregulation. We included four different treatments: an uninoculated control, inoculation with rhizobium Bradyrhizobium japonicum alone, inoculation with AM fungus Gigaspora rosea alone, and dual inoculation with rhizobium and AM fungus. In both Enrei and Kanto100, AM fungal colonization enhanced the weight and N₂ fixation of nodules, suggesting that autoregulation of host plant is not involved in the stimulatory effect of AM fungal colonization on rhizobial nodulation. In plants with the AM fungus alone, the AM fungal colonization of Enrei was comparable to that of Kanto100. In plants with dual inoculation, however, this was significantly (P?<?0.05) lower than in Kanto100. To confirm the control of AM fungal colonization by the autoregulation of host plant, a reciprocal grafting experiment was performed between Enrei and Kanto100. In plants with the AM fungus alone, AM fungal colonization was comparable among Enrei (shoot)/Enrei (root), Enrei/Kanto100, Kanto100/Enrei, and Kanto100/Kanto100 grafts. In plants with dual inoculation, however, AM fungal colonization of Enrei/Enrei and Enrei/Kanto100 grafts was significantly (P?<?0.05) lower than that of Kanto100/Enrei and Kanto100/Kanto100. These results indicate that rhizobial nodulation suppresses AM fungal colonization, and the autoregulation of host plant, initiated by nodulation, is involved in this phenomenon.     

Effects of sedimentation on an endangered benthic fish,Cobitis shikokuensis: is sediment‐free habitat a requirement or a preference?

A negative correlation between the degree of fine sediment deposition and the abundance of stream benthic fishes has often been reported, although the causal mechanisms for this effect are not fully understood. To better understand the influence of sedimentation, it is important to clarify whether it alters the habitat required by fish, or merely preferred by them. We conducted two enclosure experiments in the field to examine (i) whether an endangered benthic fish, Cobitis shikokuensis, prefers sediment‐free substrate and (ii) whether fine sediment deposition has negative effects on the physiological condition of C. shikokuensis. The first experiment, which used three types of substrates, ‘cobble’, ‘pebble’ and ‘mixture of fine sediment and pebbles’, showed that C. shikokuensis avoided the mixture. The second experiment, which compared the physiological condition of fish reared in enclosures with either a pebble or mixture substrate, showed that the condition of the fish in the latter group declined more quickly. These results suggest that, for C. shikokuensis, sediment‐free substrate is a habitat requirement, not merely a preference, and that excessive input of fine sediment due to human activities can cause a decline in the population of this species.