Only subtle protein conformational adaptations are required for ligand binding to thyroid hormone receptors: Simulations using a novel multipoint steered molecular dynamics approach

Thyroid hormone receptors (TR) are hormone-dependent transcription regulators that play a major role in human health, development, and metabolic functions. The thyroid hormone resistance syndrome, diabetes, obesity, and some types of cancer are just a few examples of important diseases that are related to TR malfunctioning, particularly impaired hormone binding. Ligand binding to and dissociation from the receptor ultimately control gene transcription and, thus, detailed knowledge of binding and release mechanisms are fundamental for the comprehension of the receptor`s biological function and development of pharmaceuticals. In this work, we present the first computational study of ligand entry into the ligand binding domain (LBD) of a nuclear receptor. We report molecular dynamics simulations of ligand binding to TRs using a generalization of the steered molecular dynamics technique designed to perform single-molecule pulling simulations along arbitrarily nonlinear driving pathways. We show that only gentle protein movements and conformational adaptations are required for ligand entry into the LBDs and that the magnitude of the forces applied to assist ligand binding are of the order of the forces involved in ligand dissociation. Our simulations suggest an alternative view for the mechanisms ligand binding and dissociation of ligands from nuclear receptors in which ligands can simply diffuse through the protein surface to reach proper positioning within the binding pocket. The proposed picture indicates that the large-amplitude protein motions suggested by the apo- and holo-RXR alpha crystallographic structures are not required, reconciling conformational changes of LBDs required for ligand entry with other nuclear receptors apo-structures that resemble the ligand-bound LBDs.

Ligand dissociation from estrogen receptor is mediated by receptor dimerization: Evidence from molecular dynamics simulations

Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17 beta-estradiol (E-2) and the selective ER modulator raloxifene (RAL)] from the human ER alpha ligand-binding domain in monomeric and dimeric forms. E-2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II`). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I`). Remarkably, ER dimerization strongly suppresses Paths II and II` for E-2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.

The effects of anticalcification treatments and hydration on the molecular dynamics of bovine pericardium collagen as revealed by (13)C solid-state NMR

This article describes a solid-state NMR (SSNMR) investigation of the influence of hydration and chemical cross-linking on the molecular dynamics of the constituents of the bovine pericardium (BP) tissues and its relation to the mechanical properties of the tissue. Samples of natural phenetylamine-diepoxide (DE)- and glutaraldehyde (GL)-fixed BP were investigated by (13)C cross-polarization SSNMR to probe the dynamics of the collagen, and the results were correlated to the mechanical properties of the tissues, probed by dynamical mechanical analysis. For samples of natural BP, the NMR results show that the higher the hydration level the more pronounced the molecular dynamics of the collagen backbone and sidechains, decreasing the tissue`s elastic modulus. In contrast, in DE- and GL-treated samples, the collagen molecules are more rigid, and the hydration seems to be less effective in increasing the collagen molecular dynamics and reducing the mechanical strength of the samples. This is mostly attributed to the presence of cross-links between the collagen plates, which renders the collagen mobility less dependent on the water absorption in chemically treated samples. Copyright (C) 2010 John Wiley & Sons, Ltd.

DAILY VARIATION OF CONSTITUTIVELY ACTIVATED NUCLEAR FACTOR KAPPA B (NFKB) IN RAT PINEAL GLAND

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h: 12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information. (Author correspondence: rpmarkus@usp.br)

Amyloid beta-peptide activates nuclear factor-kappa B through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells

Amyloid P-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappa B (NF-kappa B), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappa B activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta 1-40 (1 or 2 mu M) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappa B (1 mu M, 12 hr); both p50/p65 and p50/p50 NF-kappa B dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. AP at 1 mu M increased the expression of inhibitory protein I kappa B, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RTPCR assays. Collectively, these findings suggest that AP activates NF-kappa B by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta. (c) 2007 Wiley-Liss, Inc.

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved

Glucocorticoids Exacerbate Lipopolysaccharide-Induced Signaling in the Frontal Cortex and Hippocampus in a Dose-Dependent Manner

Although the anti-inflammatory actions of glucocorticoids (GCs) are well established, evidence has accumulated showing that proinflammatory GC effects can occur in the brain, in a poorly understood manner. Using electrophoretic mobility shift assay, real-time PCR, and immunoblotting, we investigated the ability of varying concentrations of corticosterone (CORT, the GC of rats) to modulate lipopolysaccharide (LPS)-induced activation of NF-kappa B (nuclear factor kappa B), expression of anti- and proinflammatory factors and of the MAP (mitogen-activated protein) kinase family [ERK (extracellular signal-regulated kinase), p38, and JNK/ SAPK (c-Jun N-terminal protein kinase/ stress-activated protein kinase)], and AKT. In the frontal cortex, elevated CORT levels were proinflammatory, exacerbating LPS effects on NF-kappa B, MAP kinases, and proinflammatory gene expression. Milder proinflammatory GCs effects occurred in the hippocampus. In the absence of LPS, elevated CORT levels increased basal activation of ERK1/ 2, p38, SAPK/ JNK, and AKT in both regions. These findings suggest that GCs do not uniformly suppress neuroinflammation and can even enhance it at multiple levels in the pathway linking LPS exposure to inflammation.

Population dynamics of genetically diverse Plasmodium falciparum lineages: community-based prospective study in rural Amazonia

Temporal changes in the prevalence of antigenic variants in Plasmodium falciparum populations have been interpreted as evidence of immune-mediated frequency-dependent selection, but evolutively neutral processes may generate similar patterns of serotype replacement. Over 4 years, we investigated the population dynamics of P. falciparum polymorphisms the community level by using 11 putatively neutral microsatellite markers. Plasmodium falciparum Populations were less diverse than sympatric P. vivax isolates, with less multiple-clone infections, lower number of alleles per locus and lower Virtual heterozygosity, but both species showed significant multilocus linkage disequilibrium. Evolutively neutral P. falciparum polymorphisms showed a high turnover rate, with few lineages persisting for several months in the population. Similar results had previously been obtained, in the same community, for sympatric P. vivax isolates. In contrast, the prevalence of the 2 dimorphic types of a major antigen, MSP-2, remained remarkably stable throughout the Study period. We Suggest that the relatively fast turnover of parasite lineages represents the typical population dynamics of neutral polymorphisms in small populations, with clear implications for the detection of frequency-dependent selection of polymorphisms.

Dynamics of a class of ODEs more general than almost periodic

A temporally global solution, if it exists, of a nonautonomous ordinary differential equation need not be periodic, almost periodic or almost automorphic when the forcing term is periodic, almost periodic or almost automorphic, respectively. An alternative class of functions extending periodic and almost periodic functions which has the property that a bounded temporally global solution solution of a nonautonomous ordinary differential equation belongs to this class when the forcing term does is introduced here. Specifically, the class of functions consists of uniformly continuous functions, defined on the real line and taking values in a Banach space, which have pre-compact ranges. Besides periodic and almost periodic functions, this class also includes many nonrecurrent functions. Assuming a hyperbolic structure for the unperturbed linear equation and certain properties for the linear and nonlinear parts, the existence of a special bounded entire solution, as well the existence of stable and unstable manifolds of this solution are established. Moreover, it is shown that this solution and these manifolds inherit the temporal behaviour of the vector field equation. In the stable case it is shown that this special solution is the pullback attractor of the system. A class of infinite dimensional examples involving a linear operator consisting of a time independent part which generates a C(0)-semigroup plus a small time dependent part is presented and applied to systems of coupled heat and beam equations. (C) 2010 Elsevier Ltd. All rights reserved.

Looking for intrinsic charm in the forward region at BNL RHIC and CERN LHC

The complete understanding of the basic constituents of hadrons and the hadronic dynamics at high energies are two of the main challenges for the theory of strong interactions. In particular, the existence of intrinsic heavy quark components in the hadron wave function must be confirmed (or disproved). In this paper we propose a new mechanism for the production of D-mesons at forward rapidities based on the Color Glass Condensate (CGC) formalism and demonstrate that the resulting transverse momentum spectra are strongly dependent on the behavior of the charm distribution at large Bjorken x. Our results show clearly that the hypothesis of intrinsic charm can be tested in pp and p(d)A collisions at RHIC and LHC. (C) 2010 Elsevier B.V. All rights reserved.

Sound waves and solitons in hot and dense nuclear matter

Assuming that nuclear matter can be treated as a perfect fluid, we study the propagation of perturbations in the baryon density. The equation of state is derived from a relativistic mean field model, which is a variant of the non-linear Walecka model. The expansion of the Euler and continuity equations of relativistic hydrodynamics around equilibrium configurations leads to differential equations for the density perturbation. We solve them numerically for linear and spherical perturbations and follow the propagation of the initial pulses. For linear perturbations we find single soliton solutions and solutions with one or more solitons followed by ""radiation"". Depending on the equation of state a strong damping may occur. We consider also the evolution of perturbations in a medium without dispersive effects. In this case we observe the formation and breaking of shock waves. We study all these equations also for matter at finite temperature. Our results may be relevant for the analysis of RHIC data. They suggest that the shock waves formed in the quark gluon plasma phase may survive and propagate in the hadronic phase. (C) 2009 Elseiver. B.V. All rights reserved.

Constraining the nuclear gluon distribution in eA processes at RHIC

A systematic determination of the gluon distribution is of fundamental interest in understanding the parton structure Of nuclei and the QCD dynamics. Currently, the behavior of this distribution at small x (high energy) is completely undefined. In this Letter we analyze the possibility of constraining the nuclear effects present in Xg(A) using the inclusive observables which would be measured in the future electron-nucleus collider at RHIC. We demonstrate that the Study of nuclear longitudinal and charm structure functions allows to estimate the magnitude of shadowing and antishadowing effects in the nuclear gluon distribution. (C) 2008 Elsevier B.V. All rights reserved.

Projectile breakup dynamics for (6)Li+(59)Co: Kinematical analysis of alpha-d coincidences

A study of the kinematics of the alpha-d coincidences in the (6)Li + (59)Co system at a bombarding energy of E(lab) = 29.6MeV is presented. With exclusive measurements performed over different angular intervals it is possible to identify the respective contributions of the sequential and direct projectile breakup components. The angular distributions of both breakup components are fairly well described by the Continuum-Discretized Coupled-Channels framework (CDCC). Furthermore, a careful analysis of these processes using a semiclassical approach provides information on both their lifetime and their distance of occurrence with respect to the target. Breakup to the low-lying (near-threshold) continuum is delayed, and happens at large internuclear distances. This suggests that the influence of the projectile breakup on the complete fusion process can be related essentially to the direct breakup to the (6)Li high-lying continuum spectrum.

Velocity-dependent KAM islands

We revisit the non-dissipative time-dependent annular billiard and we consider the chaotic dynamics in two planes of conjugate variables in order to describe the behavior of the growth, or saturation, of the mean velocity of an ensemble of particles. We observed that the changes in the 4-d phase space occur without changing any parameter. They occur depending on where the initial conditions start. The emerging KAM islands interfere in the behavior of the particle dynamics especially in the Fermi acceleration mechanism. We show that Fermi acceleration can be suppressed, without dissipation, even considering the non-dissipative energy context. (C) 2011 Published by Elsevier Ltd.

Magnetic resonance and conductivity study of gelatin-based proton conductor polymer electrolytes

This work report results from proton nuclear magnetic resonance (NMR), continuous-wave (CW-EPR) and pulsed electron paramagnetic resonance (P-EPR) and complex impedance spectroscopy of gelatin-based polymer gel electrolytes containing acetic acid. cross-linked with formaldehyde and plasticized with glycerol. Ionic conductivity of 2 x 10(-5) S/cm was obtained at room temperature for samples prepared with 33 wt% of acetic acid. Proton ((1)H) line shapes and spin-lattice relaxation times were measured as a function of temperature. The NMR results show that the proton mobility is dependent on acetic acid content in the plasticized polymer gel electrolytes. The CW-EPR spectra, which were carried out in samples doped with copper perchlorate, indicate the presence of the paramagnetic Cu(2+) ions in axially distorted sites. The P-EPR technique, known as electron spin echo envelope modulation (ESEEM), was employed to show the involvement of both, hydrogen and nitrogen atoms, in the copper complexation of the gel electrolyte. (C) 2009 Elsevier Ltd. All rights reserved.