S100A12, also called EN-RAGE (extracellular newly identified receptor for advanced glycation end products binding protein) or calcium-binding protein in amniotic fluid-1, is a ligand for RAGE. It has been shown that S100A12 induces adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in the vascular endothelial cell and mediates migration and activation of monocytes/macrophages through RAGE binding and that infusion of lipopolysaccharide into mice causes time-dependent increase of S100A12 in the plasma. Therefore, circulating S100A12 protein may be involved in chronic inflammation in the atherosclerotic lesion. In this study, we developed an ELISA system that uses specific monoclonal antibodies against recombinant human S100A12 to measure plasma S100A12 levels in patients with diabetes. On using our S100A12 ELISA system, the coefficients of variation of intra- and interassay were less than 4 and 9%, respectively. The analytical lower detection limit was 0.2 ng/ml. When plasma S100A12 levels were measured by this system, the concentrations were more than twice as high in the patients with diabetes, compared with those without. Using univariate analysis in all subjects, plasma S100A12 concentrations correlated with hemoglobin A1c, fasting glucose, high-sensitivity C-reactive protein and white blood cell count. Stepwise multiple regression analyses, however, revealed that only white blood cell count and hemoglobin A1c remained significant independent determinants of plasma S100A12 concentration. These results suggest that plasma S100A12 protein levels are regulated by factors related to subclinical inflammation and glucose control in patients with type 2 diabetes.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp ؊100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the ؊2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) ␤ or JEG nuclear extracts (containing RXR␣) and bacterially expressed chicken T3 receptor ␣1 (TR␣) can occupy both half-sites although the 3 half-site is dominant. T3 causes dissociation of TR␣ from the 5 half-site but increases binding to the 3 half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR؉15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3 half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp ؊700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.Type 1 iodothyronine deiodinase (D1), the product of the dio1 gene, catalyzes both the first step in thyroid hormone activation, which is outer ring deiodination of thyroxine (T4) to form 3,5,3Ј-triiodothyronine (T3), and the inactivation of T4 or T3 by removal of an inner-ring iodine from the iodothyronine nucleus (48). In the rat, T3 produced by D1 activity provides ϳ60 to 70% of the circulating hormone (35,52). D1 is one of a very small group of selenoenzymes in which selenocysteine, which is encoded by UGA, is present in the active center (8, 9). Thyroid hormone markedly increases dio1 mRNA in liver and kidney in vivo and in rat FRTL-5 and pituitary tumor cells as well as in hepatocytes in primary culture (6,7,42,47,51,67). This increase is a consequence of a direct interaction of T3 with the thyroid hormone receptor and does not require new protein synthesis as does the T3-induced increase in rat growth hormone (rGH) or spot 14 mRNA (59, 63).Triiodothyronine induction of gene expression requires the interaction of ligand with the T3 receptor (TR), which is bound to specific sequences of DNA termed thyroid hormone response elements (TREs) and usually found in the 5Ј-flanking region of T3-responsive genes. A common TRE conformation consists of two core hexamers, termed half-sites, beari...

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Type 1 iodothyronine deiodinase (D1) is a microsomal selenoenzyme which catalyzes deiodination of thyroxine to 3,5,3'-triiodothyronine. Immunoblotting showed that endogenous hepatic, renal, and transiently expressed D1 remains in microsomes after pH 11.5 treatment. In vitro translation studies using pancreatic microsomes identified a single transmembrane domain with a cytosolic carboxyl-terminal catalytic portion. The transmembrane domain is located between conserved basic amino acids at positions 11 and 12 and a group of charged residues at positions 34-39. A transiently expressed D1 protein in which residues 2-25 were deleted was inactive and not integrated into membranes. Activity was not restored by replacing these residues with transmembrane domains from a cytochrome P450 or type 3 deiodinase enzyme despite their incorporation into membranes. Elimination of the positive charges at positions 11 and 12 reduced the amount of transiently expressed protein by 70%, but the enzyme formed was catalytically normal. Similar results were found after conversion of the Lys-27 in the transmembrane domain to Met or Glu. We conclude that the amino terminus of D1 contains uncleaved signal and stop transfer sequence properties. In addition, positively charged residues at positions 11, 12, and 27 are required for optimal formation of the protein but not for catalysis.