Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
Supramolecular biovectors (SMBV) are nanoparticular drug carriers composed of an internal crossli... more Supramolecular biovectors (SMBV) are nanoparticular drug carriers composed of an internal crosslinked solid core externally grafted with fatty acids and surrounded with a phospholipid layer. We show in this paper that the internal core can be derivatized with anionic ligands such as phosphate in order to allow the efficient entrapment of cationic molecules through a process akin to ion exchange. Synthesis of SMBV involved first a cross linking and derivatization step of polysaccharides followed by a homogenization, a drying and a regioselective acylation step. Acylated polysaccharide cores are thus obtained which can be loaded with drugs and wrapped with a phospholipid layer. The SMBVs obtained are characterized through their size, 20 nm, and their ability to filter through 0.22 microns pore size membrane. Gel permeation chromatography experiments performed with various phospholipid/acylated cores ratios indicate that SMBVs form entities distinct from liposomes and that the optimum phospholipid/acylated cores ratio for this specific type of SMBVs is close to 100%. The supramolecular structure of SMBVs and in particular the spatial proximity between acylated cores and phospholipids is demonstrated through resonance energy transfer experiments. The drug loading capability of SMBVs is illustrated by the preparation of gentamicin and doxorubicin loaded SMBV. The therapeutic potential of SMBVs is then discussed notably in the light of a possible biomimetism with low density lipoproteins (LDL).
We report the solubilization of full-length single-walled carbon nanotubes into a physiological b... more We report the solubilization of full-length single-walled carbon nanotubes into a physiological buffer by sonication in presence of streptavidin. Transmission electron microscopy showed that the resultant dispersion was enriched of individual/small ropes of nanotubes. By the analysis of the crystal structure of tetrameric streptavidin and of the tryptophan emission of adsorbed proteins we hypothesized that proteins adsorbed onto SWNT sidewalls through their amine functionalities. Our results suggested using streptavidin as an interlinker between carbon nanotubes and semiconducting nanocrystals. We fabricated a supramolecular luminescent nanoassembly composed of individual or small ropes of full-length single-walled carbon nanotubes decorated with streptavidin-conjugated quantum dots. The luminescent nanoassembly was stably dispersed under physiological conditions and was readily visible by optical fluorescent microscopies.
We describe a novel tunable approach for the synthesis of carbon nanotube-silica nanobead composi... more We describe a novel tunable approach for the synthesis of carbon nanotube-silica nanobead composites. The control of nanotube morphology and bead size coupled with the versatility of silica chemistry makes these structures an excellent platform for the development of biosensors, or for optical, magnetic and catalytic applications.
Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require pr... more Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.
In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membran... more In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) β-target gene, PTPRK, promotes RA FLS invasiveness. Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The...
The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show... more The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show that PTEN transcription is upregulated by Egr-1 after irradiation in wild-type, but not egr-1-/-, mice in vivo. We found that Egr-1 specifically binds to the PTEN 5' untranslated region, which contains a functional GCGGCGGCG Egr-1-binding site. Inducing Egr-1 by exposing cells to ultraviolet light upregulates expression of PTEN messenger RNA and protein, and leads to apoptosis. egr-1-/- cells, which cannot upregulate PTEN expression after irradiation, are resistant to ultraviolet-light-induced apoptosis. Therefore, Egr-1 can directly regulate PTEN, triggering the initial step in this apoptotic pathway. Loss of Egr-1 expression, which often occurs in human cancers, could deregulate the PTEN gene and contribute to the radiation resistance of some cancer cells.
Triggering of the T cell antigen receptor/CD3 (TcR/CD3) complex leads to rapid tyrosine phosphory... more Triggering of the T cell antigen receptor/CD3 (TcR/CD3) complex leads to rapid tyrosine phosphorylation of regulatory proteins that participate in initiating T cell activation and proliferation. This signal transduction event requires the presence of the TcR/CD3-associated protein tyrosine kinase p59fyn. There is also evidence that the CD45 phosphotyrosine phosphatase is involved in TcR/CD3 signalling. We show here by capping experiments using double indirect immunofluorescence techniques that the receptor phosphotyrosine phosphatase CD45 and the intracellular protein tyrosine kinase p59fyn specifically co-distribute in functional T lymphocytes. Furthermore, we provide evidence that isolated p59fyn is a substrate for CD45 as indicated by the rapid dephosphorylation of the regulatory Tyr531 of p59fyn by CD45. This dephosphorylation is accompanied by a severalfold increase in the catalytic activity of p59fyn as measured by its autophosphorylation and phosphorylation of an exogenous substrate. We also demonstrate that CD45-mediated dephosphorylation and activation of p59fyn apparently occurs at a slow basal rate in resting T cells. This represents the first identification of a physiologic regulator of p59fyn and implies a mechanism for the role of CD45 in TcR/CD3 signal transduction.
Proceedings of The National Academy of Sciences, 1995
Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protoo... more Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting
Syk family kinases are essential for lymphocyte development and activation. Therefore the identif... more Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
Supramolecular biovectors (SMBV) are nanoparticular drug carriers composed of an internal crossli... more Supramolecular biovectors (SMBV) are nanoparticular drug carriers composed of an internal crosslinked solid core externally grafted with fatty acids and surrounded with a phospholipid layer. We show in this paper that the internal core can be derivatized with anionic ligands such as phosphate in order to allow the efficient entrapment of cationic molecules through a process akin to ion exchange. Synthesis of SMBV involved first a cross linking and derivatization step of polysaccharides followed by a homogenization, a drying and a regioselective acylation step. Acylated polysaccharide cores are thus obtained which can be loaded with drugs and wrapped with a phospholipid layer. The SMBVs obtained are characterized through their size, 20 nm, and their ability to filter through 0.22 microns pore size membrane. Gel permeation chromatography experiments performed with various phospholipid/acylated cores ratios indicate that SMBVs form entities distinct from liposomes and that the optimum phospholipid/acylated cores ratio for this specific type of SMBVs is close to 100%. The supramolecular structure of SMBVs and in particular the spatial proximity between acylated cores and phospholipids is demonstrated through resonance energy transfer experiments. The drug loading capability of SMBVs is illustrated by the preparation of gentamicin and doxorubicin loaded SMBV. The therapeutic potential of SMBVs is then discussed notably in the light of a possible biomimetism with low density lipoproteins (LDL).
We report the solubilization of full-length single-walled carbon nanotubes into a physiological b... more We report the solubilization of full-length single-walled carbon nanotubes into a physiological buffer by sonication in presence of streptavidin. Transmission electron microscopy showed that the resultant dispersion was enriched of individual/small ropes of nanotubes. By the analysis of the crystal structure of tetrameric streptavidin and of the tryptophan emission of adsorbed proteins we hypothesized that proteins adsorbed onto SWNT sidewalls through their amine functionalities. Our results suggested using streptavidin as an interlinker between carbon nanotubes and semiconducting nanocrystals. We fabricated a supramolecular luminescent nanoassembly composed of individual or small ropes of full-length single-walled carbon nanotubes decorated with streptavidin-conjugated quantum dots. The luminescent nanoassembly was stably dispersed under physiological conditions and was readily visible by optical fluorescent microscopies.
We describe a novel tunable approach for the synthesis of carbon nanotube-silica nanobead composi... more We describe a novel tunable approach for the synthesis of carbon nanotube-silica nanobead composites. The control of nanotube morphology and bead size coupled with the versatility of silica chemistry makes these structures an excellent platform for the development of biosensors, or for optical, magnetic and catalytic applications.
Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require pr... more Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.
In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membran... more In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) β-target gene, PTPRK, promotes RA FLS invasiveness. Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The...
The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show... more The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show that PTEN transcription is upregulated by Egr-1 after irradiation in wild-type, but not egr-1-/-, mice in vivo. We found that Egr-1 specifically binds to the PTEN 5' untranslated region, which contains a functional GCGGCGGCG Egr-1-binding site. Inducing Egr-1 by exposing cells to ultraviolet light upregulates expression of PTEN messenger RNA and protein, and leads to apoptosis. egr-1-/- cells, which cannot upregulate PTEN expression after irradiation, are resistant to ultraviolet-light-induced apoptosis. Therefore, Egr-1 can directly regulate PTEN, triggering the initial step in this apoptotic pathway. Loss of Egr-1 expression, which often occurs in human cancers, could deregulate the PTEN gene and contribute to the radiation resistance of some cancer cells.
Triggering of the T cell antigen receptor/CD3 (TcR/CD3) complex leads to rapid tyrosine phosphory... more Triggering of the T cell antigen receptor/CD3 (TcR/CD3) complex leads to rapid tyrosine phosphorylation of regulatory proteins that participate in initiating T cell activation and proliferation. This signal transduction event requires the presence of the TcR/CD3-associated protein tyrosine kinase p59fyn. There is also evidence that the CD45 phosphotyrosine phosphatase is involved in TcR/CD3 signalling. We show here by capping experiments using double indirect immunofluorescence techniques that the receptor phosphotyrosine phosphatase CD45 and the intracellular protein tyrosine kinase p59fyn specifically co-distribute in functional T lymphocytes. Furthermore, we provide evidence that isolated p59fyn is a substrate for CD45 as indicated by the rapid dephosphorylation of the regulatory Tyr531 of p59fyn by CD45. This dephosphorylation is accompanied by a severalfold increase in the catalytic activity of p59fyn as measured by its autophosphorylation and phosphorylation of an exogenous substrate. We also demonstrate that CD45-mediated dephosphorylation and activation of p59fyn apparently occurs at a slow basal rate in resting T cells. This represents the first identification of a physiologic regulator of p59fyn and implies a mechanism for the role of CD45 in TcR/CD3 signal transduction.
Proceedings of The National Academy of Sciences, 1995
Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protoo... more Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting
Syk family kinases are essential for lymphocyte development and activation. Therefore the identif... more Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.
Uploads
Papers by Tomas Mustelin