Functional Studies Clause Samples
Functional Studies. To determine the function of novel proteins, both in vivo and in vitro models are utilized. In both cases the research involves two major approaches; firstly, administration of purified protein at varying doses and times of treatment, secondly, administration of an antisense oligonucleotide directed at the novel gene sequence to block expression of the protein product. Subsequently, a variety of metabolic parameters are measured and gene expression of key enzyme pathways determined. The availability of real-time PCR technology in our laboratories allows us to quickly and confidently quantitate changes in expression in tissue samples following protein treatment. Each phase of the research program is currently underway and running simultaneously; different genes identified are at various stages of development throughout the program. At the end of the research and development program, we will have identified a number of new genes involved in obesity development; we will have produced their protein products and further developed this research by determining the basic function of the novel protein in tissue culture systems and in whale animal studies. The result will be key lead compounds for further development by Lipha in Stage We currently have identified eight novel genes and these genes will proceed through various stages of the above research plan during 1999/2000. The development and progress achieved with each of these previously identified novel genes and new genes uncovered will be reported to the Autogen Scientific Advisory Board under the following Milestones-,
1. Identification of gene sequences up- or down-regulated in diabetic, non-diabetic, lean and obese animals.
2. Identification of full-gene sequence and confirmation of novel character.
Functional Studies. The H24 peptide is currently being chemically synthesised and should arrive early July. It will be used to ICV treat Psammomys obesus animals to examine if it has an effect on food intake, body weight or glucose or insulin levels. 8 A, 8 B and 8 C animals will be treated with 30 ug/day for 7 days and if an effect is seen 15 ug and 3 ug doses will be administered into further animals to establish a dose response. Polyclonal antibodies will be raised to H24. H24 conjugated to KLH will be made and injected into rabbits. After 4 months (including 2-3 boosters), the antibodies will be isolated and purified, including Westerns to confirm the specificity of the antibody for H24. Altered expression/activity of [*] contributes to type 2 diabetes. Background Several methods of gene discovery simultaneously identified the various components of this system as being differentially expressed in obese, type 2 diabetic Psammomys obesus. Differential display PCR in muscle of Psammomys obesus was used to identify [*] and microarray analysis showed differential expression of calpastatin in diabetic animals. Subsequent analyses demonstrated that [*] was overexpressed in the muscle of diabetic animals and the expression of this proteolytic enzyme was associated with blood glucose concentration independent of body weight and plasma insulin levels. Both fasting and 2-week dietary energy restriction significantly reduced the expression of CAPN3. In addition, calpastatin (CAST; endogenous inhibitor of [*]) gene expression was significantly reduced by fasting and dietary energy restriction in muscle. CAST was also overexpressed in the liver of diabetic and obese animals. Together these results suggest that dysregulation within the [*] system may be involved in the pathophysiology of obesity and/or type 2 diabetes in Psammomys obesus. Research Plan