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  • br Experimental br Results br Discussion The use


    Discussion The use of Dex-BSA asa membrane-limited glucocorticoid has been met with some skepticism, mainly for its assumed instability. Here we provide conclusive evidence that Dex-BSA is stable in solution over an extended period of time and at high temperature (i.e., body temperature). It was surprising, however, to learn that Dex-BSA is manufactured with a significant concentration of free Dex not covalently bound to the BSA, which we calculated to be 4.5% of the total Dex. While the chemical purity by mass (98.2%) of commercially available Dex-BSA is within what would appear to be acceptable standards, the molar concentration of Dex well exceeded that of Dex-BSA, by nearly 2-fold. If these Dex molecules were indeed free in solution and allowed to diffuse through the plasma membrane, any effects of the Dex-BSA could, in fact, be due to the free Dex. Estradiol- and testosterone-BSA conjugates have also been shown to contain free steroid, which is released more readily under denaturing conditions [11]. Dialysis of the native sex steroid conjugates was ineffective in removing free steroid contamination, but dialysis of the denatured conjugates was not attempted in that study. These compounds have not been tested for stability over time, which is rendered difficult by their lack of fluorine substrate for the NMR biochemical analysis. Nevertheless, as shown here with the Dex-BSA compound, denaturation, dialysis and renaturation should provide a relatively easy and viable way of ensuring the purity and stability of any steroid-BSA conjugate, including corticosteroid and sex hormone conjugates.
    Acknowledgments This work was supported by NIH grants 2R01 MH066958, R01 MH104373. We thank Dr. Louis Muglia for his generous gift of the GR-GFP construct.
    Introduction Glucocorticoids (GCs) play an integral role in a wide array of physiological systems in the body, affecting lipid and glucose metabolism, immunosuppressive and anti-inflammatory reactions, growth, reproduction and Firefly Luciferase function. The effects of cortisol are largely mediated by the GC receptor (GR). In its unbound form, GR resides in the cytoplasm as part of a large multiprotein complex comprising of chaperones (hsp90, hsp70), co-chaperones, and immunophilins (FK506-binding protein) (Fig. 1) [1]. Upon binding with cortisol, conformational changes occur, leading to the dissociation of GR from the multiprotein complex. GR-GR dimers are often formed. The activated, ligand bound GR complex then translocate to the nucleus, where it functions as a transcription factor to regulate gene expression by binding at specific glucocorticoid response elements regulating both transactivation or transrepression. The mechanism of transactivation of gene expression usually involves GR dimers, binding with specific glucocorticoid response elements and activating gene expression. The transrepression mechanism involves the activated GR interacting with transcription factors, like AP-1 and NF-κB, preventing them from binding to their target genes. This interaction can sometime allow GR to indirectly regulate gene expression The transrepression mechanism involves the activated GR interacting with transcription factors, like AP-1 and NF-κB, preventing them from binding to their target genes. This interaction can sometime allow GR to regulate gene expression indirectly in the absence of direct DNA binding [2], [3].
    Glucocorticoid receptor gene The human GR gene (NR3C1) is located on chromosome 5 and consists of 9 exons (Fig. 2). Variations in the transcription and translation of the GR gene can lead to seemingly random actions of cortisol. However, these variations are necessary to allows cells and tissues to appropriately adapt to wide concentrations of GC. As an example, alternative splicing of the GR precursor mRNA can give rise to 5 GR protein subtypes- GRα,GRβ, GRγ, GR-A and GR-P. Eight more receptor proteins are produced by alternative translation initiation from GR mRNA [4], [5]. The classic GRα isoform can increase Firefly Luciferase GC sensitivity while the GRβ is transcriptionally inactive and is responsible for GC resistance. In addition to the GR isoforms generated by alternative initiation of translation or alternative splicing, four novel receptor variants (GR NS-1, GR DL-1, GR-S1 and GR-S1 (−349A)) [6], [7] with multiple amino acid replacements/truncation and unknown functional role have also been discovered. These generic variations may account for the diversity in patient presentations and responses to GC.