7 research outputs found
GM‑3 Lactone Mimetic Interacts with CD4 and HIV‑1 Env Proteins, Hampering HIV‑1 Infection without Inducing a Histopathological Alteration
Glycosphingolipids (GSLs) are involved
in HIV-1 entry. GM-3 ganglioside, a widespread GSL, affects HIV entry
and infection in different ways, depending on the concentration, through
its anchoring activity in lipid rafts. This explains why the induction
of an altered GSLs metabolism was a tempting approach to reducing
HIV-1 cell infection. This study assayed the biological properties
of a synthetic GM-3 lactone mimetic, <b>1</b>, aimed at blocking
HIV-1 infection without inducing the adverse events expected by an
altered metabolism of GLSs in vivo. The mimetic, conjugated to immunogenic
protein ovalbumin and multivalently presented, was able to bind the
CD4 molecule with high affinity and block its engagement with gp120,
thus inhibiting virus entry. Elicited antimimetic antibodies were
also able to block HIV-1 infection in vitro, with activity complementary
to that observed for <b>1</b>. These preliminary results show
that the use of GSLs mimetics can be a novel promising mode to block
HIV-1 infection and that <b>1</b> and other GSL mimetics deserve
further attention
Western blot analysis on cellular proteins separated into nuclear and cytoplasmic fractions from sNF96.2 cells.
<p><b>A</b>: Whole cell lysate (W), cytosolic (C), and nuclear (N) fractions were immunoblotted with C-19 WT1 antibody. <b>B</b>: Results were expressed as optical density (O.D.). *p<0.05 relative to whole cell lysate.</p
Time-course of siWT1 on sNF96.2.
<p>Western blot analysis on cells treated with 50 nM p-siWT1 (<b>A</b>) or s-siWT1 (<b>B</b>), compared to those treated with scrambled siNEG. siWT1 results were expressed as fold change compared to siNEG ones (<b>A1</b> and <b>B1</b> for p-siWT1 and s-siWT1, respectively). *p<0.05. <b>C</b>: Staining of WT1 protein in sNF96.2 cell treated with 50 nM siNEG, p-siWT1 or s-siWT1. Scale bars: 20 µm.</p
siWT1 on cell proliferation.
<p><b>A</b>: Effects of 25 nM and 50 nM p-siWT1 and s-siWT1 on sNF96.2 cell proliferation at 48 and 72 hours. Data represented the average value of three independent transfection experiments. *p<0.05 compared to siNEG ones at same time. <b>B</b>: Growth curves of sNF96.2 cells treated with 25 nM and 50 nM siNEG, p-siWT1 or s-siWT1.</p
WT1 expression in sNF96.2 cells determined by immunocytochemistry.
<p>Immunofluorescence positive cells to 6F-H2 (<b>B</b>) and C-19 WT1 (<b>E</b>) antibody are merged (<b>C</b> and <b>F</b>, respectively) with their own DAPI stained nuclei (<b>A</b> and <b>D</b>). Scale bars: 50 µm.</p
siWT1 on apoptosis.
<p>Effects of siWT1 on cleaved caspase-3 expression in sNF96.2 cells treated with 50 nM for 48 and 72 hours determined by Western blot analysis. As a positive apoptosis control (PC), cell lysate of human fibroblasts exposed to 0.1 mM of staurosporine for 24 hours was used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114333#pone.0114333-Johanssonn1" target="_blank">[83]</a>.</p
siWT1 on cell cycle.
<p><b>A</b>: Effects of siWT1 on PI3K/Akt/Cyclin D1 pathway in sNF96.2 cells treated with 50 nM p-siWT1 for 48 and 72 hours determined by Western blot analysis. <b>B</b>: Results were reported as fold change compared to siNEG ones (*p<0.05).</p