Synthesis of ATP by an artificially imposed electrochemical proton gradient in chromaffin granule ghosts.
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Adenosine Triphosphate↗
Biomedical subjects
D Scherman
Publications and source records attributed to D Scherman.
[Effect of transmembrane potential on noradrenaline transport in chromaffin granules].
Imposing a transmembrane potential positive with respect to the medium on chromaffin granule ghosts increased the initial rate of the delta pH-induced uptake of noradrenaline, whereas imposing a negative potential decreased this rate. The increase of the uptake rate was proportional to the potential. The ATP-induced uptake of noradrenaline was also sensitive to the potential since imposing a transient negative potential onto the positive potential generated by the membrane ATPase induced a latency in this transport.
Adrenal Glands↗
Erythropoietin secretion and physiological effect in mouse after intramuscular plasmid DNA electrotransfer.
BACKGROUND: Direct intramuscular plasmid DNA injection has recently been proposed for erythropoietin therapy, as an alternative to either systemic injection of recombinant erythropoietin or the use of viral vectors for erythropoietin gene transfer. However, direct intramuscular plasmid injection has so far been hampered by low efficiency and high interindividual variability. METHOD: We explored the use of a new method termed 'intramuscular electrotransfer' for erythropoietin gene expression in the mouse. This method is based on intramuscular plasmid injection followed by application of appropriate electric pulses. RESULTS: Intramuscular plasmid electrotransfer in mouse leg led to an increase of approximately 10- to 100-fold in circulating murine erythropoietin level, as compared to naked DNA alone. Using electrotransfer, as little as 1 microgram of an erythropoietin encoding plasmid was sufficient to induce an increase in mouse hematocrit, from 47% up to 80%. This hematocrit increase was stable for at least two months. Moreover, interindividual hematocrit variability was markedly reduced by electrotransfer, as compared with naked DNA injection. CONCLUSION: In vivo electrotransfer appears to be a convenient method for obtaining high erythropoietin expression in mice, and it could also be used for the expression of other secreted therapeutic proteins.
Animals↗
Formulations which increase the size of lipoplexes prevent serum-associated inhibition of transfection.
BACKGROUND: Cationic lipids are the most widely used nonviral vectors for gene delivery. Upon complexation to DNA, they offer a nonimmunogenic alternative to viral gene transfer. Unfortunately, their in vivo application has been limited due to a serum-associated inhibition of transfection. As a result, significant research effort has focused on overcoming this deleterious effect of serum. METHODS: To better understand this phenomenon, we investigated the influence of lipoplex colloidal stability on gene transfection in the presence of serum. In addition, conditions of the reaction medium were modulated and their effects on collidal stability and subsequent in vitro transfection efficiency were studied. RESULTS: The colloidal stability of the cationic lipid-DNA complexes, which depended on the charge ratio, determined the efficiency of in vitro transfection in the presence of serum. In particular, large-sized, colloidally unstable complexes of over 700 nm mean diameter induced efficient transfection in the presence or absence of serum. Conversely, colloidally stable complexes of less than 250 nm in size resulted in efficient transfection only in the absence of serum. Furthermore, for the same charge ratio, both colloidally stable and unstable lipoplexes could be obtained depending on the degree to which various solution parameters (NaCl concentration, cationic lipid acyl chain length, pH and DNA concentration) were altered. In each case, only those complexes lacking colloidal stability resulted in high levels of in vitro transfection in the presence of serum. This phenomenon was shown to be independent of both the percent DNA internalized and of the lamellar organization of the cationic lipid/DNA lipoplexes. CONCLUSIONS: Through the modulation of various mixture conditions, large-sized lipoplexes can be formed which are resistant to the transfection-inhibiting effect of serum.
3T3 Cells↗
Critical assessment of the nuclear import of plasmid during cationic lipid-mediated gene transfer.
BACKGROUND: Cationic lipid-mediated gene transfer is a promising approach for gene therapy. However, despite the significant amount of lipoplexes internalized by target cells, transgene expression remains too low. Obstacles to nuclear accumulation of plasmid DNA include: the passage of DNA across the cellular membrane, the dismantling of nucleolipidic particles in the cytoplasm and the nuclear import of plasmid DNA. The purpose of the present study was to evaluate the impact of cell status on cationic lipid-mediated transfer. METHODS: Cells were either growth-arrested (by aphidicolin) or synchronized (by a classical double-thymidine block protocol) and cationic lipid-mediated transfection of these cells was evaluated. For the study of the nuclear import of plasmid DNA, two techniques were developed: microinjection of plasmid DNA into intact cells, and the use of cells permeabilized with digitonin. RESULTS: When CV-1 cells were growth-arrested by aphidicolin, cationic lipid-mediated gene transfer was inhibited. Hela cells were synchronized and incubated with lipoplexes at different times after release of the block. Gene expression was greatly enhanced when cells underwent mitosis. When transfection was performed during the early period after block release, when fewer than 5% of the cells had divided, gene expression was carefully quantified and could be attributed to cells that escaped cell cycle block. However, by direct analysis of nuclear import of GFP-coding plasmid using cytoplasmic microinjection, GFP expression could be detected in a few cells that had not divided. CONCLUSIONS: Cationic lipid-mediated gene transfer efficiency increased when cells underwent mitosis. However, when cells did not divide, gene transfer was not completely abolished. Nuclear import of plasmid was greatly facilitated by a mitotic event. In non-mitotic cells, nuclear envelope crossing by plasmid DNA could be detected but was a very rare event.
Animals↗
Sterically stabilized BGTC-based lipoplexes: structural features and gene transfection into the mouse airways in vivo.
BACKGROUND: Colloidal stability of lipid/DNA aggregates is a major requirement for cationic lipid-mediated transfection which is particularly difficult to fulfil at the high DNA concentrations used for in vivo gene delivery. Thus, we have investigated the potential of poly(ethyleneglycol) (PEG) conjugates for steric stabilization of lipoplexes formed by bis(guanidinium)-tren-cholesterol/dioleoyl phosphatidylethanolamine (BGTC/DOPE) liposomes, a class of cationic liposomes we have developed over the past few years. METHODS AND RESULTS: We demonstrate that adequate lipophilic PEG derivatives can stabilize BGTC/DOPE lipoplexes formed at high DNA concentration. We also report the results of cryotransmission electron microscopy studies indicating that PEG-stabilized lipoplexes form DNA-coated structures which assemble into clusters exhibiting various complex morphologies. Finally, we report data from in vivo transfection experiments suggesting that PEG-mediated colloidal stabilization of concentrated lipoplex solutions may allow enhanced transfection of the mouse airways via intranasal administration. CONCLUSION: Our results represent an important step towards the design of multimodular BGTC-based systems for improved in vivo gene transfection.
Animals↗
Oxygen tension and a pharmacological switch in the regulation of transgene expression for gene therapy.
BACKGROUND: The combination of physiologically and pharmacologically controlled elements may provide a means to ensure both the regulation and the safety of transgene expression--two major goals in gene therapy. METHODS: A two-gene modulation system was developed that uses the following three levels of control: (i) the hypoxia-responsive element directing the transcription of the tetracycline-controlled transactivator (tTA); (ii) part of the oxygen-degradation domain limiting the production of tTA in normoxia; and (iii) the tetracycline switch of the transactivator activity (the tet-off system). RESULTS: This triple-control system allowed high expression of the gene of interest (luciferase or erythropoietin) by transfected cells upon hypoxia and low expression under normoxia or in the presence of tetracycline. This control of transgene expression was also obtained in mouse tumors. CONCLUSIONS: This multiple-control system is of interest for spatially restricting transgene expression into hypoxic tumors, and for finely adjusting the expression level of a therapeutic protein to the oxygen supply in medical applications such as neoangiogenesis or the erythropoietin-mediated treatment of anemia.
Adenoviridae↗
Neutral postgrafted colloidal particles for gene delivery.
Surface modification of cationic lipoplexes has been carried out by means of a postgrafting reaction. The original lipoplexes described comprise a cationic lipid, a neutral lipid, poly(ethylene glycol)-cholesterol (with or without a targeting ligand) and DNA. Modifying their surface via a chemical, postgrafting reaction did not alter their size (approximately 100 nm) nor their ability to compact DNA, but did give a reduced zeta potential (approximately 0 mV) to afford surface neutral particles. With the modified lipoplexes nonspecific NIH3T3 cell surface binding in vitro was inhibited. Intravenous injection of the neutralized lipoplexes in mice showed decreased accumulation of the particles in the lung as compared to PEGylated cationic lipoplexes. Tumor targeting was also achieved in vivo by the addition of an RGD-PEG-Cholesterol as a lipid-ligand in the postgrafted lipoplex formulation.
Acetates↗
Coupling of nuclear localization signals to plasmid DNA and specific interaction of the conjugates with importin alpha.
The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear targeting of proteins. We have developed a chemical strategy for covalent coupling of NLS peptides to plasmid DNA. A p-azido-tetrafluoro-benzyl-NLS peptide conjugate was synthesized. This conjugate was used to covalently associate NLS peptides to plasmid DNA by photoactivation. Reporter gene was expressed after transfection of the plasmid-NLS conjugates in NIH 3T3 cells. The conjugates interacted specifically with the NLS-receptor importin alpha, but plasmid-NLS conjugates were not detected in the nucleus, by fluorescence microscopy, after cytoplasmic microinjection.
3T3 Cells↗
Novel method for covalent fluorescent labeling of plasmid DNA that maintains structural integrity of the plasmid.
We have developed a chemical strategy for covalent coupling of fluorophores to plasmid DNA. A p-azido-tetrafluoro-benzyl-lissamine conjugate was synthesized and purified. This conjugate was used to covalently associate fluorescent molecules to plasmid DNA by photoactivation. In contrast to nick-translated plasmid DNA, plasmid-lissamine conjugates appeared on gel as supercoiled DNA. Reporter gene was expressed after transfection of the plasmid-lissamine conjugates in NIH 3T3 cells, although gene transfer efficiency was decreased by 60% as compared with unlabeled DNA. Intracellular traffic of plasmid-lissamine conjugates was studied in transfected cells. After cytoplasmic microinjection, fluorescent plasmid did not diffuse from the site of injection and appeared to be progressively degraded in the cytoplasm.
3T3 Cells↗
High-resolution magnetic resonance imaging of knee inflammatory arthritis in mice.
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Animals↗