Deliverables Profile of melatonin production and receptor distribution Melatonin (Mel) was significantly higher at night than during the day in adult fish in Spring and Autumn. Mel concentrations in juvenile fish were also significantly higher than those in adult fish. Mel contents in the tissues, i.e. small intestine, gill and kidney at 11:00 h and 23:00 h showed no significant day-night differences in melatonin concentrations and was highest in intestine. There were no daily variations in the Kd and Bmax for high affinity melatonin binding sites in different tissues. These results provide the basic information for manipulations which may be dependent on the daily light-dark cycle. Antisera to fish PTHrP Polyclonal antiserum was raised in rabbits to a 1-34 flounder parathyroid hormone-related protein peptide (PTHrP). The antiserum has been used to develop a specific and sensitive radioimmunoassay (RIA) for PTHrP. The antiserum has also been used for immunohistochemistry. Since the N-terminal region of PTHrP is very conserved the antiserum works with many fish species. cDNA clones of PTHrP responsive genes 2000 clones from sea bream larvae have been produced by suppressive subtraction hybridization of PTHrP treated and control sea bream. These clones can be used to identfy markers related to developmental processes, for functional studies and cDNA microarray production. effect of PTHrP on in vitro and in vivo vitellogenin production During reproduction E2 is the main hormone responsible for the induction of vitellogenin production, a process that induces a large increase in extracellular calcium and phosphorus. Using a PTHrP antagonist (7-34 PTHrP), we have been able to block the E2-induced rise in calcium and phosphorus, thus demonstrating that PTHrP mediates at least in part the hypercalcemic effect of E2 in calcium balance. This result may be important to understand the factors that contribute for egg quality. Optimal calcium and phosphate diet When with limited calcium access (low salinity and calcium-deficient diet) sea bream show growth arrest. A strong positive correlation was found between net calcium and phosphorus accumulation and between body weight and whole body calcium and phosphorus content. Phosphorus accumulation is strongly calcium dependent, as it decreases in parallel to calcium accumulation when the diet is calcium deficient, but phosphorus sufficient. These results highlight the importance of calcium for normal fish growth. Quantitative RT-PCR for measuring changes in PTHrP, PTHrP-R and CaSR A quantitative polymerase chain reaction method (Q-PCR)has been established to measure the mRNA expression levels of parathyroid hormone-related protein (PTHrP), parathyroid hormone receptor type 1 (PTHR1) and Calcium-sensing receptor (CaSR. This methodology can be applied to analyse gene expression in sea bream. Purified recombinant seabream PTHrP Recombinant sea bream parathyroid hormone related protein was produced and purified using an E. coli system and one step purification process with continuous elution gel electrophoresis. The bioactivity of recombinant (1-125) sbPTHrP assessed using an in vitro scale bioassay was found to be equipotent to (1-34) PTHrP in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross reacted with antisera specific for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay specific for piscine N-terminal (1-34) PTHrP, the recombinant sbPTHrP (1-125) was equipotent with (1-34) PTHrP in displacing labelled 125I (1 34) PTHrP from the antisera. The recombinant sbPTHrP allows the development of region specific assays and to carry out studies aimed at defining post-secretory processing of this protein and its biological activity in fish. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
