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Browsing by Subject "VHH"

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  • Hovi, Marianne (2012)
    The burden of diabetes is increasing globally as the number of people with diabetes reaches over 220 million. Over 90 per cent of these people are suffering from type 2 diabetes. This condition is primarily defined by the chronic increase in blood glucose level or hyperglycemia. Type 2 diabetes is characterized by insulin resistance and is usually associated with abnormal insulin secretion. Insulin resistance is a state where normal amount of blood insulin is inadequate to increase glucose uptake in the most important target tissues of insulin. Numerous reports demonstrate that oversupply of lipids leads to loss of insulin activity and the formation of type 2 diabetes. Protein kinase C (PKC) isozymes comprise a family of serine/threoninekinases, which have a regulatory role in a multiple cellular processes. PKC!-isozyme activity is known to play a role in insulin resistance and therefore in type 2 diabetes. Free fatty acid (FFA) induced insulin gene function inhibition is associated with phosphoinositide dependent kinase1 (PDK1) independent phosphorylation of PKC!-isozyme in the most important insulin target tissues. Phosphorylated PKC!-isozyme causes insulinreceptor gene expression inhibition. Present study is part of a VHH-antibodies related research where the goal is to characterize these antibodies and to find out their effects on protein kinase C. VHH-antibodies are Ilama derived antibodies which contain a single heavy-chain variable domain, that is fully capable of antigen binding. In this work, we studied VHH-antibodies binding to PKC!-isozyme and its functional domains. PKC!-isozyme and its domains were produced in Sf9-insect cells. The binding was studied using Western blot and immunoprecipitation assays. In addition, the binding of 368 VHHantibodies to PKCε-isozyme's domain 2 were studied. With Western blot, it was discovered that E7-VHH-antibody binds to PKCε-isozyme full length and to domain 3. Other VHHantibodies tested in Western blot did not bind to PKCε-isozyme. Seven VHH-antibodies bound to PKCε-isozyme in immunoprecipitation. All of these VHH-antibodies bound to the full length and to domain 3, but not to other domains. In radioligand binding assays none of the VHH-antibodies bound to domain 2 that is the binding site to the endogenous PKCε-isozyme activator diacylglycerol (DAG). The results gathered with these three different methods were in line with each other. As the results gained from Western blot and immunoprecipitation show, all the VHH-antibodies, that bind to PKCε-isozyme, bind to its domain 3. With this study, we succeeded to gather new information about the binding of VHH-antibodies to PKCε-isozyme and its domains. The exact binding site has not been studied with so many VHH-antibodies before this study. Moreover, we also exploited methods that have not been used in this context before.