Hypoxia increases adipogenesis and affects adipocytokine production in orbital fibroblasts-a possible explanation of the link between smoking and Graves' ophthalmopathy.

AIM: To assess the effects of hypoxia on human orbital fibroblasts (OF) on adipogenesis and adipocytokine production. METHODS: Human OF were derived from tissues obtained from patients with Graves' ophthalmopathy (GO) and from patients without known thyroid diseases undergoing blepharoplasty. The OF were cultured separately under normoxic and hypoxic conditions. Comparisons of adipocytokine concentrations using multiplex ELISA and lipid accumulation in the cells using Oil Red O staining were subsequently performed. RESULTS: There was increased adipogenesis in OF from GO subject when exposed to hypoxic culture conditions. This was not observed in OF from normal controls. Hypoxia led to an increase in leptin and a decrease in MCP-1 secretion in OF cultures. CONCLUSION: Hypoxia induces adipogenesis in OF and may represent a mechanism by which smoking contributes to deterioration of GO. We also found novel changes to leptin and MCP-1 production in OF cultures exposed to hypoxia suggesting important roles of these cytokines in the disease process.

A His6-SUMO-eXact tag for producing human prepro-urocortin 2 in Escherichia coli for raising monoclonal antibodies.

This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His6-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact™ affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact™ column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10⁻¹° and 10⁻¹¹ M) specific to the pro-region of Urocortin 2.