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PubMed2004

Nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins A and D and mannose-binding lectin.

Collectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant d-forms of the pentoses are better competitors than the l-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or approximately 60 kDa trimeric recombinant fragments of SP-D that are composed of the alpha-helical coiled-coil neck region and three CRDs (SP-D(n/CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)(8)(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)(8)(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues.

Animals↗
PubMed1999

Cryopreservation studies with porcine corneas.

PURPOSE: A new technique for the cryopreservation of rabbit corneas in 20% w/w dimethylsulfoxide, which has been shown to preserve significant structural and functional integrity of the endothelium, was tested in porcine corneas. METHODS: The characteristics of uptake of dimethylsulfoxide into porcine corneas were measured using proton ( 1 H) nuclear magnetic resonance (NMR) spectroscopy. The effect on structural integrity of exposure to 20% w/w dimethylsulfoxide without freezing was first assessed using vital staining (acridine orange and propidium iodide), and optimum temperature conditions for addition and removal of the cryoprotectant were derived. The effects on structural integrity of cryopreservation in 15% and 20% w/w dimethylsulfoxide, and of reducing the degree of cell swelling during cryoprotectant removal following cryopreservation, were then evaluated. RESULTS: The characteristics of uptake of dimethylsulfoxide from a 10% w/w solution fitted a single exponential, resulting in a maximum tissue concentration of 14.6% when the addition occurred on ice, and 18.5% when the addition took place at room temperature. The toxic effects of dimethylsulfoxide in porcine corneas were highly temperature dependent and only evident after removal of the cryoprotectant. Unlike rabbit corneas, cryopreservation of porcine corneas in 15% and 20% w/w dimethylsulfoxide induced substantial endothelial injury which was not improved by reducing the degree of cell swelling that occurred during removal of the cryoprotectant. CONCLUSIONS: Porcine corneas were substantially more susceptible to the toxic effects of dimethyl sulfoxide, and to cryopreservation injury, than rabbit corneas. These results underline the importance of species variation in animal studies aimed at the cryopreservation of human tissue for transplantation.

Animals↗
PubMed2001

Fixation and permanent mounting of fluorescent probes after vital labelling of cultured cells.

The use of fluorescent probes for the visualization of organelles in living cells and assessment of live/dead cells has an increasing importance in cell biology. However, rapid and irreversible morphological changes of labelled cells (due to the photosensitizing effect of most fluorescent probes) make prolonged observation and detailed analysis of living cells under continuous excitation difficult. In this study, we describe a method for fixing and mounting cultured HeLa and 3T3 cells labelled with acridine orange (lysosomes), rhodamine 123 (mitochondria), Hoechst 33342 (nuclei), and propidium iodide/acridine orange (live/dead HeLa cells subjected to nutrient deprivation). Fixation is performed with vapours of a commercially available formaldehyde solution for 0.5-1 min followed by air drying and permanent mounting in DPX. After this procedure, both the general morphology and selective fluorescent labelling of cells are well preserved. The method of vapour fixation and DPX mounting is simple, rapid and reproducible, allowing definitive preservation of the fluorescence pattern observed in unfixed cell cultures.

3T3 Cells↗
PubMed2003

Study of aluminum toxicity by means of vital staining profiles in four cultivars of Phaseolus vulgaris L.

Aluminum toxicity is a very important factor limiting crop productivity on acid soils. Early effects of aluminum toxicity comprise inhibition of cell division and effects on root elongation. The plasma membrane can be the primary target of aluminum toxicity and thus, vital staining techniques could be a powerful tool in determining effects of metal stress on the plasma membrane. In this paper. we discuss the effects of Al on growth and membrane integrity by staining root tips with a mixture of fluorescein diacetate and propidium iodide. The results show a good correlation between results from growth measurement and the vital staining. From the comparison of the luminosity patterns generated by vital staining it is easy to determine Al-resistant varieties, revealing this technique as a powerful and fast method for determining tolerance to Al in different varieties.

Adaptation, Physiological↗
PubMed2001

On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli.

The outer membrane (OM) of Gram-negative bacteria provides a protective barrier for natural occurring inhibitors. Pressure mediated OM permeabilisation therefore contributes to the elimination of Escherichia coli and Salmonella by pressure preservation processes. Pressure mediated inactivation, sublethal injury, and membrane permeabilisation of E. coli were determined using two strains differing in their barotolerance. Pressure treatment of E. coli TMW 2.427 at 300, 500 and 600 MPa for 40 min resulted in a 0, 1, and greater 6 log decrease of viable cell counts, respectively. The kinetics of OM and cytoplasmic membrane permeabilisation after pressure treatment were determined by staining of pressure treated cells with the fluorescent dyes propidium iodide (PI) and 1-N-phenylnaphtylamine (NPN), respectively. A slight increase of PI fluorescence was observed at conditions resulting in a greater 6 log decrease of viable cell counts only. In contrast, increased NPN fluorescence indicating OM permeabilisation was observed prior to cell death and sublethal injury. An on-line assay for determination of pressure mediated OM damage based on NPN fluorescence was established to distinguish between reversible and irreversible OM damage. Generally, the same degree of outer membrane damage was observed by either on line or off line determinations. However, whereas reversible membrane damage occurred fast and in thermodynamic equilibrium with pressure conditions, irreversible outer membrane damage was a time dependent process.

1-Naphthylamine↗
PubMedDate not supplied

Heparin-glutathione III: study with fluorescent probes as indicators of membrane status of bull sperm.

Sperm obtained from bull epididymes were used to validate in vitro the effect of heparin and reduced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and Triton X-100 in the presence of propidium iodide (IP) and diacetate fluorescein (FDA). The metabolic activities of treated sperm were qualitatively monitored using an alamar Blue Redox fluorescence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at 26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage. Results validated by electron microscopy of thin sections of treated sperm showed complete lack of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100. Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus, leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic activity was supported over 52 h of incubation in any of the incubation systems tested, including Triton X-100, which showed a spermaticide effect. The authors propose that membrane damage does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining can be used as a fluorescent marker of sperm plasmatic membrane permeabilization and nuclear swelling.

Acrosome↗
PubMed2001

Radiation sensitivity and apoptosis in human lymphoma cells.

PURPOSE: The impact ofapoptosis on radiation-induced eradication of clonogenic tumour cells is uncertain. The aim was to analyse the relationship of different functional stages during the apoptotic process to cell death and clonogenic cell eradication. MATERIALS AND METHODS: Apoptosis in Jurkat T-cells was studied by morphology, light scatter and caspase activation. Mitochondrial integrity was determined by the mitochondrial membrane potential (delta(phi)m). Cell death was quantified using propidium iodide exclusion. Clonogenic cell death was determined using a dilution survival assay. The influence of Bcl-2 was tested using a Bcl-2 transfected Jurkat clone. RESULTS: Irradiation induced profound apoptosis within 48 h associated with caspase activation and breakdown of delta(phi)m. Inhibition of caspases abrogated the apoptotic morphology with no influence on breakdown of delta(phi)m and survival. Over-expression of Bcl-2 abrogated all hallmarks of apoptosis; delayed cell death, however, had no influence on clonogenic survival after irradiation. CONCLUSION: Based on Bcl-2 as a positional marker, radiation-induced apoptosis can be divided into two stages: the initiation/decision phase, characterized by a breakdown of the mitochondrial membrane potential, and the execution phase, characterized by caspase activation. The execution phase had no influence on survival, whereas the initiation/decision phase controls immediate survival. However, abrogation of both phases did not influence radiation sensitivity.

Amino Acid Chloromethyl Ketones↗
PubMed1991

Differences in the DNA supercoiling response of irradiated cell lines from ataxia-telangiectasia versus unaffected individuals.

In this study the manifestation of DNA damage at the nucleoid level was examined in several AT cell lines using an image analysis system to directly visualize and measure the changes in DNA loop size which occur when increasing concentrations of propidium iodide (PI) are used to titrate the DNA supercoiling response (the 'fluorescent halo assay'). This response consists of a relaxation (0.5-7.5 micrograms/ml PI) and rewinding phase (10-50 micrograms/ml PI), the latter of which is impaired by the presence of DNA strand breaks in irradiated cells. In addition to the inhibition of DNA rewinding seen immediately after irradiation at 0 degrees C, the supercoiling response of AT diploid fibroblasts indicated an increased amount of DNA unwinding compared to fibroblasts from unaffected individuals. This difference appeared to saturate, since the excess in DNA loop size over that seen in irradiated fibroblasts from unaffected individuals remained constant after 5 Gy. These results may reflect a greater instability of the DNA-nuclear matrix attachment points in irradiated AT fibroblasts. The DNA supercoiling response in irradiated transformed AT fibroblasts and AT lymphoblasts did not differ from that observed in unaffected cells of the same type. However, all of the immortalized cell lines (AT and unaffected) had inherently larger DNA loop sizes than diploid fibroblasts and exhibited excess unwinding after irradiation.

Ataxia Telangiectasia↗
PubMed2002

By-passing of P-glycoprotein using immunoliposomes.

The over-expression of MDR1 P-glycoprotein has been associated with the development of multidrug-resistance in cancer cells. Methods used to overcome multidrug-resistance often involve the co-administration of inhibitors of P-glycoprotein. Here, we test the hypothesis that an immunoliposome-based drug delivery system may be used as an alternative approach to overcome multidrug-resistance since immunoliposomes penetrate target cells by receptor-mediated endocytosis which allows to by-pass membrane-associated P-glycoprotein. Targeting of immunoliposomes was achieved by the use of an anti-transferrin receptor monoclonal antibody (OX26 mAb). Incorporation of radiolabelled digoxin within OX26-immunoliposomes enhanced cellular uptake of digoxin by a factor of 25 in immortalised RBE4 rat brain capillary endothelial cells. Uptake of liposomal digoxin was insensitive to ritonavir, a P-glycoprotein inhibitor, and was reduced in presence of increasing free concentrations of OX26 mAb or nocodazole, a reversible inhibitor of endocytosis. In contrast, uptake of free digoxin was enhanced by a factor of 1.8 in presence of ritonavir and was insensitive to OX26 mAb or nocodazole. Cellular uptake and intracellular accumulation of liposomal digoxin (55% internalisation within 30 min) was demonstrated by acid wash of the cells and was confirmed by confocal microscopy studies. Endosomal release to the cytosol of propidium iodide loaded immunoliposomes was shown. These in vitro studies suggest that immunoliposome-based drug delivery systems can be used to by-pass P-glycoprotein and thus deliver drugs to the cytosol of a target cell.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Comparing transfection efficiency and safety for antisense oligodeoxyribonucleotide between phospholipids-based microbubbles and liposomes.

OBJECTIVE: To compare transfection efficiency and safety for antisense oligodeoxynucleotides (AS-ODNs) between two type of phospholipids-based vectors. METHODS: An AS-ODNs sequence HA824 combined with luciferase reporter plasmid was used. Under low intensity ultrasound (US), a breast cancer cell line SK-BR-3 was exposed to different concentration of microbubbles and liposomes. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the inhibitory effect of HA824 on HER-2 expression at mRNA level. Atomic force microscopy (AFM) scanning techniques was employed to observe the change of membrane pore size. RESULTS: AS-ODNs transfection efficiency showed an increasing tend with microbubble concentration, but not with liposome concentration. Maximum transfection efficiency with minimum cell viability was achieved under 2% microbubble concentration. Too strong sonoporation activity would enlarge membrane pores significantly and cause low cell viability. CONCLUSION: US-mediated AS-ODNs transfection enhanced by phospholipids-based microbubbles represents an effective and safe avenue.

Cell Line, Tumor↗
PubMed2004

The neurotoxic effect of sickle cell hemoglobin.

A growing body of experimental evidence suggests that the oxidative neurotoxicity of hemoglobin A may contribute to neuronal loss after CNS hemorrhage. Several hemoglobin variants, including hemoglobin S, are more potent oxidants in cell-free systems. However, despite the increased incidence of hemorrhagic stroke associated with sickle cell disease, little is known of the effect of hemoglobin S on cells of neural origin. In the present study, its toxicity was quantified and directly compared with that of hemoglobin A in murine cortical cell cultures. Reactive oxygen species production, as assessed by cellular fluorescence after treatment with dihydrorhodamine 123, was significantly increased by exposure to 10 microM hemoglobin S for 2-4 h. Neuronal death, as measured by propidium iodide staining and lactate dehydrogenase release, commenced at 4 h; for a 20-h exposure, the EC50 was approximately 0.71 microm. Glial cells were not injured. Cell death was completely blocked by iron chelation with deferoxamine or phenanthroline. Direct comparison of sister cultures exposed to either hemoglobin A or hemoglobin S revealed a similar amount of cell injury in both groups. A significant difference was consistently observed only after treatment with 1 microM hemoglobin for 20 h, which resulted in death of approximately one third more neurons with hemoglobin S than with hemoglobin A. The results of this study suggest that sickle cell hemoglobin is neurotoxic at physiologically relevant concentrations. This toxicity is iron-dependent, oxidative, and quantitatively similar to that produced by hemoglobin A.

Animals↗
PubMed2002

Interleukin-12 and interleukin-18 change ICAM-I expression, and enhance natural killer cell mediated cytolysis of human osteosarcoma cells.

Cytokines play important roles in the expression of adhesion molecules and the function of anti-tumor effector cells in the immune system. In this study, the influence of interleukin-12 (IL-12) and IL-18 on the expression of ICAM-1 and natural killer (NK)-cell mediated lysis in a human osteosarcoma cell line (HOS) was evaluated. ICAM-I expression of HOS cells were analyzed by flow cytometry following treatment with IL-12, IL-18 or both, and in co-cultures with peripheral lymphocytes. NK-cell activation in response to IL-12 and IL-18 was investigated by selective flow cytometry using propidium iodide. ICAM-1 expression on HOS cells was significantly enhanced by IL-12, but only when co-cultured in cell-to-cell contact with peripheral lymphocytes. Antibodies to interferon-gamma abrogated this effect. If HOS cells and peripheral lymphocytes were separated in co-cultures, IL-18 could substitute for cell-to-cell contact, facilitating IL-12-mediated enhancement of ICAM-1. Addition of IL-18 also enhanced NK-mediated cytolysis of HOS cells. These findings demonstrate that IL-12 can enhance the expression of ICAM-1 in the presence of IFN-gamma and, with IL-18, enhances NK anti-tumor activity. Immunomodulation via cytokine therapy may lead to improved eradication of chemotherapy-resistant osteosarcomas.

Cell Line, Tumor↗
PubMed2002

Immobilization of Aspergillus fumigatus colonies in a soft agar matrix allows visualization of A549 cell detachment and death.

A model to examine the effects of proliferating Aspergillus fumigatus ATCC 26933 colonies embedded in a thin layer of soft agar on a monolayer of A549 cells was developed and enabled an investigation of the response of cultured cells to fungal growth. This model simulates the conditions on the respiratory surface in patients with invasive aspergillosis and also in the mucus secretions of cystic fibrosis patients with allergic bronchopulmonary aspergillosis. Conidia of A. fumigatus adhering to A549 cells were immobilized in a thin layer of soft agar (0.6% (w/v)) and allowed to germinate at 37 degrees C. Fungal colonies greater than 5 mm in diameter caused rounding-up and detachment of A549 cells underneath the colony and towards the hyphal tips. As the fungal colony diameter increased, cell detachment occurred ahead of the hyphal tips. Cells detached for short periods (less than 6 h) showed no annexin-V (AV) or propidium iodide (PI) staining, suggesting no externalization of phosphatidylserine and an intact plasma membrane. Cells that had detached for periods greater than 6 h were positive for AV and PI indicating the rupture of the plasma membrane and cell death by necrosis. Chemical extraction and separation by thin layer chromatography of agar from zones of cell detachment around fungal colonies revealed the presence of three compounds that may play a role in inducing cell death.

Annexin A5↗
PubMed2002

Altered subcellular localization of p53 in estrogen-dependent and estrogen-independent breast cancer cells.

LCC2, an estradiol-independent tamoxifen (Tax)-resistant subline of MCF-7 human breast cancer cell line, is resistant relatively towards Tax and methotrexate (Mtx). The purpose of the present study is to evaluate the role of p53 in determining this resistance. While MCF-7 is sensitive to and undergoes apoptosis, as determined by propidium iodide stain, by Tax and Mtx, LCC2 is resistant to apoptosis induction by these agents. Both cell lines undergo apoptosis and are sensitive equally to doxorubicin (Adr). p53 cDNA of both sublines was evaluated by polymerase chain reaction (PCR) amplification and sequencing and was found to be of wild-type. p53 mRNA, as well as protein, are elevated markedly in LCC2 as compared to MCF-7 cells. p53 expression was increased by estradiol and Adr, not changed by Mtx, and decreased by Tax and estradiol-deprivation in both sublines. p53 modulation by the various agents, in both sublines, was evaluated by cytochemical staining and subcellular fractionation. This analysis showed that p53 is localized mainly in the nuclear fraction in MCF-7 cells, and in the cytoplasmatic fraction in LCC2 cells. Doxorubicin induced apoptosis in MCF-7 cells along with increase in its nuclear fraction. In contrast, LCC2 underwent apoptosis by Adr despite its cytoplasmatic sequestration. These experiments demonstrate that p53 is sequestered to cytoplasm in the estrogen-independent, Tax-resistant LCC2 cells. However, the differences in apoptotic rate between MCF-7 and LCC2 cells do not seem to be dependent on p53. The LCC2 cell line may serve as a useful model for the study of the mechanism of cytoplasmatic sequestration of wild type (wt) p53, its physiologic consequences, and its relation to estrogen-independence or Tax resistance of breast cancer cells.

Antineoplastic Agents, Hormonal↗
PubMed1989

Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes.

The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties. We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date. Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide. The attacked cells do not appear to repair the membrane lesions. Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH. In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators. Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids. Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes. It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN. This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells. The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E. coli.

Adenosine Triphosphate↗
PubMed2004

Erythropoietin attenuates post-traumatic injury in organotypic hippocampal slices.

Recent experimental evidence indicates that erythropoietin (Epo), in addition to its hormonal role in regulating red cell production, operates as a neuroprotective agent. So far, the neuroprotective effect of human recombinant Epo (rhEpo) has been mainly demonstrated in models of cerebral ischemia/hypoxia and in selected in vivo studies of traumatic neuronal injury. To further investigate the potential role of this multifunctional trophic factor in post-traumatic cell death, we examined the protective effects of rhEpo in a newly developed model of mechanical trauma in organotypic hippocampal slices. Organotypic rat hippocampal slices were subjected to traumatic injury by allowing a stylus to impact on the CA1 area with an energy of 6 microJ. Hippocampal damage was identified and measured 24 and 48 h later with the fluorescent dye propidium iodide (PI). In untreated slices, the impact induced a significant increase in the mean hippocampal PI fluorescence, co-localized with the area of impact at 24 h (primary post-traumatic injury) and progressively spread to the whole slice between 24 and 48 h (secondary post-traumatic injury). Addition of rhEpo (1-100 UI/mL) or of the NMDA antagonist MK-801 (30 microM) immediately after the traumatic injury reduced hippocampal damage by approximately 30% when observed 24 h later. At 48 h after trauma, the protective effect of rhEpo was greater (by about 47%) and significantly more pronounced than that of MK-801 (28%). Our results suggest that the neuroprotective activity of rhEpo is particularly effective against delayed, secondary post-traumatic damage. This well tolerated agent could provide a therapeutic benefit in pathologies involving post-traumatic neurodegeneration.

Animals↗
PubMed2002

Optimizing prostate cancer suicide gene therapy using herpes simplex virus thymidine kinase active site variants.

The herpes simplex virus (HSV) thymidine kinase gene (tk) forms the basis of a widely used strategy for suicide gene therapy. A library of HSV thymidine kinase enzyme (TK) active site mutants having different affinities for guanosine analog prodrugs was developed. We sought to determine the optimal combination of tk variant and prodrug specifically for prostate cancer gene therapy, using in vitro and in vivo studies of adenovirally infected CL1, DU-145, and LNCaP tumor lines carrying wild-type tk, tk30, tk75, and sr39tk mutants expressed by a strong, constitutive cytomegalovirus promoter and treated with ganciclovir and acyclovir. In vitro experiments involving prostate cancer (CaP) cell line infection were carried out with a broad range of prodrug concentrations, and cell killing was determined by limiting dilution (colony-forming), MTT, and propidium iodide assays. In vivo studies based on CL1-GFP xenograft experiments were carried out to examine the ability of each TK variant to prevent tumor formation and to inhibit tumor growth and development of metastases in established orthotopic and subcutaneous tumors in SCID mice. Both in vitro and in vivo studies suggest improved killing with the sr39tk variant. Thus, the results suggest that the use of sr39tk in future trials of prostate cancer tk suicide gene therapy may be beneficial.

Acyclovir↗
PubMed2003

Cell and animal imaging of electrically mediated gene transfer.

Electropermeabilization is a nonviral method successfully used to transfer genes into cells in vitro as in vivo. Although it shows promise in field of gene therapy, very little is known on the basic processes supporting the DNA transfer. The aim of the present investigation is to visualize gene electrotransfer and expression both in vitro and in vivo. In vitro studies have been performed by using digitized fluorescence microscopy. Membrane permeabilization occurs at the sides of the cell membrane facing the two electrodes. A free diffusion of propidium iodide across the membrane to the cytoplasm is observed in the seconds following electric pulses. Fluorescently labeled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable over a few minutes. Changing the polarity and the orientation of the pulses lead to an increase in gene expression. In vivo experiments have been performed in Tibialis Cranialis mice muscle. Electric field application lead to the in vivo expression of plasmid DNA. We directly visualize gene expression of the Green Fluorescent Protein (GFP) on live animals. GFP expression is shown to be increased by applying electric field pulses with different polarities and orientations.

Animals↗