Pb, Sr and Ba calix[6]arene hexacarboxylic acid octahedral complexation: a dramatic effect of dealkylation

<div><p>Calix[6]arene hexacarboxylic acid binds instantly and with low symmetry to Pb, Sr and Ba. Later a highly symmetric up-down alternating conformation emerges. The solution structures are identical to their p-tert-butylcalix[6]arene hexacarboxylic acid counterparts. With either receptor, an octahedral cage is formed around the metal. The transformation from low to high symmetry however proceeds at significantly faster rates for the de-t-butylated host.</p></div

Model of apoE3 containing rHDL resveratrol embedded in the hydrophobic milieu.

<p>The phospholipid bilayer with embedded resveratrol is surrounded by the amphipathic α-helices of apoE3.</p

Uptake of rHDL/res by glioblastoma cells.

<p><b>A</b>. <b>Representative confocal images of glioblastoma cells showing uptake of rHDL/res.</b> Uptake of individual components of rHDL/res was monitored by direct or indirect immunofluorescence: lipid (<i>a-c</i>), apoE3 (<i>d-f)</i> and resveratrol (<i>g-i</i>). Following exposure to rHDL/res/DiI at 37°C for 3h (<i>a-c</i>), the cells were visualized under a confocal laser scanning microscope: <i>a</i>, DAPI; <i>b</i>, DiI; <i>c</i>, merge of <i>a</i> and <i>b</i>. Following exposure to rHDL/res under same conditions (<i>d</i>—<i>f</i>), the cells were visualized by: <i>d</i>, DAPI; <i>e</i>, apoE3 monoclonal antibody, 1D7, and Alexa555-conjugated secondary antibody; <i>f</i>, merge of <i>d</i> and <i>e</i>. Following exposure to rHDL/res/NBD (5 μg) as described above (<i>g</i>—<i>i</i>), the cells were visualized by: <i>g</i>, Propidium iodide; <i>h</i>, NBD; <i>i</i>, merge of <i>g</i> and <i>h</i>. Panel <i>j</i> shows that uptake of res/NBD in the absence of rHDL. <b>B. Co-localization of res/NBD with apoE3, or with LAMP1 in late endosomal/lysosomal vesicles following cellular uptake of rHDL/res/NBD</b>. Following exposure to rHDL/res/NBD, the cells were visualized by fluorescence associated with: <i>a</i>, NBD to detect res, <i>b</i>, Alexa555-conjugated secondary antibody to detect apoE3; <i>c</i>, merge of <i>a</i> and <i>b</i>; <i>d</i>, NBD to detect res; <i>e</i>, Alexa 594-conjugated secondary antibody to detect LAMP1; <i>f</i>, merge of <i>d</i> and <i>e</i>.</p

Effect of resveratrol on LDLr binding activity of apoE3.

<p>rHDL, rHDL/res or rHDL/res/NBD (10 g protein) was incubated with 10 μg of sLDLr, followed by co-IP with anti-c-Myc-Agarose. sLDLr-bound apoE was detected by Western blot using HRP-conjugated polyclonal apoE antibody (<b>A</b>). The corresponding blot using anti-c-Myc antibody is shown for comparison (<b>B</b>). The lane assignments are: Lane 1, rHDL; lane 2, rHDL/res; lane 3, rHDL/res/NBD.</p

Characterization of rHDL and rHDL/res particles.

<p><b>A & B. Transmission electron microscopy</b>. Negative staining of rHDL (<b>A</b>) and rHDL/res (<b>B</b>) was carried with 10 μg protein. The bar represents 20 nm. Arrows draw attention to discoidal particles. <b>C. Non-denaturing PAGE</b>. rHDL and rHDL/res were electrophoresed on 4–20% acrylamide gradient gel. The far left lane bears the high molecular mass standards; the molecular mass and their corresponding Stokes’ diameters are indicated; lane 1) rHDL, and lane 2) rHDL/res. Arrows draw attention to particle heterogeneity in lane 1.</p

Quenching analysis of rHDL/res.

<p>rHDL/res (10 μg protein) was treated with increasing concentrations of KI in PBS (<b>A</b>) or 16-DSA in DMSO (<b>B</b>), and the fluorescence emission intensity recorded at each concentration. Data are plotted as F/F₀ versus quencher concentration. Representative data from 3 independent experiments are shown.</p

Synthesis scheme and fluorescence emission spectra of res/NBD.

<p>To visualize cellular uptake of res, res/NBD was synthesized (<i>Top</i>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135130#pone.0135130.s001" target="_blank">S1 File</a>. Purified res/NBD was incorporated into rHDL as described under <i>Methods</i>. Comparison of the fluorescence emission spectra (<i>Bottom</i>) of res/NBD (a) with rHDL/res/NBD (b) shows a blue shift in λ_max of NBD, indicative of incorporation of res/NBD in the lipid milieu of rHDL.</p

Spectra characteristics of rHDL and rHDL/res.

<p>(<b>A</b>) The absorbance spectra of pooled fractions of rHDL (a), rHDL/res (b), resveratrol in DMSO (c) and res/NBD in DMSO (d) were recorded. (<b>B</b>) Fluorescence emission spectra of resveratrol were recorded in: water (<b>a</b>), DMSO (<b>b</b>), 95% ethanol (<b>c</b>), isopropanol (<b>d</b>), and ethyl acetate (<b>e</b>) with dielectric constants of 80.4, 33.0, 24.3, 7.5 and 6.0, respectively, following excitation at 310 nm. (<b>C</b>). Fluorescence emission spectra of rHDL (<b>a</b>) and rHDL/res (<b>b</b>) were recorded with 10 μg protein in PBS. For comparison, emission spectrum of resveratrol in DMSO is shown (<b>c</b>).</p

Confocal microscopy images of LDLr-mediated cellular uptake of rHDL/res.

<p>Glioblastoma cells were treated with rHDL/DiI (<i>Top</i>) or rHDL/res/DiI (<i>Bottom</i>) in the absence (<i>a</i> and <i>d</i>) or the presence of 50-fold excess LDL (w/w) (<i>b</i> and <i>e</i>) or 2 mM suramin (<i>c</i> and <i>f</i>) at 37°C. for 3 h. The cells were visualized by the DiI fluorescence associated with lipids.</p