Post-doc. project by Carlo G. Ossum (AKI)
The major aim of this study is to identify the key enzymes in the stress response and in the death process in fish cell types from selected tissues, challenged with different stresses. The studies will extend the basic knowledge in fish cell biology and in the physiology of cellular stress. They will be of value for applied fishery research, such as improvement of seafood quality and analyses of fish welfare. In a previous Ph.D. project under Fishnet.dk, we identified and partly characterised ERK1 in the rainbow trout cell line RTHDF and showed that p38 and ERK1 are activated and deactivated respectively during anoxia (Ossum et al., 2004; Ossum & Hoffmann, 2004). The protective protein Hsp70 was strongly upregulated during periodic anoxia. Moreover, we have shown that the lipid oxidizing enzyme 5-lipoxygenase is upregulated during stress due to crowding of fish (F. Jessen, unpublished) and others have shown a central role for the phosphoesterase PLA2 during anoxia-induced cell death (Shinzawa & Tsujimoto, 2003). Together these observations points to a central role for lipid derived messengers in stress and cellular death.
The research plan includes a) molecular cloning of the stress kinase p38 and the growth promoting kinase ERK1 from rainbow trout, b) identification of which phospholipids-cleaving enzymes (phospholipases) are expressed in rainbow trout cells, c) definition of the exact roles of p38, ERK1 and phospholipases in stress-handling and initiation of cell death, and, d) using these results for suggestions that will improve fish food quality and for developing tests for fish welfare. We will first characterise and clone p38 and ERK1 from rainbow trout to evaluate the functional consequences of differences between fish and mammalian isoforms, using in vitro kinase assays and PCR techniques, respectively. The latter technique can also be used for identification of the expressed phospholipase- and lipoxygenase isoforms. Using knockout- and small interfering RNA techniques combined with 2D gel electrophoresis and mass spectroscopy, we will define the exact role of the mentioned proteins, as well as Hsp70 in the cellular stress response and induction of cell death. The stress situations we will concentrate on will be hypoxia, temperature changes and salt shock. Samples from food products defined as high and low quality, as well as samples from stressed and unstressed fishes will be analyzed for these key enzymes and the lipid-derived products of phospholipase activity.
The goal is to develop tests for food quality, as well for general fish welfare. The project will take advantage from the profound expertise and previous research activities of the two research groups involved, and it will constitute the basis for further exchange of knowledge and expertise, beneficial to both groups. The work at the major fields will be conducted at: (1) Danish Institute for Fishery Research, Flemming Jessen’s group (functional fish proteomics); (2) Institute of Molecular Biology and Physiology, Else K. Hoffmann’s group (signal transduction). We will continue the collaboration with Professor Niels C. Bols, University of Waterloo, around fish cell cultures and establish collaboration with Dr Øyvind Aas-Hansen, Norwegian Institute of Fisheries and Aquaculture Research, with respect to applied aspects of fish welfare.