Structural and Functional Similarity of Amphibian Constitutive Androstane Receptor with Mammalian Pregnane X Receptor

The nuclear receptors and xenosensors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) induce the expression of xenobiotic metabolizing enzymes and transporters, which also affects various endobiotics. While human and mouse CAR feature a high basal activity and low induction upon ligand exposure, we recently identified two constitutive androstane receptors in Xenopus laevis (xlCARα and β) that possess PXR-like characteristics such as low basal activity and activation in response to structurally diverse compounds. Using a set of complementary computational and biochemical approaches we provide evidence for xlCARα being the structural and functional counterpart of mammalian PXR. A three-dimensional model of the xlCARα ligand-binding domain (LBD) reveals a human PXR-like L-shaped ligand binding pocket with a larger volume than the binding pockets in human and murine CAR. The shape and amino acid composition of the ligand-binding pocket of xlCAR suggests PXR-like binding of chemically diverse ligands which was confirmed by biochemical methods. Similarly to PXR, xlCARα possesses a flexible helix 11’. Modest increase in the recruitment of coactivator PGC-1α may contribute to the enhanced basal activity of three gain-of-function xlCARα mutants humanizing key LBD amino acid residues. xlCARα and PXR appear to constitute an example of convergent evolution.     

Hepcidin Revisited, Disulfide Connectivity, Dynamics, and Structure

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys¹–Cys⁸, Cys³–Cys⁶, Cys²–Cys⁴, and Cys⁵–Cys⁷. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.Regulation of iron levels is critical to the survival of species that live in an oxygen-rich environment (1). In mammals, iron homeostasis is principally regulated by hepcidin, a 25-residue peptide hormone containing a complex network of four disulfide bonds. Hepcidin was discovered by three groups investigating either novel anti-microbial peptides or iron regulation (2–4), and subsequent genetic evidence has shown that mutation of the hepcidin gene can lead to systemic iron overload or hemochromatosis (5). Similarly, mutations in upstream control proteins HFE and hemojuvelin or mutation of the gene for ferroportin, the hepcidin receptor, cause forms of hemochromatosis of varying clinical severity (6–9). Genetic studies in mice have confirmed these relationships, identifying the hepcidin pathway as a critical component in the control of iron metabolism (10–12). Dysfunction of the hepcidin pathway and the resulting iron imbalance may play a role in multiple diseases such as anemia of inflammation (13), atherosclerosis (14), and neurodegenerative disorders (15). In anemia of inflammation, suppression of hepcidin constituted a successful treatment, suggesting that it may be an appropriate therapeutic target in the treatment of disease.³The human hepcidin gene encodes an 84-residue prepropeptide that contains a 24-residue N-terminal signal peptide that is subsequently cleaved to produce pro-hepcidin. Pro-hepcidin is then processed to produce a mature 25-amino acid hepcidin that is detectable in both blood and urine. Mass spectrometry and chemical analysis have revealed that all eight cysteines in hepcidin are involved in disulfide bonds (3) suggesting a highly constrained structure containing a precise disulfide bonding pattern.The NMR solution structure of hepcidin first reported by Hunter et al. (16) revealed a compact fold with β-sheet and β-hairpin loop elements. From structure calculations and dynamic signatures in NMR spectra, the authors inferred a disulfide connectivity of Cys¹–Cys⁸, Cys²–Cys⁷, Cys³–Cys⁶,⁴ and a rare vicinal disulfide bond at Cys⁴–Cys⁵. A later study of bass hepcidin (17) determined essentially the same fold and confirmed the same disulfide connectivity. Both studies, however, were based on incomplete NMR data because the resonances from two adjacent cysteines, Cys-13 and Cys-14 of hepcidin, were not detected, presumably due to exchange broadening.Here we demonstrate a new pattern of disulfide connectivity obtained independently from chemical and spectroscopic analysis. In addition, we present the first complete solution NMR structure of hepcidin and x-ray structure of the peptide in complex with an anti-hepcidin Fab. NMR data obtained at different temperatures reveal that hepcidin exhibits significant conformational dynamics in solution, a problem that likely occluded previous NMR studies. Data presented here show that these dynamics can be almost completely resolved by temperature variation, yielding two distinct structures of hepcidin, one at 325 K and one at 253 K in supercooled water. In addition to inferring disulfide bonds from structure calculations, we present an argument based on probabilistic interpretation of NMR data, which unequivocally establishes the same connectivity as obtained from chemical analysis.Because of the complexity of the disulfide network, hepcidin production is prone to misfolding artifacts. We demonstrate this through biophysical and biological activity characterization of hepcidin samples obtained from different sources. This information is essential for establishing accurate standards for quantitation of hepcidin levels in humans. In our experience, the highest quality material appeared to be critical for the structural studies presented here.     

The specific T-cell response to antigenic peptides is influenced by bystander peptides

T lymphocytes recognize antigens in the form of peptides presented by major histocompatibility complex (MHC) molecules on the cell surface. Only a small proportion of MHC class I and class II molecules are loaded with foreign antigenic peptides; the vast majority are loaded with thousands of different self peptides. It was suggested that MHC molecules presenting self peptides may serve either to decrease (antagonistic effect) or increase (synergistic effect) the T cell response to a specific antigen. Here, we present our finding that transfected mouse fibroblasts presenting a single antigenic peptide covalently bound to a class II MHC molecule stimulated specific mouse T cell hybridoma cells to an interleukin-2 response less efficiently than fibroblasts presenting a similar amount of antigenic peptide in the presence of class II molecules loaded with heterogenous bystander peptides.     

A universal algorithm for genome-wide in silicio identification of biologically significant gene promoter putative cis-regulatory-elements; identification of new elements for reactive oxygen species and sucrose signaling in Arabidopsis

Short motifs of many cis-regulatory elements (CREs) can be found in the promoters of most Arabidopsis genes, and this raises the question of how their presence can confer specific regulation. We developed a universal algorithm to test the biological significance of CREs by first identifying every Arabidopsis gene with a CRE and then statistically correlating the presence or absence of the element with the gene expression profile on multiple DNA microarrays. This algorithm was successfully verified for previously characterized abscisic acid, ethylene, sucrose and drought responsive CREs in Arabidopsis, showing that the presence of these elements indeed correlates with treatment-specific gene induction. Later, we used standard motif sampling methods to identify 128 putative motifs induced by excess light, reactive oxygen species and sucrose. Our algorithm was able to filter 20 out of 128 novel CREs which significantly correlated with gene induction by either heat, reactive oxygen species and/or sucrose. The position, orientation and sequence specificity of CREs was tested in silicio by analyzing the expression of genes with naturally occurring sequence variations. In three novel CREs the forward orientation correlated with sucrose induction and the reverse orientation with sucrose suppression. The functionality of the predicted novel CREs was experimentally confirmed using Arabidopsis cell-suspension cultures transformed with short promoter fragments or artificial promoters fused with the GUS reporter gene. Our genome-wide analysis opens up new possibilities for in silicio verification of the biological significance of newly discovered CREs, and allows for subsequent selection of such CREs for experimental studies.     

Iodine prophylaxis in children population on the Wielkopolska Region area from year 1992 to 2005

INTRODUCTION: The aim of the study was to evaluate of efficiency of iodine prophylaxis based on obligatory model of salt iodization. MATERIAL AND METHODS: The study included 1444 children from the rural and urban area, with the proportional sex and age (8-12 years) distribution--432 children in 1992 (before salt iodization), 558 children in 2000 and 454 children in 2005 (during salt iodization from 1996). RESULTS: The prevalence of goiter detected in children population in 1992 was 40% (recount by current criteria), in 2005 was reduced to 6% (p < 0.01). Parallel, median of urinary iodine concentration increased from 44 microg/l in 1992 to 107 microg/l (p < 0.01) in 2005. The increase of incidence of autoimmunological thyroid diseases is observed, especially chronic thyroiditis. CONCLUSIONS: The study proves high efficiency of iodine prophylaxis in Wielkopolska Region, but it is still the area with mild iodine deficiency.     

Anti-inflammatory plasma cytokines in children and adolescents with Down syndrome

Cytokines participate in many physiological processes including the regulation of immune and inflammatory responses. Production of some important cytokines in children with Down syndrome (DS) is depressed or increased. In this study we analysed the selected anti- inflammatory cytokines: interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13) in plasma of children and adolescents with DS. The study group consisted of 20 patients with Down syndrome and 33 healthy subjects at the age of 5-17 years. Levels of: IL-4, IL-10 and IL-13 in plasma samples were determined by specific enzyme- linked immunosorbent assay (ELISA) techniques according to manufacturer's instructions. IL-4 was detectable in 25% subjects with Down syndrome and in 28.6% healthy subjects. IL-13 was detectable in 15% patients with Down syndrome and in 15.2% healthy subjects, respectively. IL-10 was detectable in 1 of 20 patients with Down syndrome and in 2 of 33 healthy subjects only. No significant correlations between measurable cytokine levels and age and gender were found. No significant increased concentration of selected anti- inflammatory cytokines were detected.