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Agonist and antagonist binding to the nuclear vitamin D receptor: dynamics, mutation effects and functional implications
- Sepideh Yaghmaei1,
- Christopher Roberts1,
- Rizi Ai1,
- Mathew T Mizwicki2Email author and
- Chia-en A Chang1Email author
© Yaghmaei et al.; licensee Springer. 2013
Received: 20 June 2012
Accepted: 28 October 2012
Published: 12 February 2013
The thermodynamically favored complex between the nuclear vitamin D receptor (VDR) and 1α,25(OH)2-vitamin D3 (1,25D3) triggers a shift in equilibrium to favor VDR binding to DNA, heterodimerization with the nuclear retinoid x receptor (RXR) and subsequent regulation of gene transcription. The key amino acids and structural requirements governing VDR binding to nuclear coactivators (NCoA) are well defined. Yet very little is understood about the internal changes in amino acid flexibility underpinning the control of ligand affinity, helix 12 conformation and function. Herein, we use molecular dynamics (MD) to study how the backbone and side-chain flexibility of the VDR differs when a) complexed to 1α,25(OH)2-vitamin D3 (1,25D3, agonist) and (23S),25-dehydro-1α(OH)-vitamin D3-26,23-lactone (MK, antagonist); b) residues that form hydrogen bonds with the C25-OH (H305 and H397) of 1,25D3 are mutated to phenylalanine; c) helix 12 conformation is changed and ligand is removed; and d) x-ray water near the C1- and C3-OH groups of 1,25D3 are present or replaced with explicit solvent.
We performed molecular dynamic simulations on the apo- and holo-VDRs and used T-Analyst to monitor the changes in the backbone and side-chain flexibility of residues that form regions of the VDR ligand binding pocket (LBP), NCoA surface and control helix 12 conformation.
The VDR-1,25D3 and VDR-MK MD simulations demonstrate that 1,25D3 and MK induce highly similar changes in backbone and side-chain flexibility in residues that form the LBP. MK however did increase the backbone and side-chain flexibility of L404 and R274 respectively. MK also induced expansion of the VDR charge clamp (i.e. NCoA surface) and weakened the intramolecular interaction between H305---V418 (helix 12) and TYR401 (helix 11). In VDR_FF, MK induced a generally more rigid LBP and stronger interaction between F397 and F422 than 1,25D3, and reduced the flexibility of the R274 side-chain. Lastly the VDR MD simulations indicate that R274 can sample multiple conformations in the presence of ligand. When the R274 is extended, the β-OH group of 1,25D3 lies proximal to the backbone carbonyl oxygen of R274 and the side-chain forms H-bonds with hinge domain residues. This differs from the x-ray, kinked geometry, where the side-chain forms an H-bond with the 1α-OH group. Furthermore, 1,25D3, but not MK was observed to stabilize the x-ray geometry of R274 during the > 30 ns MD runs.
The MD methodology applied herein provides an in silico foundation to be expanded upon to better understand the intrinsic flexibility of the VDR and better understand key side-chain and backbone movements involved in the bimolecular interaction between the VDR and its' ligands.
KeywordsNuclear vitamin D receptor Vitamin D3 1α,25(OH)2-vitamin D3 Antagonist Molecular dynamics Helix 12 Conformational ensemble
The nuclear vitamin D receptor (VDR) is a member of the nuclear receptor (NR) transcription factor super family (Nuclear receptor nomenclature committee1999). Based on sequence and functional homology, NR's are partitioned into five domains (A-F), of which domain C (i.e. the DNA binding domain) contains the greatest degree of sequence homology. Domain E, the ligand binding domain (LBD), varies cross-family in sequence homology; however, the transcriptionally active conformation of NR LBDs is highly conserved (Wurtz et al1996) and is highlighted by a closed position of the C-terminal helix of domain E (helix 12), capping the ligand binding pocket (LBP) (Renaud and Moras2000). Some NRs have been observed to adopt the closed, active conformation in the absence of ligand (apoNR, e.g. Nurr1) (Wang et al2003). Alternatively other NRs more absolutely require an endogenous ligand to increase the stability of the active conformer.
NR ligands that sterically block helix 12 closure function as traditional NR antagonists by disrupting the formation of the NCoA surface/charge clamp. Three general classes of VDR antagonists have been created to date. The first class, based on extending the aliphatic side-chain of 1,25D3, function to sterically disrupt helix 12 closure (Carlberg2004); the second, based on an adamantyl side-chain chemistry, more subtly disrupt helix 12 closure (Igarashi et al2007; Nakabayashi et al2008); and the third class, which was first to be discovered, was generated by removing the chiral end of one of the terminal side-chain metabolites of 1,25D3, to generate (23S)-1α(OH)-vitamin D3-26,23-lactone (MK)] (Miura et al1999). Similar to the adamantyl-based antagonists, MK does not sterically displace helix 12 from the active position (Mizwicki et al2009a) and does mimic 1,25D3 in its' ability to stabilize other regions of the VDR LBD (Kakuda et al2010; Mizwicki et al2012). MK is a unique NR antagonist because it can be converted into a VDR superagonist (i.e. transactivation potential 10–100 fold greater than 1,25D3) by a single (H305 to F305) or double H305F/H397F (VDR_FF) point mutant (Mizwicki et al2009a; Kakuda et al2010).
The other extreme conformation of helix 12, with respect to the closed conformation, is the opened conformation. This conformation has been observed for a mutated and unliganded, apoRXR (Vivat et al1997) and 17β-estradiol bound ERα (Tanenbaum et al1998). To date, no structure has been solved that demonstrates the VDR can be stabilized in an 'opened' conformation, yet it is the conformation assumed for all ligand-activated apoNRs by the mouse-trap, induced-fit activation mechanism (Renaud and Moras2000).
Solution state limited proteolytic digest of the VDR indicates that at least three distinct helix 12 conformations exist in solution (Peleg et al1995). In specific two trypsin sites in helix 11 (R402) and helix 12 (K413), have shown differential cleavage rates when the VDR H305 and H397 residues were mutated to phenylalanine and/or following incubation with 1,25D3 or MK (Mizwicki et al2007). More over, hydrogen-deuterium exchange coupled mass spectrometry results clearly demonstrate that different VDR agonist ligands differentially alter the flexibility of helix 12 backbone atoms (Zhang et al2010). Thus it is clear that other techniques must be utilized besides crystallography to understand the molecular dynamics associated with the intrinsic flexibility of the VDR and its' bimolecular interaction with ligand and how those interactions translate into the many cellular functions of active vitamin D3 metabolites and the VDR.
Molecular Dynamics (MD) computational simulations have been employed to observe how the population of distinct conformational states fluctuate when a protein is unliganded or complexed with agonist or antagonist ligand (Gallicchio and Levy2011; Ai et al2009). MD has also been used to identify novel ligand accessible binding sites in HIV integrase (Schames et al2004). Furthermore, explicit solvent MD has been employed to demonstrate that water can play a role in allosterically regulating conformational switches (Prakash et al2012; Gorfe and Caflisch2005) and dictate 'slaved' protein motions (Fenimore et al2002). Application of MD in the VDR field has been used to a) provide a structural basis for the species specific antagonistic nature of MK (Perakyla et al2004); b) determine how ligand dissociates from the VDR (Perakyla2009); and c) design novel VDR agonists (Shen et al2012). In this work we used explicit solvent MD to assess how the backbone and side-chain flexibility of the VDR is influenced by changing helix 12 conformation, VDR primary sequence, ligand chemistry and absence of ligand.
Structures used for MD simulations
The initial coordinates of human Vitamin D receptor (VDR), VDR_H305F and VDR_FF (H305F/H397F double mutation) were obtained from the VDR-1α,25(OH)2D3 (1,25D3) x-ray crystal complex (PDB: 1DB1), VDR_H305F-MK complex (PDB: 3A2I) and the VDR_FF-MK complex (PDB: 3A2J) respectively. The three missing residues for the loop between helices 8 and 9 were added to the X-ray structure of VDR-LBD-1,25D3 using the loop refinement module of Discovery Studio 2.0 (Accelyrs Inc., San Diego). The initial structure of the VDR_H305F-1,25D3 and VDR_FF-1,25D3 complexes were constructed by aligning their x-ray coordinate to the minimized structure of VDR-1,25D3 complex and replacing MK with 1,25D3. The initial coordinates of VDR_H397F-1,25D3 were obtained by mutating the HIS397 residue, of the energy minimized VDR-1,25D3 complex, to a phenylalanine using PyMOL (Molecular Graphics System, Version 126.96.36.199 Schrödinger, LLC). The initial coordinates of the VDR-MK complex were generated by mutating the VDR_H305F-MK F305 residue to H305.
The initial structure used to simulate the MD of the helix 12 closed, apoVDR was generated by removing 1,25D3 from the X-ray coordinates of VDR-1,25D3 complex (pdb: 1DB1). The initial structure of helix 12 closed apoVDR (H305F/H397F) was constructed by mutating both H305 and H397 of pdb: 1DB1 (Additional file1:Table S1).
The initial structure of the helix 12 opened, apoVDR molecule was generated by homology modeling, in which the last twelve residues of the minimized VDR (aa414-427) were aligned to the last twelve residues of the apoRXR (PDB: 3OZJ). It is noted that an attempt to align aa401-427 failed using this method; however, the apoVDR model generated does allow for the assessment of the effect helix 12 being positioned away from the core of the VDR ligand binding domain has on the MD simulations. All models addressed in this study are listed in Additional file1: Table S1.
Molecular models and computational details
We performed molecular dynamics (MD) simulations using the Amber11 and NAMD2.6 simulation packages with the ff99sb amber force field for the proteins (Okur et al2003; Hornak et al2006; Phillips et al2005; Cornell et al1995). The parameters for the ligands were generated with the Antechamber module of the AMBER11 package using the general amber force field (gaff) (Wang et al2006). The root mean square deviation (RMSD) for all models is shown in Additional file2: Figure S1. Before solvating the system, the hydrogen atoms, side chains and the protein were each minimized for 1000 steps using a combination of steepest descent and conjugate gradient. Sodium counter ions were added to the minimized protein and the system was solvated in a cubic shell using the TIP3PBOX water model. The size of the water box for VDR protein was 82x79x89 Å3. The water box was minimized for 1000 steps while the protein was held fixed. Subsequently, the water box was equilibrated at 200K for 10 ps. The complex was gradually warmed up from 200K for 2500 steps followed by 5 ps of simulation at 250K.
The molecular dynamics simulations for the following models were performed at 300K
32ns for VDR-LBD-1,25D3 complex, 17ns for the three mutated analogues, 28 ns for the homology modeled apoVDR, 25 ns for apo VDR helix 12 closed, 15ns for apoVDR_FF helix 12 open, 21 ns for apoVDR_FF helix 12 closed, and 23ns for the VDR-MK complex (Additional file1: Table S1). The 32 ns molecular dynamic stimulation for 1,25D3 bound to VDR was performed with two different starting models: (a) the X-ray conformation of the protein with crystal water molecules 502–511 included and (b) the X-ray conformation of the protein without the crystal water molecules. The phi entropy and the side-chain entropy values were computed using the Gibbs equation and the T-Analyst program (Ai et al2010; Chang et al2008).
The pKa of the histidine residues in the protein were calculated by Multi-Conformation Continuum Electrostatics (MCCE) (Georgescu et al2002; Song et al2009). The results predict that both histidine residues near the binding site, HIS305 and HIS397, are only protonated at a pH below 6. The only histidine side-chains that are protonated at physiological pH levels are HIS140 of helix 1 and HIS326 and HIS330 of helix 7. All three of these histidine groups are exposed to solvent and reside close to an acidic side-chain. Since these histidine residues are far from the binding site their protonation state was not altered in our MD simulations.
The energy of interaction between the ligand and the protein was calculated by the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method (Tsui and Case2000; Bashford and Case2000; Hou et al2011) using an in-house script with the GBSA module of the AMBER10 package.
Effect of VDR mutations and MK side-chain chemistry on VDR charge clamp distance
Transactivation by a holo-NR requires the formation of a charge clamp that grips the LXXLL helix of nuclear coactivators. This interaction occurs on the surface of the NR ligand binding domain (LBD) (Glass and Rosenfeld2000). In CV1 cells co-transfected with VDR and VDRE-SEAP reporter constructs, 1,25D3 was measured to have a similar EC50 in the H305F and H305F/H397F (VDR_FF)-1,25D3 complexes when compared to hVDRwt-1,25D3. In VDR_H397F, 1,25D3 was a 10-fold weaker agonist ligand. Conversely, the hVDR-1,25D3 antagonist, MK, functions as a genomic superagonist in VDR_H305F and VDR_FF transfected cells (Mizwicki et al2009a).
It is noted that measuring the charge clamp distance does not directly correlate with NCoA affinity; however, it does provide a read-out for how the bimolecular interaction between the VDR and its' ligand can alter the MD of surface regions linked to macromolecular complex formation (Vaisanen et al2002). It is also noted that recent mass spectrometry coupled hydrogen-deuterium exchange results showed that when VDR binds DNA, it alters the backbone flexibility of the VDR nuclear coactivator surface in the intact VDR-RXR complex (Zhang et al2011).
Changes in helix 11 and 12 intramolecular interactions in the holoVDR models
The closed helix 12 conformation is in part stabilized by a T-shaped pi-pi interaction made between H397 and F422 and a displaced parallel pi-pi interaction between H397 and Y401 (Additional file4: Figure S2). These intramolecular interactions aid in stabilizing the closed conformation of helix 12 and are enhanced by the terminal end of the 1,25D3 side-chain forming an H-bond with H397. When the distance between H397 and Y401 in all the models was monitored, only the VDR-MK model showed significant fluctuations (Figure 2B, bottom panel). More dynamic fluctuations between V418 and Y401, and H305 and Y401 in the VDR-MK model were also observed (Figure 2B, middle and top panels). Lastly, the distance between H397 and V418 was on average 1.1 Å larger in the VDR-MK model when compared to the other models. This is important because H397 is a helix 11 residue and V418 a helix 12 residue; therefore, increase in this distance indicates that helix 12 may be moving away from helix 11. Coupled the distance calculations confirm that the residues that exclusively contact the ligands side-chain atoms in the VDR LBP (e.g. C-terminus of helix 11 and helix 12) become more flexible when complexed to MK (Perakyla et al[2004]).
In general the fluctuations in the VDR_FF models were lower in all cases when compared to the VDRwt models (Figure 2B). This is due to the enhanced pi-pi intramolecular interaction energy between F305 and Y401 and F397 and F422. F305 is towards the back of the VDR LBP (Figure 1), near helix 7, H397 and Y401 are two residues towards the C-terminus of helix 11 and F422 is part of helix 12. Thus optimizing pi-pi interactions between these residues provides an allosteric link between the regions of the VDR that bind RXR (the back), ligand (the middle) and helix 12, the front of the VDR (Figure 1).
Overall backbone and side-chain entropy changes in the holoVDR models
The dihedral entropy calculation is one way to directly measure the flexibility of protein backbone and side-chains (Zhang and Liu2006). The backbone entropy over the 23 ns VDR-MK MD increased for R296, L404 and G423, while L309 became more rigid when compared to VDR-1,25D3 (Figure 2C). Of these residues L309 and L404 have been shown by a number of laboratories to provide important hydrophobic, vdW contacts required for VDR-1,25D3 activation of gene transcription (Mizwicki et al2009a; Choi et al2003; Yamamoto et al2006).
When both H305 and H397 of VDR-MK were mutated to phenylalanine (VDR_FF), the backbone atoms of helix 7 (V297 and S298), helix 11 (S398) and helix 12 (C410) became more rigid when compared to VDRwt-MK (Figure 2D). Alternatively, the backbone atoms of helix 1 (K141), helix 3n (L219 and E220), the β-sheet (K294), helix 5 (V270), helix 7 (L309 and E311) and helix 11 (R402) became more flexible (Figure 2D). The increased flexibility of R402 was not expected given, previous proteolytic digest experiments indicated that trypsin cleavage at R402 was significantly attenuated by the VDR_FF double mutation (Mizwicki et al2007). The backbone motions revealed by the VDR_FF-1,25D3 MD simulation indicated that the β-sheet region, C-terminal end of helix 11 (Q400), helix 12 (L419 and F422) and F305 became more flexible. Alternatively, helix 7 (L309), helix 10 (E395) and helix 12 (C410) residues became more rigid (Figure 2E). Thus a common feature of the FF mutation was to increase the rigidity of C410 and residues juxtaposed to H397.
Comparison of the 1,25D3 and MK VDRwt LBP side-chain movements demonstrated that only the side-chain of R274 (helix 5 C-terminus) was significantly more flexible when bound to MK (Figure 2C). The VDR_FF mutation caused the side-chain of F422 (helix 12) to become more flexible and L230 (helix 3) and R274 (helix 5) to become more rigid when complexed to MK (Figure 2D). Thus the major difference in the VDR side-chain entropy during the VDRwt-MK and VDR_FF-MK simulations was observed in the A-ring region of the VDR LBP, not the helix 11 and helix 12 regions of the LBP. Lastly, the VDR_FF-1,25D3 side-chain entropy results demonstrated that the FF mutation caused I271 and V418 to become more flexible and L230 to become more rigid (Figure 2E). All of the residues highlighted above were shown by VDR ALA scanning analysis to be crucial to VDR agonist function (Yamamoto et al2006).
Collectively, the MD results for VDR-MK suggest that MK functions as an effective antagonist of hVDRwt because it does not stabilize the R274 side-chain and MK allows the C-terminal backbone atoms of helix 11 and helix 12 become for flexible. Thus the helix 11 and helix 12 MD results are consistent with previous flexible docking and limited proteolytic digest studies that showed increased disorder in this region is produced when MK is bound to hVDRwt (Mizwicki et al2009a). Furthermore, recent VDR-MK hydrogen-deuterium exchange coupled mass spectroscopy results demonstrated that the amide backbone exchange for peptides mapping to helix 3, residues 286–316 and 384–419 showed significantly enhanced exchange in the VDR-MK complex (Mizwicki et al2012).
Comparison of 1,25D3 and MK MD and Interactions with VDR LBP Residues
In an attempt to further our understanding for how the VDR_FF mutation converts MK, but not 1,25D3 into a superagonist, the average nearest neighbor hydrophobic and hydrogen bonding distances between residues composing the LBP of four models were compared (Figure 3C and Additional file3: Table 2b and c). In the VDR_FF-MK complex only Y143 and V234 were on average 2.64 Å and 1.68 Å further away from MK when compared to VDRwt-1,25D3. Conversely, over the 23 ns VDR_FF-1,25D3 MD run, the Y143, V418, 305 and 397 residues were on average 1.11 Å, 2.29 Å, 1.48 Å and 1.41 Å further away from 1,25D3 than in VDRwt. Furthermore, comparison of the VDR_FF-1,25D3 and MK simulations showed that MK remained on average 1.04 Å closer to F397. This interaction may contribute to the superagonist effect of MK, because it is known that the intramolecular interaction between the H397 side-chain and F422 (helix 12) is important VDR activation of gene transcription (Gonzalez et al2003).
Interaction energy between 1,25D3 or MK and VDRwt, VDR_H305F or VDR_FF
Interaction energies from MMGB/SA calculations results for the holo VDR models
Binding Affinity**(nM)
Effect of water on ligand binding pocket MD
X-ray results all show a 'kinked' conformation of R274 that is stabilized by formation of an H-bonds with the 1α-OH group and crystallographic water (Hourai et al2006), suggestive that water plays a key role in aiding in the stability of the local geometry of the active R274 side-chain conformation. The VDR MD simulations outlined above were all performed in explicit solvent and were shown to correlate quite well with 1,25D3 and MK functional results; therefore, we next used the MD technique to further investigate the relative importance of the x-ray water molecules interacting with R274. This was accomplished by running 32 ns MD simulations on two different VDR-1,25D3 starting models: (a) the X-ray conformation of the protein with crystal water molecules 502–511 included and (b) the X-ray conformation of the protein without the crystal water molecules (i.e. the technique used exclusively to this point).
The effect of helix 12 conformation on apoVDR molecular dynamics
As expected, when helix 12 was homology modeled in the opened conformation (see methods), the backbone entropy calculations showed many more changes in flexibility over the run. For example, the backbone atoms of L230, R274, F279 and the C-terminal end of helix 11 (L399, Y401 and R402) became significantly more flexible (Figure 5B). According to the side-chain entropy calculations, opening of helix 12 also increased the flexibility of L230, H397 and F422 and reduced the flexibility of R274 (Figure 5B). These results suggest that removal of the LBP lid (i.e. helix 12) increases the backbone flexibility of helicies 3, 5 and 11, but increases the rigidity of the R274 side-chain. The latter result can be potentially explained by the evidence presented herein that in the absence of ligand, R274 would bias the local conformation where the R274 side-chain interacts with H139 and T142.
Mutating H305 and H397 (apoVDR_FF) caused the helix 11 and 12 backbone residues (i.e. L399, L414, M412, K413 and E420) to become more rigid when helix 12 was opened (compare Figure 5C and D). Alternatively the backbone atoms of F406 and C410 became more rigid, and L399, Q400 and Q407 became more flexible when helix 12 was closed (Figure 5C). The side-chain entropy calculations indicated that L230 and F422 side-chains became more flexible and H397 more rigid, when helix 12 was closed (Figure 5C). Whereas, the side-chain flexibility of L404 and L414 and made V418 and F422 become more rigid (Figure 5D). Thus the results suggest that the VDR_FF mutant had a greater stabilizing effect on the opened helix 12 conformation rather than the closed conformation.
Much is known about the thermodynamically favored conformation of the nuclear vitamin D receptor (VDR) and its' role as a nuclear transcription factor; however, little is understood about its' intrinsic flexibility (Mizwicki et al2007) and how VDR molecular ensembles may influence a given ligands affinity and function. Herein, molecular dynamics simulations were used to assess a) the molecular events dictating whether MK functions as a VDR antagonist or superagonist ligand; b) whether x-ray water is required to accurately simulate the bimolecular interaction between the VDR and ligand; and c) the effect removal of ligand, the opening helix 12 and mutation of H305 and H397 have on the VDR backbone and side-chain flexibility.
Evidence that the VDR-MK MD results, performed using an explicit solvation model, correlate well with MK structure-function results included a) the side-chain of R274 is the most flexible residue in the LBP of VDR when bound to MK; b) the backbone of K246, one of the charge clamp residues (Figure 1), becomes significantly more flexible and the clamp opens (Figure 2A); and c) the backbone entropy of helix 11 and helix 12 increases (Figure 2C). Consistent with previous flexible docking results (Mizwicki et al2009a), the MD runs also show that the side-chain of MK preferentially moves closer to L227 (helix 3) and L404 (helix 11), and away from helix 12, i.e. L414 and V418 (Figure 3C). The MD results also show that the migration of MK away from helix 12 and towards the C-terminal end of helix 11 is facilitated by the pi-pi interaction made between the MK lactone ring and H305. Migration of the MK lactone side-chain places the exo-cyclic methylene as close as it could possibly get to C403 in the x-ray VDR conformation. This movement of the MK side-chain towards L404 could increase the probability for the formation of a Michael adduct C403 (Mizwicki et al2012; Kakuda et al2010).
The conversion of MK into a VDR superagonist in VDR_FF has been postulated to be triggered by the intermolecular interaction between the F305 and the lactone ring of MK (Mizwicki et al2012; Kakuda et al2010). In support of this hypothesis, the VDR_FF-MK and 1,25D3 MD results show they have similar average charge clamp distances (Figure 2A), intramolecular interactions between helix 11 (H397 and Y401) and helix 12 (V418 and F422) side-chains (Figure 2B) and backbone/side-chain entropy. Importantly, the MD simulations demonstrate that F397 is significantly further away from 1,25D3 when compared to VDRwt, but when bound to MK, F397 and F422 become closer to one another. Overall analysis of the MD frames indicates that movement of F397 (helix 11) away from 1,25D3 is caused by F397 gravitating toward F422 (helix 12). This motion provides an explanation for why VDR_FF-1,25D3 has an increased charge clamp distance and why the electrostatic interaction between 1,25D3 and the VDR_FF complex is significantly reduced, whereas it is not for MK (Table 1).
A novel finding from the MM/GBSA interaction energies was that no real change in total interaction energy is observed for 1,25D3 or MK throughout the various MD runs; however, dramatic changes in the relative contributions of each term to the total interaction energy are observed. For example, MK had a weaker hydrophobic interaction (▵Evdw), stronger electrostatic interaction (▵Eeel) and greater polar solvation free energy (▵Epol) with VDRwt, when compared to 1,25D3 (Table 1). This indicates that the reduced vdW interaction likely drives the reduced affinity of MK for the VDR in a steroid competition assay (Mizwicki et al2009a). Combining this physical characteristic with an increased ▵Epol may be a signature that can be used in the design of future VDR antagonists (Table 1).
While we were unable to homology model the apoRXR-like opened LBD conformation, peeling of helix 12 away from the body of the LBD enhances the side-chain entropy of H305 and fractures the displaced parallel pi-pi interaction between H397 and Y401. In the closed helix 12 VDR MD run, H397 maintains the intramolecular interaction with Y401, perhaps explaining why the H397 side-chain entropy is similar in the helix 12 closed apo and holo models. It is noted that a recent variant of VDR_FF, where H397 was mutated to a Tyr rather than a Phe, showed enhanced sensitivity for vitamin D3, which has no activity in VDRwt (Ousley et al2011).
Opening of helix 12 also causes the residues of helix 11 and 12 to become significantly more flexible and gravitate toward the LBP; however unlike the C-terminal portion of helix 11 in the apoRXR crystal structure (Bourguet et al1995), H11 residues did not enter the region of the LBP normally occupied by the seco-B and A-ring of the ligand. It has been proposed that the ability of the FF mutation to greatly protect the apoVDR against trypsin cleavage by increasing the rigidity of the closed helix 12 conformation by stabilizing residues proximal to the two C-terminal trypsin sites, R402 (helix 11) and K413 (helix 12) (Mizwicki et al2007). However the MD results demonstrate that the backbone entropy of residues proximal to R402 and K413 was reduced when helix 12 was opened, rather than the apo-helix 12 closed conformer (Figure 5C and D). This result suggests that the FF mutations ability to protect against helix 12 libation may be through its' altering the stability of other VDR conformational ensemble members than the helix 12 closed conformer. The fact that both the apo-helix 12 opened and closed VDR and VDR_FF MD runs show oscillation about a an average RMSD (Additional file2: Figure S1) support the theory that the closed helix 12 conformation of VDR is sampled in the absence of 1,25D3 and therefore a conformational ensemble model should be applied in rationalization/translation of VDR structure-function results (Mizwicki and Norman2009b).
Perhaps the most intriguing and novel finding in this study was that the replacement of x-ray water molecules with explicit water did not alter the accuracy in simulating the MD that exist between VDR and 1,25D3/MK.; even though, removal of the x-ray water allows the R274 side-chain to sample multiple conformational states in the presence of ligand. Given the kinked, x-ray geometry of the R274 side-chain is induced by 1,25D3 during the VDR-1,25D3 MD run, it is plausible that shifting the equilibrium of the R274 side-chain to favor the kinked geometry is key to the molecular switch underpinning the activation of VDR transcription. Screening of additional ligands and extending simulation times will allow for future testing of this hypothesis. In closing our MD, dihedral entropy and interaction energy calculations confirm and build upon previous structure-function studies and provide novel findings whose true value will be defined as we generate a greater understanding of the molecular dynamics underpinning VDR ligand recognition and dissociation and the apoVDR ensemble model.
This research was supported in part by start-up funds from the University of California, Riverside and the National Science Foundation (MCB-0919586) through TeraGrid resources provided by the National Center for Supercomputing Application under grant number (TG-MCB080039N).
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Top Definition
The erection of a male arachnid.
Observe the black widow's eraxis
by Filledebatteur4 March 20, 2010
The Urban Dictionary Mug
One side has the word, one side has the definition. Microwave and dishwasher safe. Lotsa space for your liquids.Buy the mug
The Urban Dictionary T-Shirt
Smooth, soft, slim fit American Apparel shirt. Custom printed. 100% fine jersey cotton, except for heather grey (90% cotton).Buy the t-shirt

subset: commoncrawl


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Sort listings
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Baby waker on sale
Baby Walker in good con conditions with music and playing balls
Kolkata in West Bengal (India)
Rs1000view For Babies - Infants published on 05/10 viewed 12x
Blood Pressure Chart Pdf
Although high blood stress (also know as hypertension) is a really serious wellness dilemma, there's fantastic news. Blood stress needs to be standard throughout one's life. As the volume of blood circulated via your blood vessels increases, so does the pressure on your artery walls. Not only does s...
Kranenburg in Kranenburg (India)
Rs203view For Babies - Infants published on 31/10 viewed 274x
Trying to keep Your Auto Insurance Charges Very low And Achievable
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Blood Pressure Range Chart
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Lake Burrendong in Lake Burrendong (India)
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Buying Women's Personalized Jewelry
personalised engraved bracelets Get an engagement ring and allow it to become official about your relationship. Fall on your knee with love on your eyes and provide her nuptial ring for confessing deep love towards your partner love. I assure you she tend to be on the top of the world which enables ...
Summerville in Tamil Nadu (India)
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Publisher's Description+
From GolfLogix: 4 million golfers trust GolfLogix for FREE GPS distances, 3D color course maps and digital scorecards/stats. Its easy, fast, fun and its FREE! #1 in Golf GPS for over 16 years Get the BRAND NEW 2016 GolfLogix App!You can always trust GolfLogix for:FREE GPS distances on any golf course in the worldFREE yardage book color 3D course imagesFREE 4-player scoring, stats and handicapGet GolfLogix, the #1 Golf App. Why not? Its FREEIt gets even better...Book your next GolfNow Hot Deal tee time through the GolfLogix app and get 20% off your entire foursome!Track your stats and get MyGolfDigest after every round showing personalized lessons and analysis of your game from Golf Digests top teachers including Mickelson, Harmon, Leadbetter, Haney and more.Free access to 60+ years of Golf Digests magazine archives, lessons and videos.Upgrade to a Premium member for only $4.99 and get advanced GPS features, an ad-free app experience while golfing plus an extra $20 tee time discount from GolfNow.GPS to the Front, Back and Center of GreensDaily Pin PositionsTarget distance to any point on the Fairway and GreenGPS to all Layups and HazardsPatented Club Tracking showing the location and distance of every club you hitClub Distance Grid shows where each of your clubs should land from where you are standing on the holeCalorie CounterWorks on your Apple WatchThere is so much to the GolfLogix App, youre gonna love it!GolfLogix is the world leader in Golf GPS software since 1999See why millions of golfers trust GolfLogix for their gameDownload GolfLogix, the #1 Golf App right now!Note: Continued use of GPS running in the background can dramatically decrease battery life.Below are descriptions and terms of the OPTIONAL in-app upgrades:1. GolfLogix Premium Membership with advanced GPS features and an ad-free experience on the course can be purchased for $4.99/mo or $29.99/yr.2. MyGolfDigest gives you unlimited lessons after every round and can be purchased for either $0.99/mo or $9.99/yr.Payment for in-app upgrades will be charged to your iTunes Account at confirmation of purchase. Subscriptions automatically renew unless auto-renew is turned off at least 24-hours before the end of the current period. Your iTunes Account will be charged for renewal within 24-hours prior to the end of the current period, at the original subscription amount. Subscriptions may be managed by you and auto-renewal may be turned off by going to your iTunes Account Settings after purchase. No cancellation of the current subscription is allowed during active subscription period. Any unused portion of a free trial period will be forfeited when you purchase a subscription. Privacy Policy and Terms of Use are located at: http://golflogix.com/Terms_of_Use.aspx
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Invaluable cannot guarantee the accuracy of translations through Google Translate and disclaims any responsibility for inaccurate translations.
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Lot 1475: SPECCHIERA
Design Auction
by Aste Boetto SRL
26 October 2016
Genoa, ItalyLive Auction
Past Lot
Looking for the realised and estimated price?
UNO SPECCHIO DA PARETE anni "60.
Legno di teak tinto all"anilina cristallo argentato.
Cm 67 x 54.
Browse more items


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Samir KumarSr. Account Executive at Randev Brothers Auto Pvt. ltd. Ludhiana, India | Accounts / Finance
- Sr. Account Executive at Randev Brothers Auto Pvt. ltd.
- Accounting, Taxation
Samir Kumar's Profile Overview
Current Company
Sr. Account Executive at
Randev Brothers Auto Pvt. ltd.
Department: Accounts / Finance,
Educational Information
ICWA at
Institute of Cost and Works Accountants of India ( ICWAI )
Samir Kumar's Additional information
Web Profile : http://www.peerpower.com/pub/samirkumar2
Samir Kumar's Personal Details
PeerPower.com is a networking site for top professionals enabling them to profit from networking and connect with thought leaders.
Join PeerPower >>
People Directory
Samir Kumar's Connection
Sangita DuggalMGR at Oil & Natural Gas Corporation Ltd (ONGC) Satyabrata HotaDy. Manager(F) at WESCO Distribution Shilpa PeswaniLecturer at St Francis institute of Management and Research(SFIMAR.) B AsokapatraSr. Officer(F&A) at Utility Powertech Limited Sunit AcharyaFinance & Accounts at The Louis Berger Group View All
Build your brand name by publishing a personal Newspaper on recent news events.
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The Daily Dope


text:
User-Agent: Mozilla/5.0 (Windows; U; Windows NT 5.1; en-US; rv:22.214.171.124) Gecko/20091102 Firefox/3.5.5
Build Identifier: Mozilla/5.0 (Windows; U; Windows NT 5.1; en-US; rv:126.96.36.199) Gecko/20091102 Firefox/3.5.5
This may be a duplicate of https://bugzilla.mozilla.org/show_bug.cgi?id=101276 from 2001 which doesn't appear to be resolved?
Remote browser crash if a user browses a webpage with the following HTML code:

Tested on:
Windows XP SP2:
Windows Vista SP0:
Reproducible: Always
Steps to Reproduce:
1. create new *.html file
2. upload it to a web host (optional)
4. run file
Actual Results:
browser hangs. killing the process is the only option.
Expected Results:
"this script will run forever, do you wish to terminate? y/n" warning.
Happens even when "warn me when web sites try to redirect or load the page" under tools->options->advanced->general->accessibility is selected.
I spent a few minutes debugging but couldn't get control of eip. i'm lazy.
Created attachment 413778 [details]
proof of concept
Not sure why bug 101276 never had its fix checked in, but this isn't behaving like that one. This one is chewing up a lot more memory and CPU. I _do_ get the "A script on this page may be busy, or it may have stopped responding" prompt, but it does NOT let me kill the script! Memory use goes back down to reasonable levels every time that prompt comes us. It seems to be creating recursive iframes, but only one or two per busy prompt. If they were being created due to the while loop I'd have expected a lot more.
I don't see a crash. Maybe with less memory you'd trip over one of our many out-of-memory crashes
proof of concept #1 : Firefox 3.0.x : http://pixelsinspace.net/f.html

My apologies, this causes Firefox 3.0.x to hang without prompting.
As you say, Firefox 3.5.5 reports an unresponsive script but won't allow you to continue. You also can't change tabs when the "unresponsive script" prompt is shown (also a bug?).
proof of concept #2 : Firefox 3.5.5 & Internet Explorer 8.0.6: http://pixelsinspace.net/ff.html
I've adjusted the HTML (poc url above, code below) which now causes Firefox 3.5.5 and Internet Explorer 8.0.6 to hang (IE dev team have been notified). It also causes memory exhaustion.
<iframe src="javascript:while(true) { document.write(''); }">
With WinXP SP2 @ 2.2GHz and 3GB of RAM this provides the "unresponsive script" prompt after ~10 seconds and ~800MB of memory usage. Upon clicking "stop script" the memory usage idles until the browser stop event is evoked (closed tab, or closed browser, or clicked "stop", or pressed ESC, etc).
The browser hangs and the memory usage then increases rapidly until it peaks at ~2GB. Killing the process is required.
proof of concept #3 : Firefox 3.5.5 : http://pixelsinspace.net/fff.html
For reliable denial of service repeat the previous line 100 times as it will hang the browser and exhaust memory WITHOUT prompting with "unresponsive script. continue y/n?"
Created attachment 414035 [details]
proof of concept
FYI, this is a major issue with e10s on at the very least all desktop platforms. Every 15 seconds this chews up 1GB of RAM, and closing the tab doesn't stop it. If somebody lands on a page with this and e10s they'll be crashing every time.


text:
Contact Us
To Discuss Your Project
Material Handling & Product News:The "MVD" award was launched in 1996, to recognize the totality of factors that make a particular dealer ...
Home » Associations
Steel King belongs to several industry associations in an effort to provide you with quality products and services. Associations we hold memberships with are described as follows:
Material Handling Institute - MHI
The Rack Manufacturers Institute - RMI
Frequently Asked Storage Rack Questions: www.mhi.org/rmi/faq
The Storage Equipment Manufacturers Association - SMA
Material Handling Equipment Distributors Association - MHEDA
MHEDA is an independent association of material handling equipment distributors. Steel King is an Allied (manufacturer) member. MHEDA's mission is to further improve the proficiency of material handling distributors so that they be the most effective and efficient in their respective market place.
Protective Guarding Manufacturing Association – ProGMA
American Standards National Institute - ANSI


text:
Economic Outlook
What is inflation? What is deflation? We're hearing more about inflation and deflation these days. It's as if the economy were balanced on a seesaw, tilting one way and then the other. But what are the true definitions — and consequences — of these terms?
ELC Members Honored by TNJ as Influential Women in Business
40 Under Forty Achievers - Reaching for the Stars
Members of the 2009 class of TNJ's "40 Under Forty Dynamic Achievers" can boast that their class — the twelfth in the history of the "TNJ 40 Under Forty" — comes in the first year of the first Black presidency of the United States, a year when the bar in Black achievement was hoisted to historic heights.
The Year in Review
History in the Skies: Remembering Black pioneers
The Black Political Tradition: Al Sharpton's candidacy has roots in the Reconstruction era
What Went Wrong? - Vanguarde Media and Chisholm-Mingo in retrospect.
Republican National Convention: African-Americans Making Things Happen
Building: Will minorities get a decent piece of New York's action?
Corporate Social Responsibility
With the recession sapping money from corporate social-responsibility programs at many companies, it would seem that efforts to ensure environment-friendly business operations and a diverse workplace would be in competition for the same dollars. They don't have to be, diversity advocates say.


text:
More Videos...
GHOST E-ndure Evo2 - Pedelec für den harten Geländeeinsatz
Mitte 2011 könnte dieses Pedelec von GHOST auf den Markt kommen. Die wesentlichsten Merkmale in Stichworten: * Tretlagermotor mit 150 Nm (!) max. Drehmoment * Akku: 48 V, 8.2 Ah (~394 Wh), 250 Watt Nennleistung * komplette Integration des Antriebstrangs und Akkus in den Rahmen * uneingeschränkte Linienwahl auch auf dem Trail * tiefer, zentraler Schwerpunkt - agiles und laufruhiges Fahrverhalten * geringe Leitungsverluste durch kurze Kabellängen Fotos und weitere Details auf http://www.pedelecforum.de/forum/showthread.php?t=6755
quicke spin up with newly installed chain/procket setup setup...
Audi e-bike Official - A bicycle that runs at 80 kmph HD
~ Audi e Bike - A bicycle that runs at 80 kmph .. .. .. !! Audi unveiled an extremely emotion-inspiring sports machine, the Audi e-bike Wörthersee at Wörthersee in Carinthia, Austria. The prototype cycle combines an electric drive and muscle power. Head of Design Wolfgang Egger comments: "As a high-performance e-bike for sports and trick cycling, it features the Audi core competences of design, ultra, e-tron and connect." The Audi e-bike Wörthersee puts in its first major appearance at this year's Wörthersee Tour, the 31st meet for Audi, VW, Seat and Skoda fans; trial biker Julien Dupont and downhill specialist Petra Bernhard will demonstrate their stunts and streetbike skills. As a high performance e-bike for sports and trick cycling, it features the Audi core competences of design, ultra, e-tron and connect." Wolfgang Egger, Head of Design said. The bike looks dynamic, but is also designed to be functional. All design elements are secondary to the technical features. The airy frame boasts a low centre of gravity and a compact overall volume. In this way, the e- bike is agile. The lithium-ion battery is incorporated into the frame and needs 2.5 hours to fully charge and is easily replaceable. The frame and the swinging arm that holds the back wheel are made of carbon fibre-reinforced polymer (CFRP). The same material is used for the 26" wheels, which feature broad flat spokes for an optimized transmission of pedal power. The signature Audi LED lights are also present at the front and back of the bike. For extreme tricks and stunts the seat can be lowered to run flush with the frame itself. At the press of a button, the seat then rises up and the biker can adopt a comfortable position. The electric motor is located at the lowest point on the frame and drives the bottom bracket shaft directly. The maximum torque delivered to the rear wheel is 250Nm. The electric motor generates a maximum output of around 3bhp. The complete bike excluding electrical components weighs 11kg.
RYNO Motors Full Story
This is a great story on the early days of RYNO Motors. See our latest creation pre-production show bike at our web-site and pre-order at http://rynomotors.com/store and don't forget to buy a Tee Shirt: http://rynomotors.com/ryno-tee-shirts/


text:
Showtime's period drama "The Borgias" is coming to an end — its current third season will be its last, the network announced Wednesday.
'The Borgias' Join Showtime's Demented Dynasty
By: Tracy Phillips on January 9, 2010 in Show & Tell, TV News
Showtime announced plans to order 10 episodes of a one-hour series called 'The Borgias,' based on "the world's most notorious dynasty." Set in 1492 Italy, the period drama will star Oscar-winner Jeremy Irons in his first series television role as the patriarch of the powerful Borgia family, who reigned during the Italian Renaissance. According to […]


text:
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Recensioner på andra språk
Recenserad av: Gkoo , augusti 2016
What a fabulous experience this was for my girlfriend and I. It was our first night in Paris and we booked this for her birthday dinner. Our waiter I think his name was Omar was brilliant, very friendly and a good laugh. Great with the wine recommendations and nothing was too much trouble for him. There is something here for everyone and the bar itself has a very nice welcoming feel to it.
Recenserad av: Mark R , oktober 2015
We had a ball!! The staff REALLY know their wines, which were superb by the way. And also the chef produced one of the yummiest dinners we had in France. Oh Chateau, I wish you were here!!
Recenserad av: Alexandra A , augusti 2015
The staff was excellent but it wasn't as nice as expected as far as dress code, definitely less than casual. The food was good but it wasn't quite our taste, duck liver but it is France after all. The wine was lovely that's what your paying for, it was quite a lot of wine. We walked to the love lock bridge afterwards and thank god no one was driving. The steak and dessert was excellent!
Recenserad av: Christopher F , augusti 2015
BEST PLACE!!!!! My husband and I were so happy that we decided to eat here. Wine was great and our server was not only well informative on the wines but he was also entertaining. We went there for our anniversary dinner and in the end we were surprised with candles on our dessert to celebrate our special day. Will be going there again. We highly recommend this place.
Recenserad av: Debra D , april 2015
Our tour was amazing! What a way to start off our vacation. From the service, the food, and the wines, we couldn't have gotten thing better. Would totally recommend!
Recenserad av: Andrew T , april 2015
Amazing evening. The food and wines were delicious and paired perfectly. The ambiance was chic yet comfortable. The sommelier was informative and very accommodating. Great evening out in Paris.
Recenserad av: Donna M , mars 2015
This dinner was worth every nickel I spent (or should I say Euro!)....it is a beautiful restaurant, the meal was excellent as was the "matching" of the wines. I would definitely go again. The owners are great!!!
Recenserad av: sbmeacham , mars 2015
The food and wine was wonderful. It was a great experience and we learned a lot about French wine regions.
Recenserad av: Marjorie M , september 2014
This is a definite "must" for people coming to Paris for the first time. The atmosphere, the charm and the food was outstanding. The people who run the restaurant/bar were charming and very helpful. Can't say enough about the food. The dessert was fabulous. Even my husband who does not usually eat dessert ate it and proclaimed it great. The wine served with each course was explained thoroughly and was very generous in the portions.
Recenserad av: Pamela H , september 2014
The wine and dinner were awesome. The service was excellent.
Recenserad av: jose034 , United States of America, augusti 2014
I went to this restaurant on my birthday, everything was exceptional, the food, the wine, and the services.
Recenserad av: plindsel , maj 2014
Lovely food and wines with comprehensive explanation about the wines from the staff.
Recenserad av: Kieran R , maj 2014
The food and wine was excellent and the location is easy to find; very close to the Louvre. The waiter/ maitre d' was very nice and welcoming and beginning with the glass of champagne and continuing with each course and glass of wine he was very informative of what we were about to eat and drink, which was very nice. I also liked that it was not a set menu. He asked us what we liked, didn't like and what, if anything we were allergic to and then set up the menu accordingly. The wine was so good, I brought back 5 bottles of it and the food was amazing as well. If you want a fun night out with good food, wine and service, this is a place you should go.
Recenserad av: Jens S , mars 2014
Restaurant und Ambiente sehr angenehm ,da wir kein französisch sprechen und englisch eher schlecht hat sich dafür das Personal sehr viel Mühe gegeben . Mit Hilfe des I-Pads der Gastgeber hat dann alles bestens geklappt.Das Essen swie die ausgewählten Weine zu jedem Gang waren sehr gut auf einander abgestimmt .Wie man es nicht anders kennt gab es Bagette dazu , die Anrichtung der Speisen auf den Tellern war exzellent und sehr hübsch anzusehen. Vor dem Menü gab es Champagner und nach dem Menü gab es einen Degestiv es war alles in allem eine sehr gute und runde Sache , wir würden es sehr gern weiter empfehlen und jederzeit wieder kommen.
Recenserad av: Yvette R , januari 2014
The dinner and wine pairing was a wonderful experience and great value. The food, wine, atmosphere and service were very good. I highly recommend it.
Recenserad av: Sue T , januari 2014
Great wine, great experience.
Recenserad av: Leeona R , december 2013
We really enjoyed our anniversary meal meal here. We were served excellent food which was matched by beautiful wines and we were shared the staffs expert knowledge on each wine. Would highly recommend.
Recenserad av: Julie P , december 2013
O Chateau was a great place! Olivier Magny and the waiters were friendly and informative. The food and wine were delicious and the atmosphere was warm and inviting. We even bought their Into Wine book which Olivier signed for us. We would like to go back!
Recenserad av: Donna M , december 2013
Learned about different wine and food was good. Satisfied.
Recenserad av: linda b , december 2013
We were here the last week of september 2013 and the experience takes place at O Chateau and it was great! Each course has a different wine chosen by a different sommelier to accompany your particular food choice and they explain where the wine comes from and its characteristics.The place itself is charming and the food is fantastic- we had both the fish and beef choices - both delicious,and the pear tart tatin is out of this world delicious! The owner Olivier Magny could not have been more gracious- even putting up with quite the embrace from my 80 yr old mother! She loved the place! I highly recommend this and will be back here the next time I am in Paris
Recenserad av: Lois M , september 2013
Because there were no other reservations for this dinner the evening we went - we were served a private dinner for two! Instead of a wine cellar - we were seated at a window and could people watch as we enjoyed the wonderful food and wine.
Recenserad av: Daura J , augusti 2013
I would definitely recommend this tour to everyone who visits Paris. The wine and food pairing were a perfect match. The servers were very friendly and patient. The dessert was also paired with a wine. We had a wonderful evening in Paris. We've purchased a few bottles to share with our family.
Recenserad av: Emmanuelle J , juli 2013
I bought this a part of a week-end in Paris I gave my friend as her wedding present. It was booked for their first evening in Paris. My friend was over the moon, texting me later that night how wonderful the night had been. They were expected and very well looked after. The food and wines were superb. After dinner they were welcome to stay to enjoy a couple more glasses of wine. First time I booked with Viator booking 3 experiences, all superb. Will not hesitate to book again but will not waste time looking anywhere else!
Recenserad av: Martin A , juli 2013
absolutley superb. great dinner , great wine. private seating.excellent service
we had such a good time in a private setting.
Recenserad av: skibunnymommy , juni 2013
We went here our first night. The directions on the ticket a very good. The food was excellent, the wine amazing and the staffs English also excellent. A great place to go to start getting around in Paris.


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Fabio Capello's England doctor is doing a final round of the Premier League clubs this week to make a personal check on the players whose injuries are a cause for concern ahead of next week's announcement of the 30-man provisional World Cup squad.
Click here or click the image for our guide to the England treatment room.
Dr Ian Beasley, recruited from Arsenal two years ago, is visiting at least five clubs as he seeks to speak to players and medical staff in order to give Capello an accurate idea of the state of his squad. No visit will be more important than the one to Carrington, Manchester United's training ground where Beasley will be getting updates on up to five players.
Wayne Rooney, Rio Ferdinand, Wes Brown, Gary Neville and Owen Hargreaves are those whose condition Beasley will be checking. The indications from the Capello camp were that despite having come on as a late substitute against Sunderland on Sunday after 18 months out with chronic tendonitis, Hargreaves' return to action will be too late for him to be seriously considered as an option for the World Cup squad.
The announcement will be on Tuesday and the day before Capello will sit down with general manager Franco Baldini and the rest of his staff to make a final decision. Despite the fact that the England manager's first XI has a reasonably familiar feel to it, there are some key decisions to be made as to who makes the 30-man squad, never mind the final 23 which will be announced on 1 June.
Brown is a worry for Capello who likes the United player because he can play at right-back or centre-half. Ferdinand's persistent back problems are another worry. Neville also comes into consideration if Brown is not regarded as fit enough. Yet there are also concerns over the fitness of Neville who did not feature against Sunderland on Sunday.
Beasley is expected to drop in at Fulham's Motspur Park training ground to check on the Achilles problem of Bobby Zamora who is under serious consideration for his first call-up under Capello providing he can prove his fitness. Tom Huddlestone is also pushing hard for a place, potentially at the expense of Michael Carrick, the man whose position he inherited in the Spurs midfield four years ago.
Tottenham's England contingent also present problems. Aaron Lennon has only played two games since the start of the year and Ledley King's chronic knee problems mean he has never played for Capello. But there are also concerns about a persistent groin/hamstring problem that has affected Jermain Defoe, arguably the only Spurs player who, if fit, can be certain of a place on the plane to South Africa.
The Liverpool right-back Glen Johnson, who is Capello's first choice in that position, was also absent from his club's defeat by Chelsea on Sunday. The 25-year-old has suffered a recurrence of the knee injury that ruled him out for two months.
Despite all the problems facing Capello there are still some intriguing decisions to be made and some very big names potentially being left out. It would appear that Joe Cole has not done enough to make the squad, especially with Adam Johnson's performance for Manchester City against Aston Villa on Saturday making him the one wild card for a place in South Africa.
Shaun Wright-Phillips could also miss out on the 23-man squad if Lennon's fitness is assured. Capello also has to decide whether he will take four or five strikers with Carlton Cole and Zamora competing for the last spot in the final 23-man squad.
The training camp England will use in the town of Phokeng near Rustenburg in South Africa's North West province is expected to be opened officially today by the king of the Bafokeng nation who have financed and built the project, alongside representatives from the Football Association.
- More about:
- Bobby Zamora
- Fabio Capello
- Fitness
- Ledley King


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The art that helped 1960s London swing is going on show at a new exhibition organised by Sotheby's and one of the men who helped make the careers of some of the country's most famous artists.
John Kasmin, whose gallery opened in New Bond Street opposite the auction house 50 years ago, has helped choose the work which goes on show in the exhibition called The New Situation: Art in London in the Sixties.
He helped launch the careers of names including David Hockney, Bridget Riley and Anthony Caro whose work features in the new exhibition alongside some of their now-forgotten contemporaries.
He said: "In the 60s, optimism arose in the art world and we newer people thought we could change the prevailing British culture and make the visual rival the literary attitude. We admired daring and enthusiasm. We were enemies of 'good taste'. For us, paintings were no longer adjuncts to polite living, and sculpture went wild. No more 'over the mantelpiece' or on a plinth, the artworks challenged and exulted. We were on a several year-long 'high'."
Some of the works from the exhibition, which runs from tomorrow to Wednesday September 11, will be put up for sale.
Sotheby's Specialist in Modern and Post-War British Art Simon Hucker said: "In Britain in the 60s, we see all the major movements of the post-war period - from Pop, which in many ways we can lay a serious claim to have invented, to Op, gestural abstraction to Hard-Edge and Minimalism. It was also a time when art and what an art-object could be, was redefined, with the London art scene, deeply intertwined with music, poetry and film, leading the way in installation, performance and conceptual art."
PAReuse content


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Jammin' Sound, considered to be one of the premier DJ services in the greater Buffalo and Rochester areas. With more than 20 years experience, Jammin' Sound continues to impress! If a packed dancefloor is what you envision for your Wedding, then Jammin' Sound is your DJ Service of choice. Our Service to you & your wedding is priority #1. You will definitely see more guests dancing, & more guests having the time of their lives!
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All Rights Reserved.
Copyright © 2001-2017 OneWed Inc. All Rights Reserved.


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More Videos...
Drifting - 475 Hp Ford Excursion wipeout in the snow
Drifting in an Excursion & pitching it sideways a little too much! Haha! I submitted this video to Roadkill TV show and got to be a part of the Roadkill Nation Contest and race 700hp cars around for a week! It will air on youtube November 15, 2015. Look for my team: #teamrotsun No damage happened to the truck. I slid into some snowy brush sideways next to the tree with my tire. It was in 4wd. 2004 Ford Excursion - 6.0 turbo Diesel. Cab off procedure fixing the 6.0 issues. Retuned to 475hp. Diamond Eye turbo back Exhaust, K&N high velocity air filter.
Ford Excursion "Fordzilla" Part III Nowadays 2015
ford excursion 7.3L Powerstroke 2nd place hot diesel, Orfordville, WI
orfordville, wi 9/09 hot diesel 2nd place pull. '01 7.3L excursion, stage 1 injectors, ball bearing turbo, etc.
Excursion on boggers jeep rescue 3
still pulling broken jeep..almost out Jeep had a broken front axle and broken rear driveshaft. jeeps winch was also broken excursion is V10 on 44" boggers with 8" lift ***there is no winching in this video***


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Heartwarming light rain.
distracting green wonder.
Canopy in Heaven flowing light blue garland.
Tree's standing big and ablebodied
with white aglow flower!
The sun beaming.
Making me feel" so attentive"!
Lilac's cascading in cluster's.
Transforming into different elegant variation!
Catapillar's casting their wing's.
Making me aspire to so many affirmitive thing's!
Appendages to advance.
And also appendages to adapt.
Crafting me to be an ablebodied individual.
© Copyright 2017 Robin32. All rights reserved.
Status: Finished
Genre: Poetry
Poem / Fantasy
Poem / Poetry
Poem / Poetry
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Welcome to Alabama
History suffuses Alabama, a description which could be true of many states. But there are few places where the perception of said history is so emotionally fraught. The Mississippian Native American culture built great mound cities here, and Mobile is dotted with Franco-Caribbean architecture. But for many, the word Alabama is synonymous with the American Civil Rights movement... Read More
Top experiences in Alabama
Sights in AlabamaSee all sights
Recent articles
Birmingham, Alabama: the South's newest rising star
While you're here, make sure to turn the radio dial to 107.3 for Birmingham Mountain Radio. The eclectic mix of indie, underground, soul and country this station pumps into the airwaves is a national treasure that is criminally unrecognized in the rest of the country.


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Commercial fishing and fisheries – form
Combine or separate a fishing vessel licence or entitlement (AFL19)
Use this form to combine or separate a vessel licence or entitlement and use the component parts of the licence or entitlement.
You must submit this form to the fisheries management and effort control team.
Fisheries management and effort control team
Marine Management Organisation
Lancaster House
Hampshire Court
Newcastle upon Tyne
Telephone: 0300 123 1032


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Of Cigarettes, High Heels, and Other Interesting Things
An Introduction to Semiotics 2nd Revised edition
By (author) Marcel Danesi
Format: Paperback
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Of Cigarettes, High Heels, and Other Interesting Things by Marcel Danesi
Book DescriptionWhy do people routinely risk their health by smoking cigarettes? Why do some females make walking a struggle by donning high heels? In a fully updated edition, the author attempts to answer such questions by explaining the discipline that endeavors to understand the human meaning quest: semiotics.
Buy Of Cigarettes, High Heels, and Other Interesting Things book by Marcel Danesi from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9780230605237
(216mm x 140mm x 14mm)
Pages: 235
Imprint: Palgrave Macmillan
Publisher: Palgrave Macmillan
Publish Date: 8-Aug-2008
Country of Publication: United Kingdom
Books By Author Marcel Danesi
Italian Demystified, Premium, Paperback (September 2016)
Helping you learn Italian, this edition includes hundreds of quiz and test questions, chapter-opening objectives, and specific recommendations for difficult subtopics and individual weaknesses to help you learn basic grammar structures and verb tenses, pronunciation, essential vocabulary, and how to communicate with confidence.
Complete Italian Grammar, Paperback (May 2016)» View all books by Marcel Danesi
Italian grammar comes easy with practice, practice, practice!
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Author Biography - Marcel Danesi
Marcel Danesi is Director of the Program in Semiotics and Communication Theory and Professor of Linguistic Anthropology at the University of Toronto, Canada. He is the author of many books, including Of Cigarettes, High Heels, and Other Interesting Things; X-Rated! The Power of Mythic Symbolism in Popular Culture; and The History of the Kiss: The Birth of Popular Culture. He was made a Fellow of the Royal Society of Canada in 1998, founded a research center (Center for Communication and Information Sciences), and is the Editor in Chief of Semiotica. Danesi's work has been featured in the New York Times, Toronto Star, and Psychology Today, among other print publications, and he has been a guest on broadcast outlets such as National Public Radio.
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By Charles Taylor
Published by Sports Entertainment Publishing at Smashwords
Copyright 2013 Sports Entertainment Publishing
This eBook is not an official WWE publication nor is it associated, affiliated, or otherwise endorsed by WWE. To visit WWE.com, click here.
All information contained in this eBook has been obtained by exclusive interviews, online research, and information freely available in the public domain, including Pro Wrestling Wikia under Creative Commons License
This eBook provides information that you read and use at your own risk.
Cover images are available (with attribution) royalty free and for commercial use by Tabercil (Bret Hart), Justin Moody (Vince McMahon), and Will Beardmore (Shawn Michaels).