Understanding the molecular basis of the high oxygen affinity variant human hemoglobin Coimbra.

Human hemoglobin (Hb) Coimbra (ßAsp99Glu) is one of the seven ßAsp99 Hb variants described to date. All ßAsp99 substitutions result in increased affinity for O2 and decreased heme-heme cooperativity and their carriers are clinically characterized by erythrocytocis, caused by tissue hypoxia. Since ßAsp99 plays an important role in the allosteric α1ß2 interface and the mutation in Hb Coimbra only represents the insertion of a CH2 group in this interface, the present study of Hb Coimbra is important for a better understanding of the global impact of small modifications in this allosteric interface. We carried out functional, kinetic and dynamic characterization of this hemoglobin, focusing on the interpretation of these results in the context of a growth of the position 99 side chain length in the α1ß2 interface. Oxygen affinity was evaluated by measuring p50 values in distinct pHs (Bohr effect), and the heme-heme cooperativity was analyzed by determining the Hill coefficient (n), in addition to the effect of the allosteric effectors inositol hexaphosphate (IHP) and 2,3-bisphosphoglyceric acid (2,3-BPG). Computer simulations revealed a stabilization of the R state in the Coimbra variant with respect to the wild type, and consistently, the T-to-R quaternary transition was observed on the nanosecond time scale of classical molecular dynamics simulations.

Hughes syndrome and multiple sclerosis.

Multiple sclerosis (MS) and antiphospholipid syndrome (APS) share common clinical, laboratory and radiological features. We reviewed all the English papers on MS and APS published in the literature from 1965 to 2014 using PubMed and Google Scholar. We found that APS can mimic antiphospholipid antibodies (aPL)-positive MS in many ways in its clinical presentation. Nevertheless, APS diagnosis, clinical manifestations and management differ from those of MS. aPL were found in MS patients with titers ranging from 2% to 88%. The distribution and volume of lesions on magnetic resonance imaging (MRI) may help to differentiate MS from primary APS. In addition, atypical MS presentation can guide physicians toward an alternative diagnosis, especially when features of thrombosis and/or history of connective tissue disease are present. In that case, an anticoagulation trial could be worthwhile.

Identification of a new hormone-binding site on the surface of thyroid hormone receptor.

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily of ligand-activated transcription factors involved in cell differentiation, growth, and homeostasis. Although X-ray structures of many nuclear receptor ligand-binding domains (LBDs) reveal that the ligand binds within the hydrophobic core of the ligand-binding pocket, a few studies suggest the possibility of ligands binding to other sites. Here, we report a new x-ray crystallographic structure of TR-LBD that shows a second binding site for T3 and T4 located between H9, H10, and H11 of the TRα LBD surface. Statistical multiple sequence analysis, site-directed mutagenesis, and cell transactivation assays indicate that residues of the second binding site could be important for the TR function. We also conducted molecular dynamics simulations to investigate ligand mobility and ligand-protein interaction for T3 and T4 bound to this new TR surface-binding site. Extensive molecular dynamics simulations designed to compute ligand-protein dissociation constant indicate that the binding affinities to this surface site are of the order of the plasma and intracellular concentrations of the thyroid hormones, suggesting that ligands may bind to this new binding site under physiological conditions. Therefore, the second binding site could be useful as a new target site for drug design and could modulate selectively TR functions.

Analysis of agonist and antagonist effects on thyroid hormone receptor conformation by hydrogen/deuterium exchange.

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH3. We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes.

Rotational dynamics and polymerization of C60 in C60-cubane crystals: a molecular dynamics study.

We report classical and tight-binding molecular dynamics simulations of the C(60) fullerene and cubane molecular crystal in order to investigate the intermolecular dynamics and polymerization processes. Our results show that, for 200 and 400 K, cubane molecules remain basically fixed, presenting only thermal vibrations, while C(60) fullerenes show rotational motions. Fullerenes perform "free" rotational motions at short times (approximately < 1 ps), small amplitude hindered rotational motions (librations) at intermediate times, and rotational diffusive dynamics at long times (approximately > 10 ps). The mechanisms underlying these dynamics are presented. Random copolymerizations among cubanes and fullerenes were observed when temperature is increased, leading to the formation of a disordered structure. Changes in the radial distribution function and electronic density of states indicate the coexistence of amorphous and crystalline phases. The different conformational phases that cubanes and fullerenes undergo during the copolymerization process are discussed.

Retinal nerve fibre layer thickness profile in normal eyes using third-generation optical coherence tomography.

AIMS: To establish four normal retinal nerve fibre layer (RNFL) thickness radial profiles based on third-generation optical coherence tomography (OCT) and to compare them with previously reported histologic measurements. METHODS: A total of 20 normal eyes were studied. A circular scan was adjusted to the size of the optic disc and three scans were performed with this radius and every 200 microm thereafter, up to a distance of 1400 microm. Four different radial sections (superotemporal, superonasal, inferonasal, and inferotemporal) were studied to establish RNFL thickness OCT profiles. Additionally, two radial scans orientated at 45 and 135 degrees crossing the optic disc centre were performed in six of 20 eyes, and RNFL thickness was measured at disc margin. RESULTS: Quadrant location and distance from disc margin interaction in RNFL thickness was statistically significant (P<0.001). The RNFL thickness decreased (P<0.001) as the distance from the disc margin increased for all sections. The measurements automatically generated by the OCT built-in software were thinner (P<0.001) than histologic ones close to the disc margin. CONCLUSIONS: Four normal OCT RNFL profiles were established and compared with histological data obtained from the same area. RNFL measurements assessed by OCT 3 were significantly thinner close to the optic disc margin.

Adenoviral-mediated gene transfer to the filtering bleb in rabbits.

PURPOSE: To determine if genes can be transferred to fibroblasts in the filtering bleb using adenoviral vectors. MATERIALS AND METHODS: Twelve New Zealand albino rabbits underwent bilateral full-thickness sclerostomies. On postoperative day 1 an adenoviral vector carrying a reporter gene (lacZ) was injected into the right-eye bleb and saline was injected into the left eye bleb of each rabbit. Three rabbits were euthanized on each of the after days (days 3, 7, 14, and 21). The eyes were enucleated and tissue samples from the filtering bleb were processed for beta-galactosidase activity (a marker for lacZ gene expression) and expression of vimentin (a fibroblast marker). The median number of cells per high-power field with both beta-galactosidase activity and vimentin expression on days 3, 7, 14, and 21 in the right and left eyes were counted to determine adenoviral-mediated gene expression in fibroblasts. RESULTS: In the adenoviral vector-treated eyes, the median number of cells per high-power field with both beta-galactosidase and vimentin expression on days 3, 7, 14, and 21 was 83,100, 1, and 0, respectively. No beta-galactosidase activity was noted in the saline-treated eyes. CONCLUSIONS: Adenoviral vectors can transfer genes to fibroblasts in filtering blebs after glaucoma surgery. The peak efficiency of gene transfer to fibroblasts occurred 7 days after glaucoma surgery. These studies show a potential for genetic manipulation of fibroblast activity in filtering blebs after glaucoma surgery.

Retinal nerve fiber layer thickness in normal human eyes.

PURPOSE: To measure the histologic thickness of the retinal nerve fiber layer (RNFL) in normal human eyes. METHODS: Human eyes were obtained at autopsy within 6 hours postmortem. The retina was dissected into four quadrants and serially sectioned in historesin. The RNFL thickness was measured histologically in all four quadrants at the disc margin and at regular intervals from the disc margin. Measurements of the RNFL thickness also were obtained at the fovea and in the retinal periphery. RESULTS: Ten eyes of ten white individuals were studied. Age (mean +/- standard deviation) was 53.1 +/- 19.6 years (range, 18-76 years). For the eyes studied, the disc area (mean +/- standard deviation) and cup:disc ratio (mean +/- standard deviation) were 1.92 +/- 0.1 mm2 and 0.3 +/- 0.08, respectively. Mean superior, inferior, nasal, and temporal RNFL thickness at the disc margin was 405, 376, 372, and 316 microns, respectively. In all four quadrants, the RNFL thickness decreased with increasing distance from the disc margin. The average superior and inferior RNFL thickness was inversely related to age (P = 0.033, P = 0.097, respectively). The average RNFL thickness was not related to disc area. The average RNFL thickness just superior, inferior, nasal, and temporal to the foveola was 27, 34, 26, and 12 microns, respectively. The average RNFL thickness just posterior to the ora serrata in the superior, inferior, nasal, and temporal retinal periphery was 8 to 11 microns. CONCLUSION: The peripapillary RNFL thickness in humans is thicker than that seen in nonhuman primates. The thinnest peripapillary RNFL is in the region of the papillomacular bundle. These data can be used to determine the accuracy of NFL analyzers in obtaining in vivo RNFL thickness measurements.