Steen Bennike Mortensen

Cloning and Expression of Cytokine-Inducible Nitric Oxide Synthase cDNA From Rat Islets of Langerhans

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the β-cells of interleuldn (IL)-1β–exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokineexposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH β-cells cultured in the absence of cytokines. Addition of IL-1α alone or in combination with tumor necrosis factor-α induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokineexposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the Larginine analogs, Nω-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pi values in the range of 6.8 to 7.0. In conclusion, the IL-1β-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.

Design and synthesis of novel PPARα/γ/δ triple activators using a known PPARα/γ dual activator as structural template

Abstract Using a known dual PPARα/γ activator (5) as a structural template, SAR evaluations led to the identification of triple PPARα/γ/δ activators (18–20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARα/γ/δ activation.

Purification of an Aspartic Proteinase from Aspergillus Aculeatus

Aspergilli are known to produce a number of extracellular enzymes (e.g. proteinases, carbohydrases, lipases). Several are utilized in commercial products, based on a main activity, but often containing minor side activities. Usually it is difficult to isolate small amounts of a side activity, but in such cases affinity chromatography can be a valuable tool, due to its specific action. We have succeeded in isolating a proteolytic side activity from the Aspergillus aculeatus derived carbohydrase product Viscozyme™ (Novo Nordisk A/S, Denmark). The proteinase was isolated by affinity chromatography on a bacitracin matrix, and was shown to be an aspartic proteinase.