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1.
Biochim Biophys Acta ; 1790(4): 219-30, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19714863

ABSTRACT

BACKGROUND: We recently reported an efficient formulation of siRNA targeting TNF-alpha, that was able to restore immunological balance in a mouse arthritis model following intravenous injection. METHOD: Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect. RESULTS: We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate. CONCLUSIONS: These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.


Subject(s)
DNA/chemistry , Drug Carriers/chemistry , Genetic Vectors , Lipids/chemistry , Liposomes/chemistry , RNA, Small Interfering/chemistry , Transfection/methods , Animals , Chemical Phenomena , DNA/genetics , Drug Stability , Mice , RNA, Small Interfering/genetics , Tumor Cells, Cultured
2.
J Biol Chem ; 282(1): 710-20, 2007 Jan 05.
Article in English | MEDLINE | ID: mdl-17090548

ABSTRACT

Platelet factor-4 (PF4/CXCL4) is an orphan chemokine released in large quantities in the vicinity of growing blood clots. Coagulation of plasma supplemented with a matching amount of PF4 results in a translucent jelly-like clot. Saturating amounts of PF4 reduce the porosity of the fibrin network 4.4-fold and decrease the values of the elastic and loss moduli by 31- and 59-fold, respectively. PF4 alters neither the cleavage of fibrinogen by thrombin nor the cross-linking of protofibrils by activated factor XIII but binds to fibrin and dramatically transforms the structure of the ensuing network. Scanning electron microscopy showed that PF4 gives rise to a previously unreported pattern of polymerization where fibrin assembles to form a sealed network. The subunits constituting PF4 form a tetrahedron having at its corners a RPRH motif that mimics (in reverse orientation) the Gly-His-Arg-Pro-amide peptides that co-crystallize with fibrin. Molecular modeling showed that PF4 could be docked to fibrin with remarkable complementarities and absence of steric clashes, allowing the assembly of irregular polymers. Consistent with this hypothesis, as little as 50 microm the QVRPRHIT peptide derived from PF4 affects the polymerization of fibrin.


Subject(s)
Blood Coagulation , Fibrin/chemistry , Platelet Factor 4/physiology , Cross-Linking Reagents/pharmacology , Dimerization , Fibrinogen/chemistry , Gamma Rays , Humans , Kinetics , Microscopy, Electron, Scanning , Models, Molecular , Peptides/chemistry , Platelet Factor 4/chemistry , Polymers/chemistry , Protein Binding , Static Electricity
3.
Leuk Res ; 28(12): 1329-39, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15475075

ABSTRACT

The growth factor-independent erythroleukemic cell line ERY-1 was established from the peripheral blood of a 87-year-old woman with chronic myeloid leukemia (CML) in the acute phase. Immunophenotyping showed that fresh leukemic cells were positive for CD13, CD33, CD36 and CD235a (glycophorin A), a phenotype compatible with that of erythroblastic cells. Cytogenetic and fluorescence in situ hybridization (FISH) analysis demonstrated classical t(9;22)(q34;q11) chromosomic translocation associated with a duplication of the BCR-ABL fusion gene. Other cytogenetic abnormalities were detected in all analyzed mitosis, the most frequent being a trisomy of chromosome 8. The established ERY-1 cell line retains these immunophenotypic and cytogenetic features, and light and electron microscopy confirmed the relatively mature erythroblastic phenotype of the cells. In addition, ERY-1 cell line expressed beta-globin mRNA and a non-phosphorylable form of the erythropoietin receptor, even in presence of erythropoietin. Of note, the proliferation of ERY-1 cells was inhibited by TGFbeta1 or STI-571 (Gleevec), without significant induction of further differentiation. In conclusion, ERY-1 is a new growth factor-independent human erythroleukemic cell line with a relatively mature phenotype that may be useful to study the molecular events involved in erythroblastic differentiation.


Subject(s)
Cell Line, Tumor , Leukemia, Erythroblastic, Acute/pathology , Aged , Aged, 80 and over , Antigens, CD/analysis , Benzamides , Chromosomes, Human, Pair 8 , Female , Fusion Proteins, bcr-abl/genetics , Gene Duplication , Globins/genetics , Humans , Imatinib Mesylate , Immunophenotyping , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Phenotype , Piperazines/pharmacology , Pyrimidines/pharmacology , Receptors, Erythropoietin/metabolism , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1 , Translocation, Genetic , Trisomy
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