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.     

GM1 gangliosidosis in a Japanese domestic cat: a new variant identified in Hokkaido,Japan

A male Japanese domestic cat with retarded growth in Hokkaido, Japan, showed progressive motor dysfunction, such as ataxia starting at 3 months of age and tremors, visual disorder and seizure after 4 months of age. Finally, the cat died of neurological deterioration at 9 months of age. Approximately half of the peripheral blood lymphocytes had multiple abnormal vacuoles. Magnetic resonance imaging showed bisymmetrical hyperintensity in the white matter of the parietal and occipital lobes in the forebrain on T2-weighted and fluid-attenuated inversion recovery images, and mild encephalatrophy of the olfactory bulbs and temporal lobes. The activity of lysosomal acid β-galactosidase in leukocytes was negligible, resulting in the biochemical diagnosis of GM1 gangliosidosis. Histologically, swollen neurons characterized by accumulation of pale, slightly granular cytoplasmic materials were observed throughout the central nervous system. Dysmyelination or demyelination and gemistocytic astrocytosis were observed in the white matter. Ultrastructually, membranous cytoplasmic bodies were detected in the lysosomes of neurons. However, genetic analysis did not identify the c.1448G>C mutation, which is the single known mutation of feline GM1 gangliosidosis, suggesting that the cat was affected with a new variant of the feline disease.     

Effects of testosterone on rat placental development

We investigated the morphological effects of testosterone on placental development in a rat model of polycystic ovarian syndrome (PCOS). Testosterone propionate (TP), which was subcutaneously administered to pregnant rats with 5 mg/animal from gestation day (GD) 14 to GD 18, induced a maternal weight reduction without mortality or clinical signs from GD 19 onwards. A decrease in fetal and placental weight, an increase in intrauterine growth retardation (IUGR) rates, and histological changes in the placenta were observed on GD 21 but not on GD15 or 17. Histopathologically, on GD 21, the trophoblast septa thickened, and the maternal sinusoids were narrowed in the labyrinth zone, resulting in a small placenta. Additionally, the placental weight, thickness, and histological morphology in the labyrinth zone on GD 21 in the TP-treated group were nearly identical to those on GD 17 in the control and TP-treated groups. Therefore, it was assumed that the testosterone-induced small placenta was induced in association with the developmental inhibition of the fetal part of the placentas from GD 17 onwards.     

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.     

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.     

Inhibitory effect of 2,4-dibromophenol and 2,4,6-tribromophenol on larval survival and metamorphosis of the sea urchin <Emphasis Type="Italic">Strongylocentrotus nudus</Emphasis>

ABSTRACT:   As a possible factor leading to the low recruitment level of sea urchins in kelp forests, the inhibitory effect of 2,4-dibromophenol (DBP) and 2,4,6-tribromophenol (TBP) released from the large perennial brown algae Ecklonia kurome and Eisenia bicyclis on survival and metamorphosis of eight-armed larvae of the sea urchin Strongylocentrotus nudus was examined. The percentage of larvae that underwent metamorphosis in filtered sea water after 1 h exposure to one-half dilution of saturated dibromomethane solution (∼60 ppm) as a chemical inducer reached approximately 100% after 1 h, while that in filtered sea water containing 1 ppm TBP was reduced to 73%. This was further reduced to less than 40% in the presence of 10 and 20 ppm TBP after 2 h. In filtered sea water containing 1 and 10 ppm DBP, the proportion of metamorphosed larvae was reduced markedly to 43 and 5% after 2 h, respectively. All larvae exposed to 50 ppm TBP and to 20 and 50 ppm DBP died after 1 h. These findings suggest that DBP is more toxic than TBP for sea urchin larvae, strongly inhibiting their metamorphosis.     

Immunohistochemical analysis of pseudorabies virus spread through neurons innervating the eyeball

Pseudorabies virus (PRV) was inoculated intraocularly into BALB/c mice, and the distribution pattern of cells positive for several neurotransmitters and viral antigens in the eyeball, trigeminal nerve ganglia, and superior cervical ganglia was examined immunohistochemically to clarify the neural route of the virus spread. In the eyeball, substance P (SP)- and calcitonin gene-related peptide (CGRP)-positive cells were detected in the ipsilateral iris and ciliary body, neuropeptide tyrosine (NPY)-positive cells were detected in the choloid membrane, and tyrosine hydroxylase (TH)-positive cells were detected in the ipsilateral inner nuclear layer of the retina; all these cells contained viral antigens. In the superior cervical ganglia, viral antigen-positive cells containing TH or NPY were found at bilateral sites. In the trigeminal nerve ganglia, viral antigen-positive cells containing SP or CGRP were found at bilateral sites. These findings indicated that the SP- and CGRP-positive ganglion cells of the trigeminal nerve ganglia innervating the iris or ciliary body, and the NPY-positive ganglion cells of the superior cervical ganglia innervating the iris, ciliary body, and choroid membrane served as the route for the virus spread. These findings also suggested that dopaminergic neurons, such as the TH-positive retinal cells and TH-positive ganglion cells of the superior cervical ganglia, served as the route for virus spread.     

Cytotoxicity for porcine islet cells by complement of six animal species

Complement-mediated cytotoxicity for porcine islet cells (PICs) was evaluated using sera of six animal species. Then soluble complement receptor type-1 (sCR1) as an anti-complement agent was added to those sera, and the changes in 50% hemolytic unit of complement serum (CH50) and cytotoxic effect of those sera on PICs were examined. All the sera except for that of pig showed cytotoxicity. However, the extent of toxicity was considerably different between species. In the rat and human serum, sCR1 significantly reduced CH50 and cytotoxicity, however in the dog serum, sCR1 had no suppressive effects. These results may suggest that complement contribute to humoral cytotoxicity for PICs as a main factor, and the compatibility of complement with PICs differs between animal species.     

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.