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The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3–17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.
- G-protein-coupled receptor (GPCR)
- ligand binding
- photolabelling
- urantide
- urotensin II
U-II (urotensin II, Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val) is a cyclic undecapeptide (the underlined residues represent the cyclic portion of the peptide) with activity on the cardiovascular and nervous system [1]. U-II was originally isolated from the urophysis of teleost fish [2], but the cDNA encoding its precursor has now been identified in many species [3,4]. In situ hybridization and immunohistochemistry have revealed that the peptide is localized to the medulla oblongata of the brain and the spinal cord in the central nervous system, but it is also present in tissues such as adrenal glands, kidney and spleen, suggesting neurohormonal or hormonal properties [3,5]. Although it is clear that U-II influences the physiological regulation of mammalian cardiovascular function, early reports showed conflicting results with regard to the peptide's vasoactive responses, which depend on the species or tissues used [1], whereas other studies described that administration of human U-II leads to potent vasodilator and cardiostimulant responses in human tissues [6,7]. More recently, a second peptide possessing structural similarity to U-II, while having a markedly different expression pattern, has been identified and termed U-II-related peptide (URP) [8].
U-II is the endogenous ligand of the UT receptor, a member of family A or rhodopsin-like family of GPCRs (G-protein-coupled receptors) [9]. Many features associated with this family, such as a short N-terminus, a highly conserved residue in each transmembrane domain, the presence of the D/ERY motif in the second intracellular loop, the NPXXY motif within TM7 (transmembrane domain 7) as well as potential serine/threonine phosphorylation sites in the cytoplasmic tail, are found in the UT receptor [10]. The UT receptor is detected in the central nervous system and is widely expressed in human tissues, including the left atrium and ventricle of the heart, smooth muscle cells of the coronary artery and aorta, as well as endothelial cells from several vascular beds [9]. Compelling evidence has implicated the U-II–UT receptor system in the pathophysiology of hypertension, heart failure and cardiac fibrosis and hypertrophy [1,11].
Elucidation of the molecular mechanisms by which an agonist binds to and activates a GPCR needs to be addressed using a variety of biophysical and biochemical approaches. We have sought to probe into the interactions of U-II with the UT receptor by covalently linking U-II to its receptor with a series of photoreactive analogues containing the photoactivatable amino acid Bpa (p-benzoyl-L-phenylalanine). Bpa-based photoaffinity labelling [12] has been employed successfully to identify binding sites in several peptide receptors [13–15]. In the present study, this approach was employed in order to identify contact points using two series of analogous probes that either fully or weakly activate the UT receptor. We hypothesized that photoaffinity labelling with photoprobes having different intrinsic activities may yield different contact points. Such differences may reflect the distinct ensembles of conformations that can be stabilized by both ligands [16]. We report the specific interaction of the first four positions of the full agonist, U-II, within ECL (extracellular loop) 3 of the receptor, more specifically at Met288. The first two positions of a partial U-II agonist also interact with this amino acid. However, position 6 of the partial agonist photoprobe does not appear to interact with the same binding site compared with its analogous full agonist probe [17]. The present study, coupled with our previous work [17], has allowed us to propose a model which defines for the first time the orientation of U-II within the rUT (rat UT) receptor binding pocket.
BSA, bacitracin, soya-bean trypsin inhibitor and CNBr were from Sigma. Staphylococcal V8 protease (Endo Glu-C; EC 188.8.131.52), Endo Arg-C (endoproteinase Arg-C; EC 188.8.131.52), Endo Lys-C (endoproteinase Lys-C; EC 18.104.22.168), FuGENE®-6 and Complete™ protease cocktail inhibitor were from Roche. The cDNA clone of the rUT receptor subcloned in the mammalian expression vector pcDNA3.0 was kindly provided by Dr Brian O'Dowd (Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada). DMEM (Dulbecco's modified Eagle's medium), FBS (foetal bovine serum), penicillin/streptomycin and oligonucleotide primers were obtained from Gibco Life Technologies. Human U-II was from Phoenix Pharmaceuticals. Bpa-containing U-II peptide analogues were synthesized in our laboratories using Fmoc (fluoren-9-ylmethoxycarbonyl)-protected amino acids and Wang resin (a p-benzyloxybenzyl alcohol resin from Novabiochem). Cysteine residues were protected by acetamidomethyl groups, which were removed by using mercury acetate. Disulfide bond formation was achieved by oxidation with DMSO. The synthetic peptides were purified to homogeneity by reverse-phase HPLC. 125I-U-II, 125I-[Bpa1]U-II, 125I-[Bpa2]U-II, 125I-[Bpa3]U-II, 125I-[Bpa4]U-II, 125I-[Bpa1Pen5D-Trp7Orn8]-U-II (Pen is penicillamine), 125I-[Bpa2Pen5D-Trp7-Orn8]U-II and 125I-[Pen5Bpa6D-Trp7Orn8]U-II (specific activity 400 Ci/mmol) were prepared with Iodo-Gen® (1,3,4,6-tetrachloro-3α,6α-diphenylglycouril; Pierce Chemical Co.) as described by Fraker and Speck [18]. Briefly, 10 μl of a 1 mM peptide solution was incubated with 20 μg of Iodo-Gen®, 80 μl of 100 mM borate buffer, pH 8.5, and 1 mCi of Na125I for 30 min at room temperature (25 °C) before being purified by HPLC on a C18 column. The specific radioactivity of the labelled peptides was determined by self-displacement and saturation-binding analysis.
PCR mutagenesis
Mutant receptor cDNAs were constructed by oligonucleotide-directed mutagenesis (Expand High Fidelity PCR System; Roche Diagnostics) using the rUT receptor inserted into pcDNA3.0 (Invitrogen) as a template. A set of forward and reverse oligonucleotides were constructed in order to introduce a Met→Leu mutation at position 288 of the rUT receptor to generate M288L-rUT. PCR products were re-inserted in pcDNA3.0, and mutagenesis was confirmed by nucleotide sequencing.
Cell culture and transfection
COS-7 cells were grown in DMEM containing 10% (v/v) FBS, 100 i.u./ml penicillin and 100 μg/ml streptomycin at 37 °C. Semi-confluent cells (70%) in 100-mm-diameter Petri dishes were transfected using FuGENE®-6 as described by the manufacturer. Transfected cells were grown for 48 h before photoaffinity labelling and binding assays.
Binding experiments
COS-7 cells were washed once with PBS and subjected to one freeze–thaw cycle. Broken cells were then gently scraped into washing buffer (20 mM Tris/HCl, pH 7.4, and 5 mM MgCl2), centrifuged at 2500 g for 15 min at 4 °C and resuspended in binding buffer (20 mM Tris/HCl, pH 7.4, 5 mM MgCl2, 0.1% BSA and 0.01% bacitracin). For competition binding assays, broken cells (50–75 μg of protein) were incubated for 1 h at room temperature in binding buffer with 0.1 nM 125I-U-II (400 Ci/mmol) as tracer and increasing concentrations of U-II peptide analogues in a final volume of 0.5 ml. Bound radioactivity was separated from free ligand by filtration through GF/C filters pre-soaked for at least 1 h in binding buffer. Receptor-bound radioactivity was evaluated by γ-radiation counting.
Measurement of agonist-induced production of inositol phosphates
Accumulation of inositol phosphates was determined essentially as described in [19], with the following modification. Production of inositol phosphates was induced with 100 nM U-II or each analogue for 10 min at 37 °C in buffer containing 10 mM LiCl. Inositol phosphates were sequentially eluted from the anion-exchange column by the addition of an ammonium formate/formic acid solution of increasing ionic strength. Fractions containing inositol phosphates were collected and counted for radioactivity in a liquid-scintillation counter.
Photoaffinity labelling
Photoaffinity labelling was performed as described previously [13]. Transfected COS-7 cells were incubated for 1 h at room temperature in 0.5 ml of binding buffer containing the photoreactive radioligand (5 nM), in the presence (10 μM) or absence of U-II. Cells were then washed, irradiated for 1 h at 0 °C under filtered UV light in washing buffer and centrifuged at 6000 g for 15 min at 4 °C. The pellet was solubilized on ice for 30 min in modified radioimmunoprecipitation buffer [m-RIPA; 50 mM Tris/HCl, pH 8.5, 150 mM NaCl, 5 mM sodium azide, 1% (v/v) Nonidet P40, 0.25% (v/v) deoxycholic acid and 0.1% (w/v) SDS] containing Complete™ protease inhibitor cocktail. After centrifugation at 15000 g for 30 min at 4 °C, the supernatants were kept at −80 °C until further analysis.
Partial purification of the labelled complex
Photolabelled receptors were separated by SDS/PAGE using 10% (w/v) acrylamide/Tris/glycine gels, before being exposed to BioMax® MS films (Kodak). Bands corresponding to receptor–ligand complexes were excised from the gels and submitted to electroelution. Eluted proteins were then concentrated and kept at −20 °C.
Proteolytic and chemical digestion
For V8 protease digestions, partially purified photolabelled receptors (5000 c.p.m.) were resuspended in 50 μl of digestion buffer (100 mM ammonium bicarbonate buffer, pH 8.0) at room temperature for the indicated times. Under these conditions, V8 protease cleaves at the C-terminal side of glutamate residues. When subsequent digestions were needed, products of the first digestion were run on a 16.5% (w/v) acrylamide/Tris/Tricine gel, and the corresponding band was electroeluted. Eluted proteins were concentrated and submitted to subsequent digestion. For Endo Arg-C digestion, partially purified photolabelled receptors (5000 c.p.m.) were resuspended in 100 μl of digestion buffer (100 mM Tris/HCl, pH 7.6, 100 mM CaCl2, 0.5 mM EDTA and 0.01% SDS), and samples were incubated with Arg-C (0.5 μg) for 16 h at 37 °C. For Endo Lys-C digestion, partially purified photolabelled receptors (5000 c.p.m.) were resuspended in 50 μl of digestion buffer (25 mM Tris/HCl, pH 8.5, 1 mM EDTA and 0.1% SDS), and samples were incubated with Lys-C (0.5 μg) for 16 h at 25 °C. For CNBr hydrolysis, partially purified photolabelled receptors (5000 c.p.m.) were resuspended in a 1:1 mixture of 10% trifluoroacetic acid and CNBr (100 mg/ml) and incubated at room temperature in the dark for 18 h. Reactions were terminated by adding 0.6 ml of water and the material was freeze-dried.
Analysis of the products of proteolysis and chemical cleavage
The products of proteolysis and chemical cleavage were analysed by SDS/PAGE using 16.5% (w/v) acrylamide/Tris/Tricine gels, followed by autoradiography on X-ray films (BioMax® MS) with intensifying screens. 14C-Labelled low-molecular-mass protein standards (Amersham Biosciences) were used to determine apparent molecular masses.
Molecular modelling
All calculations were performed on a Silicon Graphics Octane2 workstation. The U-II-derived ligands and the rUT receptor model were built using Insight II modules (Biopolymer, Homology, Discover; Accelrys). The molecular models of the agonist (U-II, [Bpa1]U-II, [Bpa2]U-II, [Bpa3]U-II, [Bpa4]U-II and [Bpa6]U-II) and the partial-agonist peptides ([Pen5D-Trp7Orn8]U-II, [Bpa1Pen5D-Trp7Orn8]U-II, [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II) were constructed in a linear form using the Biopolymer module from InsightII. Subsequently, a disulfide bond was added between residues in position 5 and 10 of each ligand. The potential energy of each peptide was minimized for 500 steps with steepest descents with a consistent valence force field [20] using the Discover module. All the minimized structures adopted the overall conformation reported previously for U-II [21]. A distance-dependent dielectric constant of 4 was used.
The theoretical structure of the rUT receptor (GenBank® accession number P49684) was generated by homology modelling based on the crystal structure of bovine rhodopsin (PDB code 1L9H, chain A) [22], as described previously [23]. The resulting structure represents an inactive form of the rUT receptor with an overall configuration very similar to that of bovine rhodopsin [2.35 Å (1 Å=0.1 nm) RMSD (root mean square deviation) between the backbone atoms of the transmembrane domains].
In order to allow the incorporation of the ligand within the receptor's orthosteric cleft, a cavity had to be generated. This was accomplished by rotation of the Φ angle of Thr40 from −95.69° to −23.89° (displaces the N-terminal segment outwards from the helix bundle) followed by rotation of the Φ angle of Lys247 from −133.86° to −126.66° in order to displace TM6 and TM7. Rearrangement of TM6 and TM7 following receptor activation is supported by experimental evidence [24–26]. The bonds between S–S from Cys123–Cys199, C–N from Leu186–Ala187 and C–N from Ala202–Trp203 were then broken, and the Φ angle of Cys199 was rotated from −86.83° to 136.93°, the Φ angle of Ser197 from 49.67° to 92.07°, the Ψ angle of Lys196 from −125.35° to −146.06°, and the Ψ angle of Arg193 from −25.76° to −49.76°. The broken bonds were then reformed, and a potential energy minimization was performed with transmembrane domains fixed until the maximum derivative was less than 0.1 kcal/mol (1 kcal≈4.184 kJ). The resulting structure gives an RMSD value of 0.36 Å for TM1–TM5 backbone atoms compared with the initial model and a RMSD value of 1.96 Å for all transmembrane backbone atoms.
The modelled U-II structure was placed in between the transmembrane domains of the rUT receptor, as suggested by photolabelling results obtained with the agonist ligands. Information about ligand–receptor interactions obtained in previous photoaffinity labelling and mutagenesis experiments [17,27] was considered along with the contact sites reported here, and was used as distance restraints for the energy minimization of the U-II–rUT receptor complex. A first minimization of the complex between U-II and the rUT receptor was performed using distance restraints (2 Å<d<7 Å) between the Sδ atoms of photolabelled methionine residues and the Cγ atom of the corresponding side chains on U-II. Explicitly, Glu1 was kept close to Met288, Phe6 was restrained to Met184 and Met185 [17], whereas Lys8 was restrained to Asp130 [27]. The backbone atoms of the rUT receptor transmembrane domains were held in their position during this step; the ligand, extracellular and intracellular loops were free to move and interact. The U-II–rUT receptor complex was refined by several energy-minimization steps, until the maximum derivative was less than 0.1 kcal/mol. Molecular Graphics Figures were created using PyMol (Delano Scientific).
Binding specificity of photoreactive analogues
Figure 1(A) shows the primary structure of U-II and the photoreactive U-II analogues used in the present study. For the agonist peptides, Glu1, Thr2, Pro3 and Asp4 were successively replaced by Bpa to give [Bpa1]U-II (1), [Bpa2]U-II (2), [Bpa3]U-II (3) and [Bpa4]U-II (4) respectively. We also synthesized peptides based on the structure of urantide. Although urantide was originally described as an antagonist [29], recent reports have demonstrated that this peptide acts as a weak partial agonist in various cells heterologously expressing the UT receptor [30]. Residues 1, 2 and 6 were successively replaced with Bpa in a 'humanized' version of the urantide peptide [Pen5D-Trp7Orn8]U-II (5) to give [Bpa1Pen5D-Trp7Orn8]U-II (6), [Bpa2Pen5D-Trp7Orn8]U-II (7) and [Pen5Bpa6D-Trp7Orn8]U-II (8). All peptides also contained a tyrosine residue, present in the native peptide at position 9, for radioiodination.
In competitive binding assays, all analogues exhibited high affinities for the UT receptor expressed in COS-7 cells (Table 1) with the exception of 8, which showed a slightly reduced affinity (17.7 nM). The functional properties of these analogues were then assessed by measuring the ligand-induced Ins(1,4,5)P3 production in COS-7 cells expressing the rUT receptor (Table 1). Incubation of the cells with 1, 2, 3 or 4 caused an increase in Ins(1,4,5)P3 comparable with the level produced by native U-II, suggesting the full agonist nature of these analogues. All four peptides based on urantide (5, 6, 7 and 8) elicited very weak Ins(1,4,5)P3 release in this assay, indicating their weak partial agonistic nature. It was therefore assumed that the structure probed by these peptides could differ from the one derived from the full agonist-probed receptor structure.
Photoaffinity labelling of the rUT receptor
In the photoaffinity labelling experiments, all peptides labelled a protein migrating as a 60 kDa band on SDS/PAGE (Figure 2). This adduct has been observed previously when photolabelling with 125I-[Bpa6]U-II [17] and corresponds to the intact glycosylated receptor. Incubation with 10 μM U-II abolished the labelling, indicating the specificity of the interaction. In order to identify the covalently modified receptor region, the labelled species were partially purified and treated with the chemical agent CNBr or specific endoproteinases, i.e. V8 protease, Endo Arg-C or Endo Lys-C.
V8 protease digestion of the 125I-[Bpa1]U-II–rUT, 125I-[Bpa2]U-II–rUT, 125I-[Bpa3]U-II–rUT and 125I-[Bpa4]U-II–rUT receptor complexes
In an initial attempt to map the ligand–receptor contact site for agonist peptide ligands, the 60 kDa forms of 125I-1, 125I-2, 125I-3 and 125I-4-photolabelled rUT receptor complexes were partially purified (Figure 3A, lane 1) and submitted to V8 protease digestion. This enzyme cleaves on the C-terminal side of glutamate residues (Figure 1B) and the cleavage was carried out in a time-course experiment over a period of 162 h. This digestion produced a band of apparent molecular mass 17 kDa after 2 h for each photolabelled receptor complex (Figure 3A, lane 2). One receptor domain appearing as a potential candidate is fragment Leu24–Glu147 with a calculated molecular mass of 14.8 kDa glycosylated on Asn29 and/or Asn33 (Figure 3D). Other fragments corresponding to Arg148–Glu286 or the incompletely digested Arg148–Glu293 with a calculated molecular mass of 17.5 kDa (non-glycosylated) are additional candidates. Further digestion of all four photolabelled rUT receptor complexes progressively yielded a final fragment of 3 kDa (Figure 3A, lane 6). Taking into account the molecular mass of the ligand and cleavage specificity, this small fragment can only correspond to receptor fragment Ala287–Glu293 of ECL3 for all four photoprobes (Figure 3D).
CNBr chemical cleavage of the 125I-[Bpa1]U-II–rUT, 125I-[Bpa2]U-II–rUT, 125I-[Bpa3]U-II–rUT and 125I-[Bpa4]U-II–rUT receptor complexes
To further characterize the agonist–receptor contact site, the 60 kDa forms of 125I-1-, 125I-2-, 125I-3- and 125I-4-photolabelled rUT receptor complexes were partially purified (Figure 3B, lanes 1, 3, 5 and 7) and submitted to CNBr cleavage, which specifically cleaves at the C-terminal end of methionine residues (Figure 1B). Each agonist–receptor complex (Figure 3B, lanes 2, 4, 6 and 8) when submitted to CNBr digestion produced two bands migrating at apparent molecular masses of 22 and 12.5 kDa. Owing to the large size of the fragments obtained, the best candidates for attribution of these fragments are Leu186–Met288 for the 12.5 kDa band and Leu186–Met376 for the 22 kDa band, which corresponds to an incomplete digestion at Met288. In the case of 125I-1- and 125I-4-photolabelled rUT receptor complexes, an additional band of 2 kDa was also produced. The presence of this 2 kDa band is consistent with ϵ-labelling of Met288 of the rUT receptor and corresponds to the cleaved methylthiocyanate adduct of the photoligands. Moreover, the absence of ligand release in the CNBr-digestion pattern of the 125I-2- and 125I-3-photolabelled rUT receptor complexes, together with the results obtained by the V8 protease digestion, suggests that both of these photoprobes label on the γ-carbon of Met288.
To confirm further that the first four positions of the agonist U-II are in close proximity to Met288 of ECL3 of the rUT receptor, we examined the effect of replacing this residue with leucine by expressing the M288L-rUT receptor mutant. Photolabelling of M288L-rUT with 125I-1, 125I-2, 125I-3 and 125I-4 was performed and each complex was partially purified (Figure 3C, lanes 1, 3, 5 and 7) and submitted to CNBr chemical cleavage. For all four probes, CNBr cleavage of the photolabelled mutant complex yielded a 22 kDa band corresponding to the receptor fragment Leu186–Met376 (Figure 3C, lanes 2, 4, 6 and 8). The 125I-1- and 125I-4-photolabelled M288L-rUT mutant receptor complexes failed to show any ligand release, in contrast with the wild-type receptor. Furthermore, V8 protease cleavage of the ligand–mutant complexes yielded a fragment of 3 kDa similar to that of wild-type receptor (results not shown), indicating that labelling on the mutant receptor still occurred on the Arg287–Glu293 domain. Taken together, our results strongly suggest that Met288 of ECL3 of the rUT receptor is a common contact point for all four N-terminal positions of U-II (Figure 3E).
CNBr chemical cleavage of the 125I-[Bpa1Pen5D-Trp7Orn8]U-II–rUT and 125I-[Bpa2Pen5D-Trp7Orn8]U-II–rUT complexes
In order to compare the binding mode of the full U-II agonists with that of the weak partial agonist [Pen5D-Trp7Orn8]U-II, photoprobes incorporating Bpa in positions 1, 2 and 6 were used to determine peptide–receptor contact sites. Initially, the peptides 125I-6 and 125I-7 containing Bpa in the first two positions were used. The 60 kDa forms of 125I-6- and 125I-7-photolabelled rUT receptor complexes were partially purified (Figure 4, lanes 1 and 5) and submitted to CNBr cleavage. Each ligand–receptor complex (Figure 4, lanes 2 and 6) following CNBr digestion produced two bands migrating at apparent molecular masses of 22 and 12.5 kDa, with the 125I-6-photolabelled receptor complex additionally producing ligand release. This CNBr-digestion pattern is identical with that obtained with their full agonist counterparts 125I-1 and 125I-2, indicating that labelling for both partial agonist photoprobes also occurred on Met288 of ECL3 of the rUT receptor (Figure 3E). To corroborate this result, photolabelling of M288L-rUT with 125I-6 and 125I-7, was performed, both photolabelled receptor mutant complexes were partially purified (Figure 4, lanes 3 and 7) and submitted to CNBr chemical cleavage. Following this cleavage (Figure 4, lanes 4 and 8), ligand release for 125I-6 was completely abolished. These results indicate that labelling of the N-terminal portion of both agonist and partial agonist U-II ligands occurs on Met288.
CNBr chemical cleavage of the 125I-[Pen5Bpa6D-Trp7Orn8]U-II–rUT receptor complex
We had shown previously that the full agonist [Bpa6]U-II labelled Met184/Met185 of the fourth transmembrane domain of the rUT receptor [17]. The photoprobe [Pen5D-Trp7Orn8]U-II incorporating Bpa in the sixth position, a weak partial agonist, was used to assess whether labelling would also occur at Met184/Met185. First, the 60 kDa form of the 125I-8-photolabelled receptor complex was partially purified and digested with V8 protease. The resulting 17 kDa fragment (Figure 5A, lane 1), which corresponds to Arg148–Glu286 [17], was partially purified. Cleavage of this fragment with CNBr (Figure 5A, lane 2) led to the production of a 12.5 kDa fragment, which could be attributed to the fragment Met185–Glu286 (Figure 5C). More importantly, the lack of any detected ligand release (Figure 5A, lane 2) indicates that 125I-8 did not cross-link to Met184/Met185 as was observed for [Bpa6]U-II. To identify further the binding domain of 125I-8, the 17 kDa V8 protease fragment (Figure 5B, lane 1) was submitted to Endo Lys-C digestion, which cleaves specifically at the C-terminal end of lysine residues (Figure 1B). Endo Lys-C digestion produced two bands migrating at apparent molecular masses of 10 and 7.5 kDa (Figure 5B, lane 2). Since the 10 kDa band is progressively cleaved to give the 7.5 kDa band after prolonged digestion times (results not shown), the former is believed to originate from partial enzyme cleavage. Taking into account the region delimited by the V8 protease digestion, the only candidate for the 7.5 kDa band is the fragment Ser197–Lys247. This fragment encompasses part of ECL2, TM5 and part of intracellular loop 3 (Figure 5D). To narrow down the domain of interaction of 125I-8 further, the 17 kDa V8 protease fragment was submitted to Endo Arg-C digestion, which specifically cleaves at the C-terminal end of arginine residues (Figure 1B). This digestion produced a single band migrating at a molecular mass of 4 kDa (Figure 5B). Taking into account the results obtained from the Lys-C digestion, the only possible candidate for this band is the fragment Thr210–Arg233, therefore localizing the labelling of 125I-8 to TM5 of the rUT receptor (Figure 5E).
Identifying the molecular determinants involved in the bimolecular recognition of ligand and receptor is of great importance in order to understand the initial steps leading to receptor activation. In the present study, photoaffinity labelling was used as a strategy to investigate the interactions of U-II peptides with the rUT receptor. This approach depends on the spatial proximity between radioactive photoligands and receptor domains to form a covalent conjugate that is subsequently mapped to identify the interaction sites. We hypothesized that the receptor conformation stabilized by a U-II agonist might differ from a conformation stabilized by a weak partial agonist and that these changes may be detected by photoaffinity labelling. Such an approach has recently been exploited for the cholecystokinin and secretin receptors [31,32].
To allow for the most optimal comparison between both N-terminal and C-terminal positions of U-II, a humanized version of urantide containing the first four residues of human U-II was designed. Four analogues of U-II containing Bpa in the variable N-terminal part of U-II at positions 1, 2, 3 and 4, as well as three humanized urantide analogues containing Bpa in both the variable (positions 1 and 2) and cyclic (position 6) were prepared. These peptides were characterized for their capacity to recognize and activate the UT receptor and the photolabelling patterns of agonist peptides were compared with those of the partial agonists. A recent report showed that N-terminal deletions or substitutions of U-II had little or no effect on agonistic properties or binding affinities of the peptides [33], therefore we expected that Bpa substitution within this region should be permissible. Indeed, all U-II photoligands with Bpa in positions 1, 2, 3 and 4 demonstrated full agonist properties with high affinity for the UT receptor. Replacing positions 1, 2 and 6 in humanized urantide ([Pen5D-Trp7Orn8]U-II) with Bpa led to the production of partial agonists, as demonstrated by their capacity at producing low levels of Ins(1,4,5)P3, which is in line with the reported partial agonist nature of urantide [30]. It is noteworthy to mention, however, that studies have demonstrated that urantide is a partial agonist towards heterologously expressed hUT receptor (human UT receptor), but an antagonist towards rat aorta preparations [35], thereby suggesting that differences probably arise because of the cellular or tissue preparation used. Also, previous studies from our group indicated that substituting Bpa at position 6 in the cyclic portion of U-II analogues did not greatly affect its affinity [17]. Introduction of Bpa at position 6 in the humanized urantide peptide lowered its affinity for the UT receptor somewhat when compared with humanized urantide itself (5). Nevertheless, on the basis of their pharmacological properties (Table 1) and their complete displacement by the parent UII peptide in the photolabelling experiments, it is reasonable to assume that each analogue, agonist or partial agonist, would adopt a orthosteric binding mode similar to that taken by its parent peptide.
Previously, it has been shown that position 6 of U-II photolabelled Met184/Met185 of the rUT receptor and that a U-II analogue containing Bpa in position 7 failed to bind the receptor [17]. Analogues containing Bpa in position 8 and 9 of U-II also failed to bind the rUT receptor (results not shown). This is not surprising in the light of structure–activity studies on U-II [33]. Our present results from enzymatic and chemical degradation of the photoligand–receptor complexes incorporating Bpa in an N-terminal position for both full and partial agonists have identified Met288 of ECL3 as the sole contact point. It could be reasoned that the N-terminal part of U-II analogues can interact weakly with the extracellular loops of the UT receptor; this would explain why these residues are less important for affinity and receptor activation. It is noteworthy to mention that the rUT and hUT receptors share 90% sequence similarity. Interestingly, whereas most residues of ECL3 are conserved between the rUT and hUT receptor, the hUT receptor lacks Met288 in ECL3. Because it has been shown using surface plasmon resonance that ECL3 from the human receptor specifically binds U-II [36], it is reasonable to hypothesize that the interaction mode of the N-terminus of U-II with either the rat or the human receptor will be similar.
Interaction of benzophenone residues with methionine residues of receptors has been widely reported [15,37]. This type of interaction appears to be favoured, since the free radical is stabilized as a charge-transfer complex by the presence of the thioether moiety found in methionine. Such a complex would drive the equilibrium towards the formation of covalent products with the adjacent methyl or methylene groups. A benzophenone-containing ligand could thus be attracted to methionine residues located in close proximity to the binding site, but beyond the van der Waals radius, although, if no methionine is present, Bpa is still able to covalently link side chains of other residues [38–40]. Photolabelling of methionine residues can lead to two different labelling patterns: ϵ-labelling, which leads to ligand release following CNBr cleavage, and γ-labelling, which does not lead to ligand release [23]. This property can be exploited to refine the mapping studies. Our results show that CNBr cleavage gives rise to ligand release for the 125I-1-, 125I-4- and 125I-6-photolabelled rUT receptor complexes, whereas ligand release is not observed in the case of photolabelling with 125I-2, 125I-3 and 125I-7. CNBr cleavage of these latter complexes generates only traces of the 12.5 kDa fragment and mostly the complete 186–375 fragment of 22 kDa; this is indicative of γ-labelling. Our results show that the CNBr reaction is particularly difficult to drive to completion within the Met288 region. This can be explained in two ways: first, it has been observed that peptide bonds containing proline residues are difficult to cleave (residue 289 is proline), and, secondly, as reported above, the close proximity of ligand attachment to Met288 could restrict the formation of CNBr reaction intermediates, thus preventing the reaction from proceeding optimally [41]. Such steric hindrance can be induced by label incorporation into the γ-methylene group of Met288 and would thus demonstrate that 125I-2, 125I-3 and 125I-7 also interact with Met288. Exclusive γ-labelling of 125I-2, 125I-3 and 125I-7 indicates that the Bpa moiety of these peptides is in closer proximity to the Met288 backbone than that of 125I-1, 125I-4 and 125I-6 which shows ϵ-labelling. Molecular modelling, based on the reported U-II structure which conserved the type II' β-turn [21] (Figure 6A), suggests further that Bpa would indeed occupy distinct conformational spaces in the receptor's orthosteric binding pocket depending on the residue's position within either an agonist or partial agonist peptide. Although these models cannot be seen as experimental structures, they nonetheless offer a reasonable structural framework to explain the position-dependence of the photolabelling of Met288.
It is generally accepted that binding of ligands with different potencies to a given receptor should lead to the stabilization of distinct receptor conformations [42]. We had found previously that photolabelling with a full agonist U-II peptide containing Bpa in position 6 led to the identification of Met184/Met185 of TM4 of the rUT receptor as a contact site. We now show that photolabelling with the weak partial agonist 125I-8, which also incorporates Bpa in position 6, identified a different labelling site which was subsequently mapped to TM5. Thus these differences may reflect changes in the conformations adopted by the receptor during the activation process and more particularly the different relative orientations adopted by TM4 and TM5 within the receptor core. We propose that, during activation, TM4 may move closer to the receptor's orthosteric binding pocket and/or that TM5 may move away from the receptor's binding pocket, allowing labelling of TM4 by the agonist. At this point, it is not possible to determine the exact nature or extent of these movements. Our results support the notion that full agonist and partial agonist U-II peptides bind to the UT receptor differently [36] and hence may stabilize different rUT receptor conformations.
In order to assess the relative position of the full agonist peptide in the binding pocket of the rUT receptor, we sought to model the receptor in complex with the U-II peptide. To correctly position U-II within the receptor binding pocket, distance restraints needed to be defined. A first point used was the putative ionic interaction between Lys8 of U-II and Asp130 of the rUT receptor (Figure 6B, inset). In support of this interaction, the corresponding Asp122 of the somatostatin receptor subtype 2, a member of the same GPCR subfamily as the UT receptor, was found to interact with Lys9 of somatostatin, a peptide analogous to U-II [43]. Furthermore, substitution of alanine for Asp130 in the rUT receptor totally abolished receptor binding (results not shown), and structure–activity studies of U-II consistently show that Lys8 is critical to U-II binding and function [33]. Our photolabelling results were used to define a second interaction point: an area defined as a mean position between the Cα of Glu1–Asp4 of U-II with Met288 of the rUT receptor. A third anchoring point is position Bpa6 which is in close proximity to Met184/Met185 of TM4, which we had identified previously. Using these anchoring points, a U-II–rUT receptor complex was modelled (Figure 6B, see also animated Supplementary Figure 1 and Supplementary pdb file at http://www.BiochemJ.org/bj/402/bj4020051add.htm). Following minimization of the complex with distance restraints defined, the structure of docked U-II has a RMSD value of 1.4 Å compared with the initial U-II structure and the rUT receptor transmembrane domains have a RMSD value of 1.5 Å compared with the initial model. These values indicate that the structure of both ligand and receptor varied little during the docking process. In our model, the critical Trp7, Lys8 and Tyr9 residues which compose the pharmacophore of U-II [34] are well oriented within a pocket formed by the cytoplasmic side of the transmembrane domains of the rUT receptor, highlighting their importance for receptor binding and activation.
In conclusion, our results support the hypothesis that agonist or partial agonist ligands may stabilize different receptor conformations upon binding. For the N-terminal portion of U-II peptides, we show that binding determinants are present in ECL3 of the rUT receptor. We also show a possible contribution of TM4 and TM5 during receptor activation. Although most studies to date implicate either TM6 or TM3 movements during receptor activation [25,26], it is increasingly becoming recognized that TM5 may play a more important role during the GPCR activation process than appreciated previously [44]. Further studies combining mutagenesis, SCAM (substituted cysteine accessibility method) and molecular modelling should provide further information crucial to the development of models defining the binding pocket, and perhaps the activation mechanism of this receptor. Because of the putative role of U-II in several pathophysiologies, including cardiovascular disease, the design of novel ligands could be facilitated by the knowledge brought on by these models.
We thank Dr Brian O'Dowd (Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada) for the rUT receptor clone and Marie-Reine Lefebvre (Department of Pharmacology, University of Sherbrooke) for synthesis of the U-II analogues. We also thank Stéphane Martin and Martin Clément (both at the Department of Pharmacology, University of Sherbrooke) for helpful discussions. This work is supported by grants from the Canadian Institutes for Health Research and from the Heart and Stroke Foundation. This work is part of the Ph.D. Thesis of B. J. H. C. D. P. is a recipient of a Heart and Stroke Foundation studentship. E. E. is the recipient of the J. C. Edwards Chair in cardiovascular research. R. L. is a Chercheur National of the Fonds de la Recherche en Santé du Québec.
Abbreviations: Bpa, p-benzoyl-L-phenylalanine; DMEM, Dulbecco's modified Eagle's medium; ECL, extracellular loop; Endo, endoproteinase; FBS, foetal bovine serum; GPCR, G-protein-coupled receptor; Pen, penicillamine; RMSD, root mean square deviation; TM, transmembrane domain; U-II, urotensin II; UT receptor, U-II receptor; hUT, human U-II receptor; rUT receptor, rat U-II receptor
- The Biochemical Society, London


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More than 1,000 people — mostly grown-ups with kids in tow — grabbed peppermint bannisters and hopped aboard the Kansas City Southern Holiday Express train when it stopped Friday in Baton Rouge.
The colorfully decorated six-car train brightened a lackluster area of Baton Rouge near the tan Kansas City Southern Lines building and the gray BREC Memorial Stadium. Kansas City Southern has railroads running across the country and the company's workers, dressed as elves, presented Baton Rouge Salvation Army representatives with a box filled with gift cards to help families this Christmas season.
Lexi Menard, 25, came from Lafayette with her 4-year-old son, Kason, to tour the train. She read about the event on Facebook and arrived more than an hour early to secure the first spot in line.
"They're ready to see Santa," she said about her son and his friend, who came along.
They walked through car after car of the train — the first one glittering with winter wonderland figurines, the second whistling with toy trains, the third shining with Santa statues. Christmas music played in the background, and spirited Kansas City Southern workers greeted them along the way and helped them navigate each train car.
Kason kept his eye on the prize. He and his mother finally made it to Santa, who sat beside the train to greet visitors. Kason posed for photos, completing the tour with a smile on his face.
Those waiting in line also could visit Salvation Army workers serving hot drinks, though they quickly ran out of hot chocolate. Baton Rouge Salvation Army Capt. Brett Meredith said they didn't know how many people to expect, and he was wowed by the turnout.
Meredith said he was unsure of the amount of gift card money given to Baton Rouge specifically, but Kansas City Southern will distribute more than $150,000 nationwide.
"It's a lot of gift cards; it'll help a lot of kids," Meredith said.
The Holiday Express travels across the country annually, and Baton Rouge was its first stop this year.

subset: commoncrawl


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Louis VuittonBrown Damier Coated Canvas Leather Trim "Berkeley" Bag
The Louis Vuitton "Berkeley" bag is timeless and spacious, making it an ideal choice for everyday use. Constructed of brown Damier coated canvas with leather trim. Large logo plaque at the front. Dual rolled top handles. Side pockets. Top zipper closure. Gold toned hardware. Red suede lined interior features two side pockets. Comes with dust bag. Retails for $1,970.
Made in: France Color: Brown Fabric Content: Coated Canvas and Leather Condition: Pre-owned. This item is in good condition. Faint wear onedges of bag along corners of leather trim. Light marks on hardware. Light dust in interior.
Measurements & Sizing
- Total Height: 9"
- Total Length: 12"
- Depth: 7.25"
- Handle Length: 23.25"
- Handle Drop: 7"
Returns & Shipping
Luxury Garage Sale guarantees that all items sold on our site are 100% authentic. All products we accept are inspected for brand-specific guidelines and are put through a rigorous authenticity test process.
LGS is not affiliated with Louis Vuitton. We guarantee this Louis Vuitton item to be authentic. Louis Vuitton® is a registered trademark of Louis Vuitton.
Item #: 140048


text:
A Bankruptcy Brawl, Set to Music
At 76-years old, Peter Nero has two Grammys, a 50-year old recording career and 60 albums under his belt. But the man behind "Peter Nero and the Philly Pops" wants just one more thing, according to his lawyer: respect.
Paul Rosen, Nero's attorney, seized center stage in bankruptcy court Wednesday, aiming to end what he says is a history of neglect suffered by Nero at the hands of the Philadelphia Orchestra Association and carve out a piece of the non-profit's newly launched case for one of its stars.
"He should not be cast aside in this bankruptcy," Rosen declared, espousing warnings about how Nero–1961's "Best New Artist"–has been treated like "an orphan stepchild" by the orchestra.


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Goran Visnjic
Maturity Number 7
Goran Visnjic is an Actor.
He was born on Saturday September 9th 1972, in Sibenik, Croatia.
A 7 Maturity Number marks a life lived following a path that is divergent from that of others. Goran Visnjic will spend his life seeking the truth behind everything. In his later years he will continue to pursue knowledge of his inner-self and universal truths, and people will seek him out for his knowledge.
As {+FirstName+] matures, he will become increasingly occupied with the larger questions of life, reading, contemplating and searching for a deeper understanding of who he is and what this life is all about. Visnjic may turn to philosophy or religion, but will likely examine a wide array of areas, broadening his knowledge in many disciplines.
Goran's intuition grows stronger, as does his ability to look beneath the surface of every subject he studies. Goran Visnjic is able to analyze abstract questions and live according to his higher ideals.
Visnjic may find himself needing more time alone as his need for privacy increases.
If Goran already has several sevens in his chart, he will have to guard against becoming withdrawn, and alienated. However, if there are no other 7s in his numerological chart, Goran Visnjic will now have the ability to deepen his understanding of one particular field, and will have the opportunity to become grounded in a particular area of study.
You and Goran
About Goran's Maturity number
Goran Visnjic's Maturity number indicates an underlying wish or desire that gradually surfaces around age thirty to thirty-five. This underlying goal begins to emerge as Goran gains a better understanding of himself.
With self-knowledge comes a greater awareness of who Visnjic is, what his true goals in life are, and what direction he wants to set for his life.
Read more about our numerological interpretation of Goran Visnjic's maturity


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I don't want lectures about me losing weight I just want some suggestions on what I should wear this summer! I always go to stores to find clothes but it seems none of them look flattering or okay on me. Maybe I'm looking for the wrong clothes? Can anyone give me advice for fashionable clothing for a big girl?
This is what I look like. Please give me some suggestions on clothing to wear especially for this summer it's going to be hot and I don't want to cover up!
Most Helpful Guy
Hey, you have great legs, an attractive curvy figure and cute shoulders - it depends how brave you are, and to some extent how well your chest copes without a bra, but something strapless and floaty would suit you perfectly - elasticated or fitted at the bust so you get a waist (you don't want it to hang from your chest like a small tent!) but then floaty and pretty from there down. Any length from above the knee down would work, but you don't look too tall so probably a shorter dress would work best. I just googled 'strapless summer dress' and there are hundreds of great dresses that would work brilliantly for you - get shopping, be brave and enjoy! :)11|00|0
Thanks! :)
Most Helpful Girl
Look, I'm gonna be honest here, most clothes won't flatter you, because you're pretty overweight. It's not overly attractive to most people, and usually, clothes won't hide that. If you want everything to look good on you, eat better and exercise.
That being said, you can dress to hide your body type, without covering up completely. Figure hugging clothing will not be your friend. Floaty, hippy-style clothing will disguise your frame better. Think gypsy pants, floaty maxi skirts and dresses, and cute, non-restrictive tops.00|00|0
Thanks for the MHO.
I'd like to add, though, since I just saw your update, that you will never lose weight while you keep making excuses for being overweight. While hypothyroidism can make it easier to gain and harder to lose, it does NOT force you to be overweight. Nor does birth control. You have choices as to what you eat and what you do, and you've made bad ones. THAT is why you are overweight. Don't make excuses. Accept it, and if you're unhappy with it, fix it. Get treatment for your hypothyroidism, and take better care of your body.


text:
These are from the original driver disk included in the box for my Abit KD7-RAID mainboard. Unzip files to A \Win_XP and use during the Windows XP installation if needed by pressing F6 for a third party RAID controller. No clue if this will work for you, but here it is. Good luck!
|Discussion Thread
|HIGHPOINT HTP374 (also known sold as RocketRaid454) (ide controoler) (DOS) [IDE] 3 replies
|Mar 8, 2011
|Highpoint pci ide (Windows XP Professional) [IDE] 1 reply
|Mar 19, 2008
|Highpoint ATA 100 Disk Accelerator (Windows Vista) [IDE] 1 reply
|Sep 13, 2007
|Highpoint rocketraid 1740 (Linux) [PCI / ISA]
|Jul 29, 2007


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Global & European Recruitment/Staffing Market Trends, Growth Drivers, Market Dynamics, Competitive Landscape & Analysis Forecasts 2016-2018
Recruitment is the process of attracting, screening, and selecting a qualified person (from within or outside of an organization) for a job opening. The global recruitment/staffing industry comprises of recruitment or employment agencies providing for services of recruiting staff/employees for various business enterprises in different sectors. Such companies hire these employment agencies to recruit personnel to carry out their business operations. The recruitment market can be segmented into temporary and permanent staffing markets on the basis of job duration.
The Global Recruitment Market Report: 2016 Edition research of 86 pages with 69 Charts and 11 Tables, 4 company profiles to the banking and financial services industry segment of its online data and intelligence library, now available at http://www.marketreportsonline.com/447239.html.
The temporary staffing market is cyclical in nature and establishes high correlation with the global GDP. Permanent staffing market also maintains a positive relation with economic conditions. Both permanent and temporary staffing markets have a positive relation with the deregulation of labor market. The staffing market varies considerably from one country to another with the U.S., Japan and the UK showing the highest degree of fragmentation. North America represented the single largest market measured in terms of revenues followed by Japan, the UK, France and Germany.
The EU region continues to struggle with its economic revival, especially France, Spain, Greece, among others. Income growth has also come under pressure from rising unemployment, putting downward pressure on real wages in many advanced economies. With the economic revival in the coming years, sources of new employment opportunities can be expected; however, the continuing challenge in overcoming the problem of skill gap in both advanced and emerging economies will continue to affect the revenue growth of the staffing industry.
Most of the mergers and acquisitions that took place in the IT & Healthcare segment and involved middle sized staffing firms as they proved to be attractive acquisition targets, offering geographic coverage and differentiated services. The staffing industry acquisition activities remain primarily driven by privately held strategic buyers, as the larger and better known public staffing companies' account for only a limited number of transactions each year. People are increasingly using social media to network and identify employment opportunities.
Moreover, the global recruitment market is fiercely competitive and highly fragmented with numerous players vying for market share. However, consolidation in the staffing industry is ongoing, particularly in the fragmented markets and is prompted by several factors. Adecco Group is the largest company operating in this industry, followed by Randstad and Manpower. The present report profiles these four companies along with a discussion of their respective business strategies.
The report provides a comprehensive overview of the global staffing industry along with the coverage of major regional markets like North America (the U.S. and Canada), Europe (France, Germany, Italy, the UK,), and Asia Pacific (Australia and Japan), discussing the market size, segmentation, and employment/unemployment rates.
Purchase a copy of this Global Recruitment/Staffing Market research report at USD 800 (Single User License) http://www.marketreportsonline.com/contacts/purchase.php?name=447239.
By combining SPSS Inc.'s data integration and analysis capabilities with our relevant findings, we have predicted the future growth of the industry. We employed various significant variables that have an impact on this industry and created regression models with SPSS Base to determine the future direction of the recruitment market. Before deploying the regression model, the relationship between several independent or predictor variables and the dependent variable was analyzed using standard SPSS output, including charts, tables, and tests.
Major Points from Table of Contents:
Table of Contents
1. Market Overview
1.1 Recruitment
1.2 Types of Recruitment
1.3 Recruitment Strategies
1.4 Industry Definition
2. Global Recruitment Market
2.1 Global Recruitment Market by Value
2.2 Global Recruitment Market by Regions
2.3 Global Recruitment Market by Services
2.4 Global Recruitment Market by Employment Type
2.5 Global Temporary Recruitment Market by Value
2.6 Global Temporary Recruitment Market by Regions
3. North American Recruitment Market
3.1 The U.S. Recruitment/Staffing Market by Value
3.2 The U.S. Staffing Market by Segments
3.3 The U.S. Temporary Staffing Market
3.1.1 The U.S. Temporary Staffing Market by Value
3.3.2 The U.S. Temporary & Contract Staffing Market by Volume
3.3.3 The U.S. Temporary Staffing Market by Segments
3.4 The U.S. Permanent Staffing Market
3.4.1 The U.S. Permanent Staffing Market by Value
3.4.2 The U.S. Permanent Staffing Market by Types
3.5 Canada
3.5.1 The Canadian Staffing Market by Value
3.5.2 The Canadian Staffing Market by Segments
3.5.3 The Canadian Temporary Staffing Market by Segments
4. European Recruitment Market
4.1 The U.K. Recruitment Market by Value
4.2 The U.K. Recruitment Market by Segments
4.3 Recruitment Market of France by Value
4.3.1 Temporary Recruitment Market of France by Value
4.4 Recruitment Market of Germany by Value
4.4.1 Temporary Recruitment Market of Germany by Volume
5. Asia-Pacific Recruitment Market
5.1 Recruitment Market of Japan by Value
5.2 Australian Recruitment Market by Value
6. Market Dynamics
Browse All Latest Banking and Financial Services Market Report at http://www.marketreportsonline.com/cat/banking-services-market-research.html.
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text:
According to a latest report from a source verified by BGR tech blog , the 6th generation iPhone is under Engineering Verification Stage 3 – EVT3, which is the third in line revision phase of the mobile's engineering test stage. After EVT3, the new design will be put to measure in the "Design Verification Test" phase. This is the final stage after which iPhone 5 will go into mass production. The assembly of the parts have already started at Foxconn . Read here .
Update – How to make Nano Sim at Home for iPhone 5 .
Image below : 19 Pin USB Port on alleged iPhone 5 prototype
Nano Sim Card Support :
Nano SIM cards are are approximately 40% smaller than the current generation Micro SIM that are used in Apple's iPhone 4 and iPhone 4S . BGR's exclusive report confirms that iPhone 5 will indeed support Nano- Sim cards. The report also claims that multiple carriers including AT&T have received these Nano-SIM card adapters from Apple for compatibility testing on their respective networks using standard test devices before they are widely deployed when the new iPhone 5 is launched .
The BGR report also confirms that the iPhone 5 will have 1GB RAM and 4G LTE support for high-speed data transmission . The hardware is upgraded to support the high speeds .
NFC : Not Released in 2012
In the confirmation list there is a mention of NFC technology . Most high-end devices from competing vendors like Samsung have already introduced this feature on their phones . With Samsung mobile increasing its hold in the smart phone market share, this is the first real test of Apple. The iPhone has enjoyed the authority since the very first phone hit the market in year 2007. But now smart devices from Samsung is rivaling its long dominance with cheaper, high selling and built with stronger hardware than the iPhone.
Get Updates as the story develops . Stay tuned by subscribing to our News Channel below .


text:
Page 10 of 104
Ren Zhong Yuan exhibits paintings at Liangyou Books in Qingdao from January 12 to February 12, 2014.
Lazy 1st play Brit Pop at Downtown Bar in Qingdao on January 18, 2014.
Laowai gets free ride on old minibuses in Qingdao. Nice sandals.
Strong Ale brews better beer in Qingdao at La Villa on Hong Kong Road.
Download back issues of Arteffect Qingdao online before they disappear.
Old Jimo Lu in Old Town Qingdao, circa 1992, courtesy Derek Walter.
Qingdao Sculpture Park is near the Hyatt in Laoshan district off Donghai Lu.
Qingdaonese member Da Wei in Qingdao for Christmas.
When in Qingdao one is never far from the coast. Check out photos of Qingdao's coast.
CBA Basketball in Qingdao
Qingdao Metro subway Line 3 to be complete at the end of 2015.
The Qingdao Double Star Eagles shoot for improvement on last year's last place finish in the CBA when they play at home on December 6, 2013.


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Guilin Central Hostel brings a whole new meaning to hostels in China. We are the newest hostel in town and we\'ve got... Plus d'info »
Cancellation Policy: Bookings will need to be cancelled via email by 2pm of the previous day the guest is supposed to... Plus d'info »
FACEBOOK/TWITTER is accessible. Skyhome is a new hostel with a roof garden which overlooks all the beautiful scenery... Plus d'info »
A playful hostel where you can chill! Guilin Travelling With Hostel (the former Green Forest Hostel) is ideally... Plus d'info »
Guilin Riverside Hostel is an attractive property offering hotel-quality accommodation at hostel prices. We have a... Plus d'info »
Guilin 68° Hotel North Train Station Branch is just a 500-meters walk from Guilin North Train Station ,North Bus... Plus d'info »
Beer Friends Hostel is located in Guilin, distance to Guilin bus terminal 5 minutes walking distance, distance Guilin... Plus d'info »
Helen's Home With more than 10 year of experience in hospitality, having managed two luxury boutique hotels in... Plus d'info »
Relax and enjoy the scenic beauty of Yangshuo County at the Yangshuo moonlight hostel. We offer a range of spacious... Plus d'info »
CyanBox Hostel is opposite the famous Seven Star Park, which is located in NO.2 Jian Gan Road, Guilin. The location... Plus d'info »
our reception is onpen from 6:00---24:00, and only suitable for young people, students, backpackers.breakfast is not... Plus d'info »
Junshe Boutique Guest House offers comfortable accommodation in Guilin with 1 km away from Seven Star Park. It... Plus d'info »
Ease Hostel (two rivers and four lakes branch) is located in No.7 Xinyi Road,Guilin. backed by the Lijiang River is... Plus d'info »
Guilin Innsight Hostel is a a family-run hostel by the side of the beautiful Seven Star Park. The hostel, decorated... Plus d'info »
Perfectly located in the heart of Guilin, Ming Palace is located on a quiet street between famous Jinjiang Prince... Plus d'info »
L'hôtel Oasis de Guilin Le sommaire de l' hôtel : Concernant les affaires avec l'étranger, l'hôtel Oasis gère... Plus d'info »
Inside the Inn, you can enjoy our awesome bar, which is a perfect blend of Chinese and Western style. A perfect place... Plus d'info »
New Plaza Hotel is a three star joint-Venture hotel, Located in the city of the hearts of the most senior three-star... Plus d'info »
Welcome to Buddy's Hostel.it''''''''''''''''s a safe, comfortable, economical, sanitary, private and... Plus d'info »
旅舍位于城市绝对中心,榕湖之Ĭ... Plus d'info »


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Why settle for "almost the right shade" of blush? This perceptive one-shade-fits-all powder blush reacts with your skin's pH to create a one-of-a-kind, customized shade perfect for you! The ultra-blendable, velvety soft formula magically smoothes the appearance of fine lines, pores and pigmentation, leaving skin with a luminous, flawless flush*. The weightless powder can be layered to achieve just the right amount of color and coverage. * No more guessing or wearing the wrong color blush * Reacts to the wearer's pH levels to create the perfect shade every time * Harmonizes with your own skin tone to enhance your natural beauty, not overwhelm it A blush built truly for everyone, a one-of-a-kind customized color is just a brush stroke away. * The blush contains a spherical silicone elastomer treated powder that provides a soft focus effect and diffuses light. There is a silky feel leaving skin soft and smooth. The treated silicone powder fills in fine lines and wrinkles on the skin, making it appear more youthful.
stila products are non-returnable.
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Special Shipping Requirements: ORM-D Class
ORM-D classified products such as aerosol sprays, nail polish, perfume and products with high alcohol content may only be shipped by UPS Ground within the Continental US.
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text:
|Half Baths
|2,000 sq. ft.
|Property type
|Minimum Stay
|2 - 7 nights
Lovingly restored, this beautifully decorated home retains many of its original features, yet has the added conveniences you have come to expect while on vacation. Amenities include central A/C, an over-sized refrigerator, microwave, dishwasher, laundry room with washer and dryer, large flat screen TV, cable and Internet. The first floor is open and welcoming, with several areas to sit and relax. All bedrooms are upstairs, including the master 'tower room' with a queen-size bed, high ceiling and 6 tall windows to optimize the view. The grand wrap around porch is perfect for dining outside or just sipping a cool drink while watching the sunset over the ocean. There is a large backyard, a gas grill and a hot outdoor shower in back. The house has everything you need for a memorable vacation experience!
This was a great property for two families to stay. The house was in great condition and the location was good either to go to the local beach or to visit the rest of the cape. Most anything you would need was in the house, including entertainment for young children, chairs for the beach and all the kitchen utensils for cooking your own meals.
We would definitely recommend and stay here again.
Seven family members stayed four days and we all fell in love with the house the minute we opened the door. It's a lovely old home beautifully renovated and delightfully furnished. Beds were very comfortable and we all had good night sleeps.The beach is across the road, and there is a bakers, ice cream parlor and a Cumberland Farms all within walking distance. A number of towns are easily reached by car and larger stores are within 15 mins drive. Owner was great to work with. We would definitely recommend it to rent.
Would most definitely return.
A group of eight of us stayed here while attending a wedding in the area and absolutely loved it. The home is spacious and beautiful, and has an amazing wraparound porch. The master bedroom has an excellent view and lovely high ceilings. It wasn't quite cold enough for a fire but the fireplace makes for a cozy ambiance. I was a bit apprehensive at first about having only one shower for eight people, but it worked out just fine, and there are outdoor showers that can be used, weather permitting. The owners are friendly, responsive, and accommodating. There is a gas grill, the kitchen is stocked, and was great for cooking meals at home. Would definitely stay here again.
This historic home was a wonderful addition to our family vacation. The location couldn't be beat. Almost directly across from the beach. The wrap around porch was a delight in the morning. We enjoyed being able to walk to the beach, bakery, ice cream shop, pizza place, and playground. We rented a stand up paddle board and a kayak from the business behind the ice cream shop. The home was centrally located to take day trips to Plymouth, Woods Hole, and Hyannis. The owners were great to deal with and easily accessible by email. I would recommend this home to all.
We LOVED this house and location! Our Family of seven + mother in law, means eight total - had an amazing time here. We are from the Pacific Northwest and wanted to do an East Coast trip. Part of our stay included 5 nights in this lovely home.
Our children (1-10 years old) enjoyed the park and beach that are a close walk from this location. My wife who LOVES to bargain shop, had a great time visiting all the thrift shops located in the basements of old churches or small farm houses (you'll need a car and at least half a day to do this) spread around this part of the Cape.
Plymouth Rock, within an hours drive away, allowed some history to be part of this trip. We traveled down the Old Kings Hwy and went all the way to the tip of the Cape and back in one day. I'd strongly recommend Four Seas Ice Cream as some of the best I've ever had.
One of the best recommendations came from our hostess - First Encounter Beach in Eastham. At low tide the ocean literally recedes one mile out, you can only imagine what a pack of kids would think of all the shell hunting.
The pictures are a very accurate representation of the home. The house is charming and well restored. This is where we will stay if we get a chance to visit the Cape again.
This gorgeous antique home was fully renovated a few years ago. It is located in Monument Beach, a quiet, quaint area of the Cape just 5 minutes past the Bourne Bridge. Monument Beach is the perfect location to relax and get away from it all, yet still be close to everything the Cape has to offer. The house is walking distance to the public beach, tennis courts, playground, convenience store, local ice-cream shoppe, bakery and pizza restaurant. You can take a short drive to several restaurants including the Lobster Trap and the Chart Room, the canal bike path, hiking trails, mini golf, water park or golf course. Sandwich, Falmouth, Woods Hole and Ferries to the Islands are close by for short day trips.
|Cleaning Fee
First half of the payment is due to reserve the week. The second half plus security is due 60 days prior to arrival.
We accept checks and PayPal.


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STAVELOT, BELGIUM - MAY 7: (EDITORIAL USE ONLY) In this handout image provided by Red Bull, The race winning #8 Audi Sport Team Joest LMP1 car driven by Oliver Jarvis of the UK enters the pits with fellow drivers Lucas Di Grassi (top) of Brazil and Loic Duval (L) of France on the car during the 6 Hours of Spa-Francorchamps, the second round of the 2016 FIA World Endurance Championship's at Spa-Francorchamps Circuit on May 7, 2016 in Stavelot, Belgium.
(Photo : Dean Treml/Red Bull via Getty Images)
Audi News revealed that it was a devastating day for the car company as they fell behind Toyota at the WEC 6 Hours in Fuji. In fact, the carmaker even admitted that what they took to the tracks was their quickest package.
Loci Duval, the #8 car driver during the WEC 6 Hours of Fuji along with Oliver Jarvis and Lucas di Grassi lost to the #6 Toyota who finished first during the final hour.
Duval was only 13 seconds behind the winner during the final 45 minutes and came closer to 1.4 seconds before the checkered flag.
The defeat was not the carmaker's first for this year. In fact, during the Circuit of the Americas, Duval also expressed his distraught for losing with a powerhouse vehicle on the tracks.
According to Motor Sport, Duval felt the race was quite close to the finish. However, Duval was expecting the Porsche to outrun it, as news have manifested before the race even began.
In other Audi News, the Grand Prix Times reported that driver of the #6 Toyota, Kamui Kobayashi, finished the FIA World Endurance Championship first too.
Its performance during races was owed to the team's short-fueling service car and the last but one stop to move into second gave it the best chance at finishing first.
The #26 G-Drive Racing car of Will Stevens was also fighting to end the race as champion. However, Alex Brundle and Roman Rusinov claimed the team's first victory in all history of the race.
Despite the loss of Loci Duval during the WEC 6 Hours of Fuji race as was revealed by Audi news, the team was able to defeat the Porsche candidate - a development since they previously expected to be outrun by the vehicle. Despite the loss, Duval still admitted that the Toyota candidate deserved the well-earned win.


text: Editor's Note: Any reference to TheStreet Ratings and its underlying recommendation does not reflect the opinion of TheStreet, Inc. or any of its contributors including Jim Cramer or Stephanie Link. All three major indices are trading up today with the Dow Jones Industrial Average ( ^DJI) trading up 67 points (0.4%) at 16,436 as of Friday, Aug. 8, 2014, 12:55 PM ET. The NYSE advances/declines ratio sits at 1,992 issues advancing vs. 961 declining with 169 unchanged. The Real Estate industry currently sits up 0.5% versus the S&P 500, which is up 0.4%. A company within the industry that fell today was CoStar Group ( CSGP), up 0.6%. Top gainers within the industry include China HGS Real Estate ( HGSH), up 46.4%, Howard Hughes ( HHC), up 2.5%, Altisource Portfolio Solutions ( ASPS), up 2.5%, CubeSmart ( CUBE), up 1.8% and Corrections Corp of America ( CXW), up 1.5%. TheStreet would like to highlight 3 stocks pushing the industry lower today: 3. Alexander & Baldwin ( ALEX) is one of the companies pushing the Real Estate industry lower today. As of noon trading, Alexander & Baldwin is down $0.56 (-1.4%) to $38.68 on average volume. Thus far, 128,356 shares of Alexander & Baldwin exchanged hands as compared to its average daily volume of 196,800 shares. The stock has ranged in price between $38.60-$39.49 after having opened the day at $39.20 as compared to the previous trading day's close of $39.24. Alexander & Baldwin, Inc., together with its subsidiaries, primarily provides real estate development and leasing services. The company operates through four segments: Real Estate Development and Sales, Real Estate Leasing, Natural Materials and Construction, and Agribusiness. Alexander & Baldwin has a market cap of $1.9 billion and is part of the financial sector. Shares are down 6.0% year-to-date as of the close of trading on Thursday. Currently there are 2 analysts that rate Alexander & Baldwin a buy, no analysts rate it a sell, and 1 rates it a hold. TheStreet Ratings rates Alexander & Baldwin as a hold. The company's strengths can be seen in multiple areas, such as its robust revenue growth, compelling growth in net income and largely solid financial position with reasonable debt levels by most measures. However, as a counter to these strengths, we also find weaknesses including a generally disappointing performance in the stock itself, poor profit margins and weak operating cash flow. Get the full Alexander & Baldwin Ratings Report now. STOCKS TO BUY: TheStreet Quant Ratings has identified a handful of stocks that can potentially TRIPLE in the next 12 months. Learn more.


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Ok I met this girl at my universty and she's cool. And i think there is a such thing as being confident. Like for example, realistically I don't think I'm super hot like Chris Hemsworth or Chis Evans, So I would never say I'm a 10 out of 10. I'm probably like a 7/10 but I'm still confident because I won't hard to stay in shape, I work on my qualities, manners, presenation, my skills and personality.
This girl I know, she claims she's a 10/10, she thinks she has the most perfect genetics, she thinks most guys wants her, she says it all the time how much she sas it going on, how guys want her left and right, how she is so sexy and how perfect her looks are.
Am I wrong to be bothered by this? Is she just being confident or cocky?
Most Helpful Girl
I'd have no issue dating someone who's goodlooking, and is aware of that. I mean, let's be honest- most people have a good idea of where they stand in terms of physical attractiveness, whether that be high, low, or somewhere in the middle.
I'd be majorly turned off if this person straight up told me that they think they're a 10/10 (such a thing doesn't exist), that they have perfect genetics, that most of the opposite sex want them, etc..
Sounds like her ego is taking up way too much space to leave room for your being in the picture. Plus, that screams her having a very entitled approach to things.
So you can't really say that you weren't warned. :P00|00|0
"I mean, let's be honest- most people have a good idea of where they stand in terms of physical attractiveness, whether that be high, low, or somewhere in the middle."
- Except that you often have people who think they 'stand' on the side of beauty or sexiness but are really just very plain or average. And you have the ones who don't think they're special but are extremely attractive.
- Show AllShow Less
@ManOnFire I've never encountered girls or guys who were so confident that they thought they were 'god's gift'.
Most Helpful Guy
Lol we call them narshores , narcissist slutty whores.
Stay away from them, no need to burst their bubble.
Thats a serious problem though, world compliments women so much that they get big head but forget that the world compliments almost every woman. Its just being polite.00|00|0


text: Our Family Guy The Quest For Stuff Hack Tool is now available to easy download to all our loyal fans and subscribers. With our Family Guy The Quest For Stuff cheat tool you can unlock all the stuff in the game that you've always wanted but didn't really want to pay for. We feel your


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Find out how NPU can save you £1000's TODAY
Here at Nationwide Placements (UK) we have a team of highly experienced consultants who knows exactly how important delivery is. Our team will strive to deliver the right candidates in a speedy and accurate manner. We are fully transparent and we promise simplicity from the start to the end of the recruitment process.
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This qualification is aimed at anyone working, or wishing to work, as a safety steward at live events, such as sporting or musical events.
The qualification also provides a mandatory component of the Intermediate Apprenticeship in Spectator Safety and meets the requirements laid out in the Football Licensing Authority's Guide to Safety at Sports Grounds (known as the Green Guide), along with other related safety guides.
Planned start dates for this course are in:
Bespoke training dates are also available on request.
You are required to have Level 1 Maths and English or equivalent qualifications, plus good communication skills and a commitment to the role.
This qualification provides an assessment of knowledge and competence across the following areas:
Because this is a competency-based qualification, the majority of evidence will be gathered through learner observation, products of work and questioning. However, for some units contained within the qualification, simulation is permitted.
There are no additional costs involved.
You can utilise this certificate to work as a qualified and competent live event steward. However, if you wish to continue your educational development, the following qualifications may be of interest:
Derby College offers a range of courses relating to the security industry. Please see our prospectus for full listings and details.
* Tuition - This figure is the tuition fee to be paid - There are no concessionary fees available.


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Kristina24. Kiev, Ukraine
- 126 lb - 57 kg
- 5'9" - 175 cm
- light brown hair
- brown eyes
- I don't drink
- I don't smoke
- 07/04/1993
- Christian
- still studying
- Model and student
- I don't speak English
- I'm single
- I don't have children
I am very purposeful, kind, persistent person. I like sport. I practiced in the past judo and kickboxing, so I can protect us if it will be necessary! Kiding.. I think that my man will protect us! Constantly working on myself. I like to read a books about the life and psychology.I decide to try this new dating site, because i am one of that ukraine mates that want to get married with a serious man!
I like to read books, i go to dance courses and i like to do sport
He must be very handsome like Apolon!I am looking for kind, communicative, serious, responsible man who can laugh about the problem and not make it complicated but to solve at once. And who will choose me from all single ladies that are in the world! Caring and simple man who desires to love and be loved:)


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
How do I fix a height issue on the custom thank you page?Asked by opencrm on January 12, 2015 at 05:04 AM
I have a form which uses a custom thank you page. The height of the iframe works fine through out the form, but on the custom thank you page, it cuts off before the thank you message ends. I have tried shortening the message, but the height of the page seems to be responsive and still cut off too soon.
The iframe height in the embed code is huge (2550px) which is not the same as the actual height of the iframe, so I am quite puzzled as to where this height is being read from?
Could you please advise me on how I can fix this issue please?
I found the answer to this on the forum at: http://www.jotform.com/answers/492493-Bottom-Part-of-the-Thank-You-Message-is-Getting-Cut-Off.
Thank you
JotForm Support
Hello opencrm,
I am glad to know that you found the answer your self. I hope that your issue is resolved.
Do get back to us if you have any questions.
Thank you!
Hi Paul,
You can also integrate all contacts by using a crm software. You can try our best crm solution uk. Give me a shot to help you.
JotForm Support
Hi, I have opened a separate thread for your concern. Please refer to this link instead: https://www.jotform.com/answers/976222. Thank you.


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Disclosure: This post was provided by eAccountable and contains affiliate links that may yield commissions earned at no cost to you.
I am not one to rush Christmas and skip the holidays and important moments in the months leading up to it. However, more than once this "live in the moment" attitude has left me spending a premium at the last minute to have Christmas cards made and sent out. In fact, I've completely skipped it a couple of years because the stress of rushing it just wasn't worth it.
That's why this year I put ordering Christmas cards on my calendar early. And since my main goal here on Wondermom Wannabe is to help you learn from MY mistakes, I'm posting this as a reminder to you to add it to your list too. I'm even incentivizing this task by sharing a great offer on Christmas cards from MagnetStreet. From now until 11/20/15, you can save 25% off holiday cards with code LHOLIDAY25.
Starting your own holiday tradition can begin with a beautiful, custom-designed holiday greeting from MagnetStreet. Connect with family and friends (colleagues, too) with a card that speaks to the season and is easy to personalize. Browse beautifully designed layouts and then change the colors to fit your style or theme (even custom colors). Your photos will print with crystal clarity and you can word your card just the way you want!
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Corinne Schmitt
Latest posts by Corinne Schmitt (see all)
- Strawberry Stuffed Pork Loin with Sweet Strawberry Mashed Potatoes - August 23, 2017
- 15 Fun Educational Activities for Kids - August 22, 2017
- Lemon Ricotta Waffles with Mascarpone and Blueberries - August 21, 2017


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As you saw in Flop's daily update (Luke Warm Linkage), it is Video Friday. And there's nothing else like a political nut to add to the deliciousness that is Video Friday.
In every election cycle, we get a few crazies who end up garnering a lot of attention via blogs and John Stewart's The Daily Show. Well, the 2010 Gubernatorial Election in Tennessee brings us the next great office-seeking psycho: Basil Marceaux.
Such a name immediately reminds me of creole Louisiana, but unfortunately this guy is running for the governor's office of my beloved home state of Tennessee.
Since we are the Volunteer State, we would not want to keep Basil Marceaux away from the rest of the country. Enjoy (and hopefully you can understand his ramblings).
In general:
On gun rights:
And for more of the above, visit his website found here. Basil Marceaux 2010.


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Urjanet's utility data platform deploys Bots that visit the world's largest global network of utilities to collect every data point on all of your utility bills.
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Related Resources


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Chappy here, coming to you live from Athens, Greece. I know the insurance company (and my mother) have told me never to reveal the fact that I am out of town, but nobody reads this blog and (note to would-be burglars) my cat loves the taste of stranger-flesh.
Anyway, it's been a while, and I just felt like sharing thoughts, so here is Chappy's big ballin' review of Athens:
First off, both of my parents (separately, I might add) have reminded me that this was the last place my Grandfather visited before he passed away. Since he was too ill from late-stage cancer, Ed had to take a taxi to all the sites and peer out the window. This, as you might imagine, adds a little gravity to scaling the hill up to the Acropolis and looking out over one of the world's most historically significant cities. Throw in the fact that he was a) the primary genetic source of any humor I may have and b) apparently part of why I am now noticing the onset of male-pattern baldness, and it really hits home. In his honor, I want to throw up a warm dedication to the incomparable Edwin Leroy Chapman.
I'm Surrounded by Cats
Any other travel guide might start out by mentioning the history, the unibrows, important Olympic tie-ins. You know what the most obvious thing to my untrained eye was? Cats, man. There are stray cats everywhere. We had lunch the other day (inside a building) and there was a black cat at the foot of our table. At first, it was amusing, as I fancy myself somewhat of an animal lover, but dadgummit, there could not be more unattended felines in this city. If you want a free cat, drop me a line and I'll bring one back for you.
If They Hate Americans, I Blame Snooki
Due to jetlag, I was up much later on Monday than a Chappy should ever be. I turned on the television, and what to my wondering eyes should appear – the first ever episode of Jersey Shore. Imagine what a Greek person thinks when they read the translation of "I just want to hook up with hot Guidos" on the bottom of the screen and see an orange midget bouncing around the boardwalk. Hey John Kerry, you want to improve our foreign relations? Tell MTV to shut it down…
The Greeks Will Work it for a Euro
One of my British friends set the bar high by declaring I would live "like a King" in Greece. True to form, I'm pretty much impressed with every bill I get (outside of the Hilton). If you have weathered the Euro or Pound elsewhere, a five-course dinner and wine for 25€ is legit. As soon as I speak, an English menu is delivered. The people are also quite friendly, despite their pissed-off disposition. No joke, I haven't seen a person smile yet without provocation.
Cops are Everywhere
Confession: I jaywalked earlier…but only after 5 other people went first. The police-to-civilian ratio has to be closer to 1:1 than anywhere I have ever been. There are so many cops that they must share motorcycles. Imagine getting pulled over by one motorcycle with two cops on it (wearing protective armor no less). The other day, we were walking through a major park and a police dog tried to eat our faces off through the bars of his nearby cage. This (and the 4 officers smoking nearby) merely confirmed that we had better not exercise any unwelcome freedoms. Point made: I would recommend not rioting in Athens if you can help it.
Greek Food is Delish
I've been to many a place that holds back on spices in an effort to not offend discerning patrons. Not here. Put that spice on that meat and get it into my belly as quickly as possible! I'm telling you – if you like assorted meats and a flavorful mouth journey, then hop a plane to this part of the world. Double bonus, they deliver plates of food with all the ingredients (you foodie types might call this "deconstructed") which means you get to make your own personalized mini pita sandwiches. Thumbs in the air, and pass that taziki sauce!
All Ruins Need a "Before" Pic
I love me some history. It has always been one of my best subjects. But I also do a bit of sales in my day job, and if you want to put the historical "spin" on a pile of stones, give me a high quality picture and a backstory of what I'm looking at. To give you an example, below you will find the current state of Zues' house. Ummm…who drank too much and knocked down all the columns?
So there you have it. Thinking of heading to Greece? Give me a shout and I'll offer up an even more comprehensive review.


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Introducing Premium T-shirts. The wearable canvas art deserves.
Newcastle Nsw Australia.Nikon D7000Sigma 10-20mm @ 20mm.(please view large)
newcastle baths sunset newcastle nsw
nice one mate. up to the big smoke then?
Hey Bazza,Went out to Wollembi pub for lunch & come home via Newi..thanks mate.
Cool reflections there mate. Nice work.
Outstanding capture Waz:o)
Thanks Mark..Im starting to think Im the only Aussie on RB that hasnt been down the Cradle Mt..cheers mate.
Nice reflections, lines and tones Waz…..well done !!
Thanks Ian..had the ISO on 400 from an early shoot at Terrigal the morning before..live & learn.lol.
Cool great reflections and colour!
Those chains are magic for adding interest to a shot – look at the reflections!! Love it Waz. Well done. xx
Nice work Wazza, love the reflection and light!
Hey Simone, many thanks.
Time to let the moths out of the wallet and bring Julie down for a holiday Waz. If not for this years Fagus week, plan on next year. Bring Baz with you as well…..sounds like he doesn't mind a beer:o)
See what I can do,pretty cheap flights at the moment..maybe winter ! I"ll bring some port .just about to have one ..hope Claire,you & your family are well..CHEERS.
Very nice image Warren. I really like the reflections and the curvature.
G"day Hany..thanks very much mate..cheers.
Beautiful image Warren♥
Thanks Bev.
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Mitt Romney Seems To Be Holding Up
"Is that an A&W? I will suck your fucking dick…"
reddit user mbk95 captured this pic of an apparently security-less Mitt Romney pumping gas in La Jolla last night where he's been holed up since the election. But before you go accusing him of looking like a man who just finished axe-murdering his… More »


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Welcome to the second showcase of Moonlight Studios. This game is Paper Mario: Cardboard Kings, for the Wii U.
The game will be ver similar to its predecessors in terms of appearence and gameplay, but introduces new ideas and concepts.
- Some enemies, and most bosses, will be made from cardboard, which is much tougher than paper.
- New playable characters such as Rosalina, Toadette and Baby Mario.
- The Koopalings make an appearance for the first time in the Paper Mario series, as Paper and Cardboard Bosses.
- There will be new partners including: -
- Sir Stingley, who is based off of a Stingby, can help the character fly.
- Burrows Jr., who is based off of Major Burrows, can help the player dig.
- and Luma. who is based off of the Luma who helps Mario in Super Mario Galaxy 2, can help the player by creating sling stars and making the player shoot them in a certain direction.
- Each character has different stats, which can effect how they perform in the game (e.g: how easy they defeat enemies, how fast they go ect.)
- There will be many new locations to the series, including the main hub world: Boxborough.
- Each location will have one boss, and one to two mini-bosses, excluding the final world; which has four Mini Bosses, one Boss, and one final boss.
Thanks for reading, Have aVery MerryChristmasFromBaby Yoshi.


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7th heaven theme song MP3
Theme song from the television show 7th Heaven.
7th heaven theme song MP3
7th Heaven, Betch MP3
acoustic performance of the classic 7th heaven theme song brought to you by lindsay and katie mourand.
ALBEDO- Dancing on Mercury (7th HEAVEN) MP3
Coldplay - Hymn For The Weekend (Official video) MP3
The second single to be taken from Coldplay's acclaimed new album, A Head Full Of Dreams (out now). Download the song from http://smarturl.it/AHFOD or ...
Sidewalk - 7th Heaven MP3
Sidewalk on Facebook: https://www.facebook.com/sidewalk.symbiosis Written and performed by Sidewalk / $tr8 "Outta Control" Instrumental taken from "Allure" ...
Mary-Jess - Glorious (7th Heaven Club Mix) [HQ] MP3
Mary-Jess - Glorious (7th Heaven Club Mix)
7th heaven season finale MP3
this is my very first video ever so sorry for the shitty quality.. it's scenes from the series finale of 7th heaven.
7th Heaven- This Summers Gonna Last Forever MP3
No copyright is intended. This video is simply for entertainment purposes. All material belongs to it's rightful owner(s). Artist: 7th Heaven Album: U.S.A.-U.K. Year: ...
7th Heaven - 30 songs in 30 minutes part1 - Schaumburg Septemberfest 09-05-2011 MP3
7th Heaven - 30 songs in 30 minutes part1 - Schaumburg Septemberfest 09-05-2011.
7th Heaven - She MP3
Jane McDonald-One Night Only-7th Heaven remix-video edit MP3
The Fabulous Dreamgirls track remixed by seventh Heaven..
The White Stripes - 'Seven Nation Army' MP3
Directed by Alex & Martin http://www.whitestripes.com http://www.xlrecordings.com.
7th Heaven Dear Lord Let It Be Song MP3
A great song from a wonderful singer.
7th Heaven (Inspired by Final Fantasy 7) MP3
A song I came up with, inspired by the Playstation game Final Fantasy VII. I'm quite proud of it, especially since I'm not the best at piano. (I can't read music worth ...
MHD - 7th Heaven MP3
Lyrics & performance by M.H.D. Beat copyrights go to Rapitfly Beats. --- [Bridge] Face to face, chest to chest, lip to lip, now that's hot I don't wanna wait or a date, ...
7th Heaven- Rock Medley MP3
I DO NOT OWN ANYTHING*** I am a fan of this band, and instead of listening to 3 separate parts of the medley, I created one long version. ***I DO NOT OWN ...
Kara no Kyoukai - Shiki's Theme Compilation MP3
Garden of sinners - Kara no Kyoukai - Shiki's Theme Compilation I compiled about 6 or 7 different variations of shiki's theme from Garden of Sinners. Only 1 ...
Gwen Guthrie - Seventh Heaven MP3
Gwen Guthrie - Seventh Heaven (extended mix) 1983 on Fourth & Broadway Records Funk/ Soul.


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Dogeared X Frankies Bikinis - Exclusive Collaboration
The versatile gold Solstice belly chain with diamond shaker accents can be styled a variety of ways and can be worn as a long necklace, double strand choker, belt, or belly chain. The perfect pieces for all your Wanderlust adventures.
- Adjustable spring ring closure
- 30" chain + 2" extender
- Chain Sterling Silver 14 karat gold dipped
- Jump rings 3mm gold filled
Care Instructions
To keep your jewels as bright and amazing as you are we have provided some cleaning tips. Your gems might tarnish faster if you wear them while you're working up a sweat, enjoying the ocean waves, or sometimes if they bathe in your favorite perfume or oils. But no worries! With these tips your jewels will be ready to shine with you, wherever your story takes you! Keep your jewelry beautiful!


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Tag Archives: Paws
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text: A couple of days ago i was low on memory so i decided to take the plunge and root my device with a custom rom and take advantage of apps2sd+ This all worked out fine and gave me the the space i needed. The phone was showing around 140mb of free space with most of my apps installed and from titanium backup i could see that the partition was being used. However after a couple of days i have noticed that my internal memory is back down to 16mb. I have cleared the caches and dalvik cache. The only thing i have done since the change in memory is move my sim contacts to the phone, moved the apps that can be moved to my sd card and installed a few new apps. Is there an app i can use to see what is taking up all the memory? if not how can i rectify this problem?