Co-staining of calreticulin with APP and presenilin at the cell surface of live hippocampal neurons.

<p>Live hippocampal neurons were incubated with goat calreticulin antibody T-19 recognizing the N-terminus (CRT-N) (<b>A, D, F</b>) or C-17 recognizing the C-terminus (CRT-C) (<b>B, C, F</b>) and with rabbit APP antibody A8967 directed against the N-terminus (<b>A, B</b>), rabbit L1 (<b>C</b>) or calmodulin (CaM) (<b>D</b>) antibody. After fixation, cells were incubated with the calreticulin antibodies (<b>E</b>) or the rabbit presenilin antibody 2953 and fluorescent-labeled secondary antibodies. Superimposition of immunostainings (merge) shows co-localization of calreticulin and APP (yellow). Phase contrast shows the cellular structures. Scale bar, 5 µm.</p

Calreticulin affects the production of Aβ₄₀ and Aβ₄₂.

<p>(<b>A, B</b>) CHO cells overexpressing APP were mock-transfected (mock) or transfected with constructs encoding the P-domain (P), the N-domain (N) or the N- and the P-domain (NP) of calreticulin or full-length calreticulin (CRT). (<b>B</b>) Transfected cells were subjected to cell surface biotinylation, biotinylated proteins were isolated and Western blot analysis of cell lysates and biotinylated cell surface proteins with the APP antibody A8717 was performed. (<b>C</b>) CHO cells overexpressing APP were incubated with GST-calreticulin (CRT), GST/C-domain (C) or GST/P-domain (P). (<b>D</b>) Amino acids 706–710 of APP comprising the γ-cleavage site for generation of Aβ₄₀ and Aβ₄₂ the sequence of the corresponding antisense peptide and the inverted sequence of the antisense peptide are shown. Similarities between the inverse antisense peptide sequence and amino acid 330–344 of calreticulin are indicated (bar, colon and dot represent identical, highly conserved and weakly conserved amino acids). NT: N-terminus; CT: C-terminus. (<b>E</b>) Label-free binding assay using substrate-coated Aβ₄₀ or Aβ₄₂ peptides and soluble calreticulin peptide comprising amino acids 330–344 (CRT pep) or antisense peptide (anti pep). Mean values ± SD from triplicates of a representative experiment are shown. (<b>F</b>) GST/C-domain fusion was incubated with a detergent extract of mouse brain homogenate in the absence (-) or presence of calreticulin peptide (CRT pep) or antisense peptide (anti pep). Proteins bound to the GST/C-domain were precipitated with glutathione-agarose (pull-down) and subjected to Western blot analysis with the APP antibody A8967. Aliquots taken from the samples before precipitation were used as input control. (<b>G</b>) CHO cells overexpressing APP were incubated in the absence or presence of the calreticulin peptide (CRT pep) or antisense peptide (anti pep). (<b>A, C, G</b>) Cell culture supernatants were collected and the amounts of Aβ₄₀ and Aβ₄₂ in cell culture supernatants were determined by ELISA. The levels obtained after mock-transfection were set to 100%. Mean values ±SEM from 3 independent experiments with biological duplicates per experiment are shown (Students t-test * p>0.05, ** p>0.005).</p

Co-immunoprecipitation of calreticulin and APP.

<p>(<b>A, B</b>) A synaptosomal fraction was subjected to immunoprecipitation using the rabbit antibody CRT283 against calreticulin (CRT) (<b>A, B</b>), the rabbit APP antibody WO2 (APP) (<b>A</b>) or B63-4 (<b>B</b>) or non-immune control rabbit antibody (rIg) (<b>A, B</b>). (<b>C</b>) CHO cells were mock-transfected or transfected with a construct encoding the C99 APP stump and used for immunoprecipitation with the goat calreticulin antibody C-17 (CRT), the rabbit APP antibody ED-APP (APP) or non-immune control goat antibody (gIg). (<b>A, B, C</b>) Immunoprecipitates were subjected to Western blot analysis using the APP antibody 22C11 (<b>A</b>), the goat calreticulin antibody C-17 (<b>B</b>) or the rabbit polyclonal APP antibody B63–4 recognizing the C99 APP stump (<b>C</b>).</p

Binding of calreticulin to the synthetic peptide containing the γ-cleavage site of APP.

<p>Recombinant calreticulin (<b>A</b>) or synthetic biotinylated peptides comprising the γ-secretase cleavage site (APP- γ) or the β-secretase cleavage site (APP-β) (<b>B</b>) were coated as substrates and incubated with different concentrations of biotinylated APP-γ or APP-β peptides (<b>A</b>) or recombinant calreticulin (<b>B</b>). Detection of bound proteins was carried out using HRP-coupled streptavidin (<b>A</b>) or the rabbit calreticulin antibody CRT283 and HRP-coupled secondary antibodies (<b>B</b>). Mean values ± SD from three independent experiments are shown.</p

The γ-secretase activity is altered by calreticulin and the P-domain of calreticulin.

<p>Immunoprecipitated γ-secretase was incubated with C100 APP stump as substrate and GST or the GST-fusion protein with full length calreticulin (GST-CRT) or the P-domain of calreticulin (CRT-P) in the absence (<b>A-C</b>) or presence of the γ-secretase inhibitor DAPT (<b>C</b>). Aβ was detected by Western blot analysis using the rabbit antibody 2D8 (<b>A</b>) and the amount of Aβ was determined by densitometry (<b>B</b>). (<b>C</b>) Western blots showing uncleaved C100 substrate and Aβ in the presence of GST and GST-P and in the absence or presence of DAPT. Mean values ±SD are from 4 experiments (Students t-test * p>0.005, ** p>0.0005).</p

Co-localization of APP and calreticulin in primary cultures of live hippocampal neurons.

<p>(<b>A</b>) Hippocampal neurons maintained for one week in culture were incubated with the rabbit APP antibody WO2 (green). After fixation, the neurons were immunostained with the goat calreticulin antibody C-17 (red). Superimposition of immunostainings (merge) shows co-localization of calreticulin and APP (yellow). Phase contrast shows the cellular structures. Scale bar, 10 µm. (<b>B</b>) Cell surface biotinylation of HEK cells overexpressing wild-type (WT) or mutated (mut) presenilin. Biotinylated surface proteins were isolated and subjected to Western blot analysis with the rabbit APP antibody B63-4, the goat calreticulin antibody C-17 (CRT) or an actin antibody. Cell lysates were used as input control.</p

Photoexpulsion of Surface-Grafted Ruthenium Complexes and Subsequent Release of Cytotoxic Cargos to Cancer Cells from Mesoporous Silica Nanoparticles

Ruthenium­(II) polypyridyl complexes have emerged both as promising probes of DNA structure and as anticancer agents because of their unique photophysical and cytotoxic properties. A key consideration in the administration of those therapeutic agents is the optimization of their chemical reactivities to allow facile attack on the target sites, yet avoid unwanted side effects. Here, we present a drug delivery platform technology, obtained by grafting the surface of mesoporous silica nanoparticles (MSNPs) with ruthenium­(II) dipyridophenazine (dppz) complexes. This hybrid nanomaterial displays enhanced luminescent properties relative to that of the ruthenium­(II) dppz complex in a homogeneous phase. Since the coordination between the ruthenium­(II) complex and a monodentate ligand linked covalently to the nanoparticles can be cleaved under irradiation with visible light, the ruthenium complex can be released from the surface of the nanoparticles by selective substitution of this ligand with a water molecule. Indeed, the modified MSNPs undergo rapid cellular uptake, and after activation with light, the release of an aqua ruthenium­(II) complex is observed. We have delivered, in combination, the ruthenium­(II) complex and paclitaxel, loaded in the mesoporous structure, to breast cancer cells. This hybrid material represents a promising candidate as one of the so-called theranostic agents that possess both diagnostic and therapeutic functions

Photoexpulsion of Surface-Grafted Ruthenium Complexes and Subsequent Release of Cytotoxic Cargos to Cancer Cells from Mesoporous Silica Nanoparticles

Ruthenium­(II) polypyridyl complexes have emerged both as promising probes of DNA structure and as anticancer agents because of their unique photophysical and cytotoxic properties. A key consideration in the administration of those therapeutic agents is the optimization of their chemical reactivities to allow facile attack on the target sites, yet avoid unwanted side effects. Here, we present a drug delivery platform technology, obtained by grafting the surface of mesoporous silica nanoparticles (MSNPs) with ruthenium­(II) dipyridophenazine (dppz) complexes. This hybrid nanomaterial displays enhanced luminescent properties relative to that of the ruthenium­(II) dppz complex in a homogeneous phase. Since the coordination between the ruthenium­(II) complex and a monodentate ligand linked covalently to the nanoparticles can be cleaved under irradiation with visible light, the ruthenium complex can be released from the surface of the nanoparticles by selective substitution of this ligand with a water molecule. Indeed, the modified MSNPs undergo rapid cellular uptake, and after activation with light, the release of an aqua ruthenium­(II) complex is observed. We have delivered, in combination, the ruthenium­(II) complex and paclitaxel, loaded in the mesoporous structure, to breast cancer cells. This hybrid material represents a promising candidate as one of the so-called theranostic agents that possess both diagnostic and therapeutic functions

Photoexpulsion of Surface-Grafted Ruthenium Complexes and Subsequent Release of Cytotoxic Cargos to Cancer Cells from Mesoporous Silica Nanoparticles

Ruthenium­(II) polypyridyl complexes have emerged both as promising probes of DNA structure and as anticancer agents because of their unique photophysical and cytotoxic properties. A key consideration in the administration of those therapeutic agents is the optimization of their chemical reactivities to allow facile attack on the target sites, yet avoid unwanted side effects. Here, we present a drug delivery platform technology, obtained by grafting the surface of mesoporous silica nanoparticles (MSNPs) with ruthenium­(II) dipyridophenazine (dppz) complexes. This hybrid nanomaterial displays enhanced luminescent properties relative to that of the ruthenium­(II) dppz complex in a homogeneous phase. Since the coordination between the ruthenium­(II) complex and a monodentate ligand linked covalently to the nanoparticles can be cleaved under irradiation with visible light, the ruthenium complex can be released from the surface of the nanoparticles by selective substitution of this ligand with a water molecule. Indeed, the modified MSNPs undergo rapid cellular uptake, and after activation with light, the release of an aqua ruthenium­(II) complex is observed. We have delivered, in combination, the ruthenium­(II) complex and paclitaxel, loaded in the mesoporous structure, to breast cancer cells. This hybrid material represents a promising candidate as one of the so-called theranostic agents that possess both diagnostic and therapeutic functions