Transgene manipulation in rainbow trout using Cre recombinase

Cre/loxP-mediated cell targeting is considered to be a powerful tool for biotechnology in farmed fish. As a first step toward establishing cell targeting in salmonids, we analyzed the functionality of the Cre/loxP system in rainbow trout. We first established stable transgenic strains carrying the DsRed gene, which was flanked by loxP sites and further spliced with the EGFP gene. By microinjecting Cre complementary RNA (cRNA) into fertilized eggs of the transgenic trout, the functionality of the Cre/loxP system was evaluated. The results showed that all of the embryos exhibited green fluorescence in at least some of their cells. While 19 out of 20 embryos comprised cells showing both green and red fluorescence, the remaining embryo showed only green fluorescence. Polymerase chain reaction (PCR) using primers designed to recognize sequences outside of the two loxP sites revealed that, in addition to long intact fragments, the 19 individuals carried short fragments that were equivalent in length to the loxP-excised fragments. The remaining green embryo carried only this short fragment. DNA sequencing of the short fragment revealed that it lacked the DNA fragments flanking the loxP sites and the spliced fragments did not contain any sequence rearrangements. These results suggest that the Cre/loxP system is functional in rainbow trout.     

Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase

Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.     

Mechanism-based CYP2D6 inactivation by acridone alkaloids of Indonesian medicinal plant Lunasia amara

Fourteen acridone alkaloids isolated from Lunasia amara Blanco were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). Among the 14 compounds, 5-hydroxygraveroline (1), 8-methoxyifflaiamine (2), lunamarine (3), and lunine (12) increased their inhibitory activity with increasing preincubation time. Then, we further examined the possibility of mechanism-based inhibition on 5-hydroxygraveroline (1) and lunamarine (3), which showed the potent inhibition. Further investigations on 1 and 3 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. Thus, 1 and 3 were concluded as mechanism-based inactivators of CYP2D6.     

Accumulation of constitutive diterpenoids in the rhytidome and secondary phloem of the branch bark of Larix gmelinii var. japonica

The quantitative compositions of the major constitutive diterpenoids in the rhytidome and secondary phloem of the branch bark of Kuril larch (Larix gmelinii var. japonica) were investigated. The eight major diterpenoids were isolated from a diethyl ether extract of the branch bark of L. gmelinii var. japonica and identified as 13-epimanool (1), larixol (2), larixyl acetate (3), 13-epitorulosyl acetate (4), abietic acid (5), neoabietic acid (6), dehydroabietic acid (7), and isopimaric acid (8). The amount of each diterpenoid was subsequently quantified in both the rhytidome and secondary phloem. All of the diterpenoids were present in both bark tissues, but the amounts were significantly higher in the rhytidome than in the secondary phloem. Developed fusiform resin cavities containing oleoresins were commonly observed in dead secondary phloem captured into the rhytidome of a bark transverse section. The accumulation and distribution of these constitutive diterpenoids in the bark tissues can probably be attributed to terpenoid biosynthesis in the living secondary phloem and the rhytidome formation process. From the viewpoint of constitutive chemical defense in conifers, it is suggested that the tree body may be more effectively defended against natural enemies by the higher amount of diterpenoid amount in the outermost and dead bark tissue, the rhytidome, where the potentially poisonous and easily oxidizable diterpenoids can be safely and stably maintained.     

Basal levels and responses to glucose infusion of plasma glucose and insulin in beef steer before and after the end of the growing phase on pasture

We examined the effect of grazing during the growing phase on plasma glucose and insulin behavior at the end of the growing phase and at the early stage of the subsequent fattening phase in beef steers. From 13 to 45 weeks of age (growing phase), crossbred beef steers were grazed with minimal supplement (group G: n = 5) or housed while being fed on hay and concentrate (group H: n = 5). Following this phase, both groups were housed for finishing (fattening phase). At the end of the growing phase, group G showed faster plasma glucose disappearance after intravenous glucose infusion, with a smaller plasma insulin response, compared with group H. At the third week of the fattening phase, group G still showed higher glucose tolerance, although they experienced abrupt changes in nutritional and environmental factors. The results suggest that grazing during the growing phase probably improves the glucose tolerance and insulin response to glucose infusion in steers compared with animals that were housed during the corresponding period, and the improved properties may persist at least a few weeks after the commencement of fattening.     

Changes in molecular species composition of triacylglycerol in subcutaneous fat from beef cattle during fattening

The composition of the molecular species of triacylglycerols (TG) in subcutaneous fat biopsied from Japanese Black steers was studied during the fattening period. An analysis of the fatty acid (FA) composition of the TG showed that palmitic, palmitoleic, stearic and oleic acids were the major FA, together accounting for over 80% of the total FA. The concentrations of C14:0, C16:0 and C18:0 decreased between 10 and 30 months of age, while those of C16:1 c9 and C18:1 c9 increased during the fattening period. The major molecular species among more than 40 compounds were palmitoyl‐dioleoyl‐glycerol (POO) and dipalmitoyl‐oleoyl‐glycerol (PPO). POO showed the highest concentration during the fattening period. The levels of palmitoyl‐oleoyl‐linoleoyl glycerol and some tentatively identified molecular species increased during the fattening period, while the levels of tripalmitoyl, dipalmitoyl‐stearoyl‐glycerol, palmitoyl‐distearoyl and palmitoyl‐stearoyl‐oleoyl‐glycerol decreased with growth. A comparison of the experimental values of TG molecular species with theoretical values derived from the experimental FA contents showed that the FA distribution in TG was non‐random. TG synthesis favors the formation of TG molecular species containing at least one C16 FA, rather than three C18 FA.     

Immature T cell neoplasms in three young cattle

Immature T cell neoplasms in three young Holstein cattle with neoplastic involvement of the thymus are described. Case 1, with a precursor T lymphoblastic leukemia (calf form of leukosis), was an 86-day-old female calf. The leukemia was characterized by replacement of the bone marrow and spleen by leukemia cells, but preservation of epithelial frameworks throughout the thymus. The other two neoplasms were thymic γδ T cell lymphomas, which were observed in a 246-day-old steer (case 2) and a 16-month-old heifer (case 3). Histological examination revealed obliteration of the normal thymic architecture and stromal fibrosis, with the spleen and liver far less severely affected than in case 1. There were cytological differences bewteen the tumors in case 1 and cases 2 and 3. Additionally, WC1 and CD8 were expressed only in the latter. Thus, the leukemia and these lymphomas should be regarded as independent disease entities on the basis of histological and immunohistochemical characteristics.