A novel HPLC method for glutathione-insulin transhydrogenase assay using fluorescence labeled insulin

The activity of rat liver glutathione-insulin transhydrogenase (GIT) was measured by HPLC. The degradation of fluorescein isothiocyanate-I (FITC-I)-labeled insulin is separated into several peaks, which are bound different amount of FITC-I. We selected mono-fluorescein-thiocarbamylated insulin to estimate the decrease of insulin content and it became possible to assay GIT activity. This novel method was time-saving and simple, and this system could utilize instead of previous method.     

Interannual variability in stock abundance of the neon flying squid, Ommastrephes bartramii, in the North Pacific Ocean during 1979–1998: impact of driftnet fishing and oceanographic conditions

Variability in catch-per-unit-effort (CPUE) was examined for the autumn cohort of Ommastrephes bartramii collected with research driftnets during 1979–1998 along five longitudinal transects passing through the Subarctic, Transitional and Subtropical Domains in the North Pacific. CPUE was generally low during the period of intensive commercial driftnet fishing (1980–1992) and increased following the 1992 moratorium on the use of large-scale driftnets. However, CPUE levels were low for the cohorts hatched in 1992 and 1996 (captured in subsequent years owing to a one-year life of O. bartramii ) that experienced low sea surface temperatures from hatching to recruitment. Among similar-aged squid collected from 180° and 179°30'W in June, mantle lengths were significantly greater in 1997 than during 1995–96. These findings suggest that the driftnet fishery and sea surface temperatures from hatching to recruitment strongly affected stock abundance and possibly growth.     

Relationship between zooplankton abundance and the early marine life history of juvenile chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> in eastern Hokkaido,Japan

Interannual variations in abundance, timing of outmigration from rivers, growth rate and condition of juvenile chum salmon (Oncorhynchus keta) were studied in the Nemuro Strait (eastern Hokkaido, Japan) during 1999–2002 to establish a possible relationship to zooplankton abundance. The otolith microstructure of juveniles was examined each year in late June to determine their time and size at sea entry (i.e., outmigration), and to estimate the early marine growth rates. Salmon outmigration peaked in mid- or late May, which coincided, in three of the four study years, with the peak release of juveniles into rivers within the study area. Abundance, growth rate and condition of fish were higher in 2001, when—compared to other years—smaller fish experienced higher growth rates, coinciding with greater zooplankton abundance for that year. Our results suggest that high zooplankton abundance positively influenced juvenile chum salmon growth and the condition of the fish during their early marine life despite their small size at sea entry.     

Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-M?), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-M? from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-M? population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.     

A role for balance of interferon-gamma and interleukin-4 production in protective immunity against Neospora caninum infection

A suitable balance in the production of Th1/Th2-type cytokines has a crucial role in the control of microbial infections. We investigated cytokine production patterns and effects during Neospora caninum infection, based on two mouse models and an in vitro system. In the acute infection of N. caninum, BALB/c-background IFN-gamma-deficient mice that were sensitive to the N. caninum infection showed high levels of IL-10 production, whereas significant levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production were observed in resistant wild type mice. BALB/c mice vaccinated with recombinant vaccinia virus expressing N. caninum surface protein NcSRS2 resisted parasite spread throughout the body, low levels of IFN-gamma production and high levels of IL-4 production were observed compared to unvaccinated animals. The treatment of N. caninum-infected cells with IFN-gamma or IL-10 decreased the host-cell viability in an in vitro system using mouse macrophage J774A.1 cells. On the other hand, IL-4, but not IL-10 administration, increased the viability of N. caninum-infected and IFN-gamma-treated cells. In the light of the balance of Th1/Th2-type cytokine production, an IFN-gamma/IL-4 balance may have a crucial role for the control of cellular responses against the parasite invasion.     

Comparison of macrophage scavenger receptor-A knockout mice with wild type ones in the immune response against repeated infestation with Haemaphysalis longicornis

Using macrophage scavenger receptor-A knockout (SRKO) mice, we examined the role of macrophage class A scavenger receptors (MRS-A) on the immune response and acquisition of host resistance against repeated infestation with Haemaphysalis longicornis. Except for one batch of nymphs that infested one of the SRKO (SR-/-) mice and showed no appreciable reduction in body weight, all the other groups of nymphs manifested significant decrease in body weight. Both SR-/- and wild type (SR+/+) mice showed a sustained increase in anti-tick antibody titers, but SR+/+ mice showed significantly higher titers. The IFN-gamma assayed in SR-/- mouse immune sera was substantially less compared with that in SR+/+ mice. Immune sera from SR-/- and SR+/+ mice recognized the 51 and 44 kDa, and 44 kDa proteins, respectively, of the salivary gland antigen. The difference in the level of anti-tick resistance manifested by both groups of mice may be influenced by less efficient trapping and processing of tick antigens by macrophages in mice lacking for the macrophage scavenger receptors, and consequently affected the cascade of Th1 and Th2 responses. We have thus obtained valuable data that strongly infer the role of MSR-A in enhancing host defense against repeated infestation with H. longicornis.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Novel species specific antigens of trypanosoma congolense and their different localization among life-cycle stages

Seven monoclonal antibodies (mAbs) were raised against Trypanosoma congolense procyclic form (PCF). Localization of the antigens recognized by the mAbs was determined in bloodstream form (BSF), PCF, epimastigote form (EMF) and metacyclic form (MCF) by confocal laser scanning microscopy (CLSM). Two mAbs (10F9 and 20H12) showed different fluorescent patterns among different life-cycle stages of the parasite. The 10F9 recognized a 76 kDa antigen of all life-cycle stages of the parasite and the antigen localization corresponded with that of a mitochondrion. While the 20H12 recognized 119 and 122 kDa antigens of all the life-cycle stages and the antigen localization corresponded with a flagellum in BSF and MCF, tip of a flagellum in PCF, and part of cytoplasm in EMF. Moreover, the 20H12 did not react to T. brucei gambiense, T. b. rhodesiense and T. evansi antigens in both CLSM and immunoblotting. Therefore, the antigens recognized by the 20H12 seem to be T. congolense specific. Although, further studies will be required for a full characterization of the T. congolense specific 119 and 122 kDa antigens, the mAb 20H12 and the specific antigens may be useful in not only establishment of T. congolense specific diagnosis methods but also studies on molecular mechanisms regulating differentiation of the parasite during life-cycle.     

Homoeological relationships between the f chromosome of Brassica rapa and the e chromosome of Brassica oleracea

Eight plants of the putative double monosomic addition line (DMAL, 2n= 20) were developed by crossing a monosomic chromosome addition line of radish [f(A)‐type monosomic addition line (MAL) (2n= 19)] carrying the f chromosome of Brassica rapa (2n= 20, AA) with another [e(C)‐type MAL (2n= 19)] having the echromosome of Brassica oleracea (2n= 18, CC). The homoeological relationships between the two alien chromosomes were investigated by morphological, cytogenetic and random amplified polymorphic DNA (RAPD) analysis. Seventeen morphological traits that were not present in the radish cv. ‘Shogoin’ were observed in both MALs and these traits were substantially exhibited in DMAL plants. At the first metaphase of pollen mother cells (PMCs), the two parental MALs showed a chromosome configuration of 9II +1I, demonstrating impossibility of recombination between the R and the added chromosomes. The DMALs formed 10II in approximately 73% of PMCs, with one bivalent showing loose pairing between two chromosomes differing in size. In an attempt to identify the two MALs by RAPD‐specific markers using 26 selected random primers, 13 and 20 bands were specific for the f(A)‐type and the e(C)‐type MALs, respectively; 12 bands were common to both MALs (26.7%). In conclusion, the f chromosome of B. rapa is homoeologous to the e chromosome of B. oleracea. The genetic domain (genes) for 17 morphological traits are linked to each homoeologous chromosome bearing 27% of the corresponding RAPD markers.     

暴雨径流期间前次洪水对悬移质传输影响的研究

对日本北海道西南部的两个不同土地利用方式流域的几场降雨中的悬移质输沙量进行了观测。一个流域完全被森林所覆盖 ,另一个流域农业用地占 35%。这个地区主要种植日耕型的高原作物 ,该作物系喂养菜牛的饲料。从 1 997年 5月到 1 998年 1 0月 (不包括 1 997年 1 1月至1 998年 4