No abstract

Supplies of marine fish oils (FO) are limited, and sustainable production in aquaculture dictates that alternatives that do not compromise fish health and product quality, such as vegetable oils, must be found. Nutrigenomics will increase our understanding of how nutrition influences metabolic pathways and homeostatic control, and may be used to measure and validate subtle changes in organ-specific, metabolic gene expression signatures. We compared 2 groups of Atlantic salmon fed diets containing 100% FO or 75% rapeseed oil (RO) for 42 wk. A small-scale cDNA microarray was constructed to screen for changes in the expression of lipid metabolism genes in the liver resulting from this partial substitution of RO for FO. Delta5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor gamma, as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.

In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314-BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription-polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.

Ovine uterine artery (UA) endothelial cells (UAEC) maintained in culture to passage 4 retain pregnancy-specific changes in vasodilator production, which in turn is associated with differences in Ca(2+) and ERK 1/2 signaling. The question remains whether this is an accurate portrayal of the situation in vivo, or more simply whether these same signaling responses seen at passage 4 accurately reflect those functioning in the cells in vivo. Small groups of endothelial nitric oxide synthase-positive cells from both pregnant and nonpregnant ewes were freshly isolated and used to image changes in the intracellular free calcium concentration ([Ca(2+)](i)) using fura 2 and to detect ERK 1/2 phosphorylation by immunocytochemistry. Furthermore, detailed comparisons of mRNA species were made between freshly isolated and cultured (passage 4) cells using cDNA microarray analysis and verified, where possible, using PowerBlot analysis. Freshly isolated cells showed no detectable [Ca(2+)](i) elevation in response to angiotensin II, epidermal growth factor, basic fibroblast growth factor, or vascular endothelial growth factor but did respond to ATP in a dose-dependent (1-300 microM) manner. At higher doses of ATP, [Ca(2+)](i) elevation was sustained longer and showed a high incidence of regular oscillations in cells from pregnant compared with nonpregnant ewes. Also, ATP and basic fibroblast growth factor treatment caused activation of ERK 1/2 in significantly greater numbers of freshly isolated cells from pregnant than from nonpregnant ewes. cDNA microarray analysis showed results consistent with endothelium but revealed few differences in mRNA species and levels between freshly isolated and passage 4 cells or between the pregnant and nonpregnant ewes. In conclusion, our data show for the first time that pregnancy-specific changes in Ca(2+) and ERK 1/2 signaling are indeed observed in freshly isolated UA endothelium. This suggests in turn that such pregnancy-specific changes in UA endothelial function in vivo in response to a variety of agonists during pregnancy are both programmed at the level of cell signaling and retained in culture.

Diapause-destined embryos of the crustacean Artemia franciscana cease development as gastrulae, encyst, and enter a resting stage characterized by greatly reduced metabolic activity and extreme stress resistance. To better understand diapause induction and maintenance in Artemia embryos gene expression was analyzed by subtractive hybridization at two days post-fertilization, a time early in this developmental process. Eighty-five of 264 cDNA clones sequenced matched GenBank entries and they fell into categories designated as environmental information processing, cellular processes, genetic information processing and metabolism. Semi-quantitative RT-PCR of cDNAs populating the subtractive library identified seventeen up-regulated and four down-regulated transcripts, the former including those encoding a human transcription cofactor homologue, three small heat shock proteins, putative cell growth suppressor proteins and several enzymes. As examples, p8 may modulate gene expression during diapause in Artemia embryos. BRCA1 associated protein-1 (BAP1) and other functionally related proteins may influence cell growth and division during transition into diapause, a time when these processes are inhibited, whereas small heat shock proteins protect embryos from stress. This study represents the first systematic molecular characterization of diapause in crustaceans. Several differentially expressed genes were identified, expanding the repertoire of proteins potentially modified during diapause and suggesting mechanistic pathways indigenous to the initiation and maintenance of this physiological state.