Transient transfection of CHO cells using linear polyethylenimine is a simple and effective means of producing rainbow trout recombinant IFN-γ protein

A practical method was developed for the transient transfection of Chinese hamster ovary (CHO) cells with 25 kDa linear polyethylenimine (PEI) then optimal culture conditions determined for the production of rainbow trout (Oncorhynchus mykiss) IFN-γ recombinant protein. We found that culture temperature had a significant impact upon recombinant protein yield, with best results being obtained at 32 °C. However the amount of serum added to the culture medium had no effect upon recombinant IFN-γ (rIFN-γ) production. In this study maximal rIFN-γ yields and minimal PEI toxicity were achieved using a DNA/PEI ratio of 1:8, where the amount of PEI did not exceed 10 µg per 5 ml of RPMI1640 culture medium, with cells subsequently cultured at 32 °C for 7 days. Thus, linear PEI is a technically simple and cost-efficient method for the transient transfection of CHO cells and is compatible with serum-free operations.     

Cloning and characterization of the trout perforin

The pore-forming protein, perforin is one of the effectors of cell-mediated killing. A perforin cDNA clone was isolated from rainbow trout (Oncorhynchus mykiss) after screening of a spleen cDNA library. The full-length cDNA is 2070 bp in size, encoding for a polypeptide of 589 amino acids. The predicted amino acid sequence of the trout perforin is 64, 58 and 40% identical to those of Japanese flounder, zebrafish and human perforins, respectively. Although its membrane attack complex/perforin (MACPF) domain is conserved, trout perforin shows low homology to human and trout terminal complement components (C6, C7, C8 and C9), ranging from 19 to 26% identity. Expression analysis reveals that the trout perforin gene is expressed in the blood, brain, heart, kidney, intestine and spleen. Phylogenetic analysis of proteins which belong to the MACPF superfamily clusters the trout perforin in the same group with other known perforins.     

Tissue damage in organic rainbow trout muscle investigated by proteomics and bioinformatics

The response to tissue damage is a complex process, which involves the coordinated regulation of multiple proteins to ensure tissue repair. In order to investigate the effect of tissue damage in a lower vertebrate, samples were taken from rainbow trout (Oncorhynchus mykiss) at day 7 after damage and proteins were separated using 2DE. The experimental design included two groups of rainbow trout, which were fed organic feed either with or without astaxanthin. In total, 96 proteins were found to be affected by tissue damage, clearly demonstrating in this lower vertebrate the complexity and magnitude of the cellular response, in the context of a regenerative process. Using a bioinformatics approach, the main biological function of these proteins were assigned, showing the regulation of proteins involved in processes such as apoptosis, iron homeostasis, and regulation of muscular structure. Interestingly, it was established that exclusively within the astaxanthin feed group, three members of the annexin protein family (annexin IV, V, and VI) were regulated in response to tissue damage.     

Copper exposure and the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss)

Abstract

Sublethal concentrations of copper in water cause the degeneration of olfactory receptors in rainbow trout (Oncorhynchus mykiss). Receptor cell loss has been correlated to the loss of olfaction in fish and may cause difficulties in olfactory mediated behaviors such as migration. This study investigated the effects of three levels of copper (100, 75 and 50 mg L^?1) on the olfactory epithelium of rainbow trout. Twenty fish randomly allocated between three exposure groups and one control were exposed for 24 hours under static renewal conditions. Light and scanning electron microscopic observations of olfactory tissue were taken to determine the extent of degeneration of receptors. In addition, levels of copper and zinc in the brain tissues were analyzed to determine if the olfactory route was a significant route of copper exposure and transfer to fish brain tissue. Results indicate that degeneration of receptors is related to the concentration of copper. Levels of copper in brain were found to be below detection of the instrument. Levels of zinc were extremely variable ranging from 52 to 132 ng zinc g^?1 brain tissue.     

Induction of cytochrome P450 1A and DNA damage in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes by 2,3,7,8 tetrachlorodibenzo-p-dioxin

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatocytes isolated from immature rainbow trout (Oncorhynchus mykiss) by collagenase perfusion were investigated with respect to induction of cytochrome P450 1A (CYP1A) enzyme activities and protein contents as well as DNA damage. Exposure of primary rainbow trout hepatocytes to TCDD resulted in increased CYP1A contents, as determined by immunoblotting, enhanced activities of 7-ethoxyresorufin-O-deethylase (EROD) and increased DNA damage as determined by the comet assay. By means of electron microscopy, no symptoms of cytotoxicity could be observed except for slight increases of lysosomal components and the smooth endoplasmic reticulum. Whereas CYP1A contents constantly increased over the duration of the entire experiment, EROD activities remained constant from day 3 of exposure to 1 nM TCDD; maximum induction of CYP1A activities was reached with 0.1 nM TCDD after 5 days. DNA damage increased in a time- and dose-dependent fashion until day 3. After 5 days, DNA damage was less pronounced, and the number of damaged nuclei declined in all TCDD concentrations. Since TCDD has been shown to not directly react with DNA, metabolism of TCDD or TCDD-induced changes in other metabolic pathways are suspected to result in the production of DNA-reactive (endogenous) substances.     

Gastro-intestinal transport of calcium and cadmium in fresh water and seawater acclimated trout (Oncorhynchus mykiss)

Transport of calcium (Ca) and cadmium (Cd) was examined along the gastro-intestinal tract (GIT) of freshwater and seawater Oncorhynchus mykiss irideus (FWT and SWTies respectively) using in vitro and in vivo experiments. Based on known physiological differences between FWT and SWT which aid in regulating ion levels and osmolarity, we hypothesized that SWT would have lower rates of Ca uptake. Also, we predicted that Cd rates would also be lower because Cd is known to share a common transport mechanism with Ca. Kinetics of Ca and Cd transport were determined using mucosal salines of varying concentrations [1, 10, 30, 60, and 100 (mmol L^− 1 for Ca, μmol L^− 1 for Cd)]. Linear and saturating relationships were found for Ca for FWT and SWT, but overall SWT had lower rates. Linear and/or saturating relationships were also found for Cd uptake, but rates varied little between fish types. Elevated Ca had no inhibitory effect on Cd transport, and Ca channel blockers nifedipine and verapamil had little effect on Ca or Cd uptake. However, lanthanum reduced Ca transport into some compartments. A 21 day in vivo feeding experiment was also performed where FWT and SWT were exposed to control diets or Cd-spiked diets (552 μg Cd g^− 1 food). Whole body Cd uptake between fish types was similar, but the majority of Cd in SWT remained in the posterior intestine tissue, while FWT transported more Cd through their gut wall. Overall it appears that large differences in Ca and Cd uptake between FWT and SWT exist, with SWT generally having lower rates.     

Ultrastructure of the physostomatous swimbladder of rainbow trout (Salmo gairdneri)

Summary Rainbow trout swimbladder epithelium consists of non-ciliated and ciliated cells in the ratio of greater than 21. Non-ciliated cells contain vesicles filled with a mucus-like material and similar material is found lining the surface of the swimbladder lumen. Morphological evidence for discharge of the vesicle contents was obtained. In addition, nonciliated cells contain osmiophilic lamellar bodies which resemble the cytosomes of lung alveolar cells of air-breathing vertebrates. The non-ciliated cells do not appear to be involved in a process of active gas secretion.Supported by a research grant from the American Cancer Society, Oregon Division, Inc.     

A biomechanically derived minimum work model of the fish gill lamellar system exhibits its exquisite morphological arrangement and perfusate regulation for oxygen uptake from water

To evaluate the efficiency of oxygen (O₂) uptake from water through the fish gill lamellar system, a cost function (CF) representing mechanical power expenditure for water ventilation and blood circulation through the gill was formulated, by applying steady-state fluid mechanics to a homogeneous lamellar-channel model. This approach allowed us to express CF as the function of inter-lamellar water channel width (w) and to derive an analytical solution of the width (w_min) at the minimum CF. Morphometric and physiological data for rainbow trout in the literature were referred to calculate CF(w) curves and their w_min values at five intensity stages of swimming exercise. Obtained w_min values were evenly distributed around the standard measure of the width (w_s = 24 μm) in this fish. Individual levels of CF(w_min) were also fairly close to the corresponding CF(w_s) values within a 10% deviation, suggesting the reliability of approximating [CF(w_min) = CF(w_s)]. The cost-performance of O₂ uptake through the gill (η_g) was then assessed from reported data of total O₂ uptake/CF(w_s) at each intensity stage. The η_g levels at any swimming stage exceeded 95% of the theoretical maximum value, implying that O₂ uptake is nearly optimally performed in the lamellar-channel system at all swimming speeds. Further analyses of O₂ transport in this fresh water fish revealed that the water ventilation by the buccal/opercular pumping evokes a critical limit of swimming velocity, due to confined O₂ supply to the peripheral skeletal muscles, which is avoided in ram ventilators such as tuna.     

硬头鳟巨噬细胞的体外长期培养(简报)

鱼类的巨噬细胞和高等脊椎动物的一样,在吞灭入侵的病原体方面起着极其重要的作用。探索在体外长期培养巨噬细胞的方法,有助于研究巨噬细胞的机能。Braun-Nesje等虽已从硬头鳟(Salmo gairdneri)等鲑科鱼类的头肾中分离出大量的巨噬细胞,并在体外培养了3个月之久,但因细胞不分裂,无法传代。本研究改用组织块培养法和饲养层技术探索长期培养巨噬细胞的方法,并获得成功。迄今巨噬细胞已在体外培养22个月,传代48次。细胞的原代培养分为三组。前两组是单独的脾或头肾的组织培养;第三组是脾与头肾的混合组织培养。培养液是Leibovitz’s L-15,外加20%胎牛血清,100 IU/ml青霉素和100μg/ml链霉素。巨噬细胞的吞噬活力用酵母菌Candida