Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure, N ⑀ -(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-B and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.Receptor for AGE 1 (RAGE), a member of the immunoglobulin superfamily, was first described as a cell surface interaction site for advanced glycation end products (AGEs), products of glycation and oxidation of proteins and lipids (1-2). AGEs are a heterogeneous class of compounds, whose accumulation in disorders such as diabetes, renal failure, Alzheimer's disease, and, indeed, natural aging, albeit to a lesser degree, has suggested their potential contribution to the pathogenesis of complications that typify these conditions (3-7). Our previous studies demonstrated that both in vitro and in vivo derived heterogeneous AGEs ligate cell surface RAGE on endothelium (ECs), mononuclear phagocytes (MPs), vascular smooth muscle (VSMC), and neurons to activate cell signaling pathways such as ERK1/ERK2 kinases and NF-B (8 -9), thereby redirecting cellular function in a manner linked to expression of inflammatory and prothrombotic genes important in the pathogenesis of chronic disorders as apparently diverse as diabetic macrovascular disease and amyloidosis (10 -20).Our recent studies suggested that interruption of the interaction of AGEs with RAGE in vivo, by administration of soluble RAGE (sRAGE), the extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerotic lesion development and complexity in diabetic rodents (19 -20). In the latter studies, analysis of plasma demonstrated evidence of an sRAGE⅐AGE complex; immunoprecipitation of plasma obtained from diabetic sRAGEtreated mice with anti-RAGE IgG yielded species immunoreactive with both anti-RAGE IgG or affinity purified anti-AGE IgG, suggesting that sRAGE might bind up AGEs and limit their interaction with and activation of cell surface RAGE. The beneficial effects of sRAGE were independent of alterations in other risk factors, such as hyperglycemia and hyperlipidemia, implicating a role for AGE-RAGE interaction in the development of vascular dysfunction in diabetes (20).These past studies, however, did not elucidate ...

Separate genes encode thyroid hormone receptor subtypes TRalpha (NR1A1) and TRbeta (NR1A2). Products from each of these contribute to hormone action, but the subtypes differ in tissue distribution and physiological response. Compounds that discriminate between these subtypes in vivo may be useful in treating important medical problems such as obesity and hypercholesterolemia. We previously determined the crystal structure of the rat (r) TRalpha ligand-binding domain (LBD). In the present study, we determined the crystal structure of the rTRalpha LBD in a complex with an additional ligand, Triac (3,5, 3'-triiodothyroacetic acid), and two crystal structures of the human (h) TRbeta receptor LBD in a complex with either Triac or a TRbeta-selective compound, GC-1 [3,5-dimethyl-4-(4'-hydroy-3'-isopropylbenzyl)-phenoxy acetic acid]. The rTRalpha and hTRbeta LBDs show close structural similarity. However, the hTRbeta structures extend into the DNA-binding domain and allow definition of a structural "hinge" region of only three amino acids. The two TR subtypes differ in the loop between helices 1 and 3, which could affect both ligand recognition and the effects of ligand in binding coactivators and corepressors. The two subtypes also differ in a single amino acid residue in the hormone-binding pocket, Asn (TRbeta) for Ser (TRalpha). Studies here with TRs in which the subtype-specific residue is exchanged suggest that most of the selectivity in binding derives from this amino acid difference. The flexibility of the polar region in the TRbeta receptor, combined with differential recognition of the chemical group at the 1-carbon position, seems to stabilize the complex with GC-1 and contribute to its beta-selectivity. These results suggest a strategy for development of subtype-specific compounds involving modifications of the ligand at the 1-position.

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs ␣ and ␤) that bind triiodothyronine (T 3 , 3,5,3-triiodo-L-thyronine) with high affinity, and its precursor thyroxine (T 4 , 3,5,3,5-tetraiodo-L-thyronine) with lower affinity. T 4 contains a bulky 5 iodine group absent from T 3 . Because T 3 is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5 substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T 4 affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T 4 complexes adopt a conformation that differs from TR-T 3 complexes in solution. Nonetheless, T 4 behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T 3 does not contribute to agonist activity. We determined x-ray crystal structures of the TR␤ LBD in complex with T 3 and T 4 at 2.5-Å and 3.1-Å resolution. Comparison of the structures reveals that TR␤ accommodates T 4 through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5 iodine and complete the coactivator binding surface. While T 3 is the major active TH, our results suggest that T 4 could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5 extension should be considered in TR ligand design. Thyroid hormone (TH)1 plays important regulatory roles in metabolism, homeostasis, and development by binding and altering the transcriptional regulatory properties of two related nuclear receptors (NRs), the thyroid hormone receptors (TRs) ␣ and ␤ (1, 2). Most TH produced in the thyroid gland is secreted in the form of thyroxine (T 4 ; 3,5,3Ј,5Ј-tetraiodo-L-thyronine) (2, 3). The thyroid gland also produces smaller amounts of triiodothyronine (T 3 ; 3,5,3Ј-triiodo-L-thyronine) and reverse T 3 (rT 3 ; 3,3Ј,5Ј-triiodo-L-thyronine), and 80% of T 4 is converted to T 3 and rT 3 in peripheral tissues by two selenium deiodinases, which are tissue-specific (4). Current beliefs are that T 3 is the dominant active form of TH; T 3 binds the TRs with an affinity about 20 -30 times higher than that of T 4 (5-9), and some studies suggest that T 3 is present at higher concentrations in the nucleus than T 4 (10, 11). Nonetheless, the question of whether T 4 is simply a prohormone or an active TH species is not completely resolved. T 4 exerts rapid nongenomic effects at several loci distinct from TRs (12). Moreover, saturating levels of T 4 activate transcription of TH-responsive genes in cell culture (see for example Ref. 5). Whereas it is possible that at least some of this activity is due to T 3 generated from T 4 in the cell, these results suggest that T 4 may act as a TR agonist. Normal concentrations of plasma-free T 4 are about 4 -6-fold higher than th...

In vitro engineering of autologous fatty tissue constructs is still a major challenge for the treatment of congenital deformities, tumor resections or high-graded burns. In this study, we evaluated the suitability of photo-crosslinkable methacrylated gelatin (GM) and mature adipocytes as components for the composition of three-dimensional fatty tissue constructs. Cytocompatibility evaluations of the GM and the photoinitiator Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) showed no cytotoxicity in the relevant range of concentrations. Matrix stiffness of cell-laden hydrogels was adjusted to native fatty tissue by tuning the degree of crosslinking and was shown to be comparable to that of native fatty tissue. Mature adipocytes were then cultured for 14 days within the GM resulting in a fatty tissue construct loaded with viable cells expressing cell markers perilipin A and laminin. This work demonstrates that mature adipocytes are a highly valuable cell source for the composition of fatty tissue equivalents in vitro. Photo-crosslinkable methacrylated gelatin is an excellent tissue scaffold and a promising bioink for new printing techniques due to its biocompatibility and tunable properties.

We report the first photochemical protocol for the generation of sequence defined macromolecules employing two hetero bifunctional photoreactive synthons, exploiting the orthogonal nature of photochemical - via the use of caged dienes - and thermally driven ligation protocols. We demonstrate that the iterative alternating synthon addition to an initial bifunctional core under irradiation at ambient temperature enables the generation of a macromolecule with up to 10 units (M = 3231.58 g mol(-1), Đ = 1.00). The resulting macromolecules are monodisperse and feature absolute chain end fidelity. The unit-by-unit construction of the macromolecule is evidenced by Nuclear Magnetic Resonance Spectroscopy, Electrospray Ionization Mass Spectrometry and Size Exclusion Chromatography. The fundamental principle demonstrated herein paves the way for employing photochemical strategies for the design of sequence defined polymers.