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Structure and Dynamics of Electric Field-Induced Membrane Pores as Revealed by Rapid-Freezing Electron Microscopy
... The diameters of the transient electropores as large as 2 0 -1 2 0 nm should be sufficient to allow most macromolecules to pass through. The life time of these membrane pores (in the range of seconds) was also long enough to allow elongated molecules (such as DNA) to diffuse in the cell (Chang, 1992). ...
... These results suggest that membrane-cytoskeletal interactions may play an important role in the shaping of electropores (Chang, 1992). ...
... It was suggested by Chang (1992) that the membrane pores are not only shaped by the primary effects of interactions between the applied electric field and the cell membrane, but also by a secondary effect of material flow following the initial permeabilization of the cell membrane induced by the electric field. The diameters of their observed membrane openings were larger than those predicted in many theories that consider principally the primary effects (Chemomordik et a l , 1983;Powell & Weaver, 1986). ...
This thesis is concerned with studies on two aspects of membrane fusion. The first aspect is the mechanism of membrane fusion in electrically-induced cell fusion. The second aspect is the mechanism of membrane fusion induced by fusogenic viral peptides. Evidence was found from both fluorescence microscopy and freeze-fracture electron microscopy for the occurrence of hemi-fusion in the electrofusion of human erythrocytes. The conditions that favour hemi-fusion as opposed to complete fusion were characterised, and the possibility that hemi-fusion might precede complete electrically-induced cell fusion are discussed. The procoagulant activity of human erythrocytes, which provides a measure of the translocation of phosphatidylserine from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on erythrocyte fusion induced by electrical breakdown pulses under differing experimental conditions. It seems possible that a localised, surface exposure of phosphatidylserine may contribute to the "long-lived fusogenic state". Divalent cations in the pulsing medium may interact with phosphatidylserine molecules, translocated from the inner to the outer monolayer of the erythrocyte plasma membrane by breakdown pulses, to stabilise the pulsed erythrocyte membrane against haemolysis, and to assist the formation of pearl chains of pulsed cells. It was found that the entry of sugar molecules, via electropores in the plasma membrane, facilitated the rounding-up of electrically fused erythrocytes into giant cells, while impermeable molecules e.g. poly (ethylene glycol) or dextran inhibited this process. The secondary structures and orientations of the fusion peptides (corresponding to the N-terminus of HA2 protein of strains A/PR/8/34 and X31 influenza viruses (HA and WT peptide, respectively), a peptide (G1E peptide) with a substitution of glutamic acid for the glycine residue at the N-terminal of the WT peptide, and the fusion peptide corresponding to the N-terminus of gp41 protein of ARV2 strain HIV virus were investigated in egg PC phospholipid bilayers with polarized, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR). Both dried and hydrated samples were studied. The relationships between the observed secondary structures and orientation of these fusion peptides and how they may induce membrane fusion are discussed.
... Each of the three methods of activation tested by us utilize a different mechanism for the elevation of intracellular calcium. Electroactivation opens temporary pores in the oolemma [33], and a consequent calcium influx provided only that a positive calcium gradient exists [34]. ...
... These results confirm previous observations on the effects of 6-DMAP on pronuclear formation in both bovine and ovine oocytes matured in vitro [25]. Interestingly, while ungulate oocytes almost invariably form a single diploid pronucleus after 6-DMAP inhibition, mouse oocytes form 2 normal-sized pronuclei but, in common with the other species, do not extrude a polar body [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. It is postulated that 6-DMAP inhibits MAP-kinase with a consequent disruption of spindle organization in metaphase II oocytes and irregularities in the gross morphology of the oocyte [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. ...
... Interestingly, while ungulate oocytes almost invariably form a single diploid pronucleus after 6-DMAP inhibition, mouse oocytes form 2 normal-sized pronuclei but, in common with the other species, do not extrude a polar body [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. It is postulated that 6-DMAP inhibits MAP-kinase with a consequent disruption of spindle organization in metaphase II oocytes and irregularities in the gross morphology of the oocyte [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. Although 6-DMAP treatment enhances the speed of pronuclear formation and suppresses polar body extrusion, this treatment has no measurable effect on the levels of MPF-kinase in ovine oocytes after chemical activation [40]. ...
Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (approximately 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst stage in higher percentage after 6-DMAP treatment (83% vs. 25%). We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protocol develop readily to term.
... The memory effect of electroporation needs to be included on this model. [12][13][14] Thus, it was observed that the parameters E p2 and σ t should be adjusted dynamically. E p2 should decrease while σ t should increase pulse after pulse. ...
... Electroporated cell membranes cannot fully recover in the intervals between pulses, so in each new pulse the conductivity has a gradually increasing value. 13,14 But, conductivity does not increase indefinitely, being limited by the extracellular medium. This is reported in the work of Kinosita et al as saturation. ...
... These include the route of entry (transcellular or via the lipid lamellae), the maximum molecular weight of the delivered molecule, the extent of the skin that is electroporated, the maximum amount of current that can be safely applied, and the reversibility of the pores generated in cells. Since cell membranes are more difficult to electroporate than lipid lamellae (Chang, 1992), and since the cells of the stratum corneum have very thick cell envelopes, the pores are probably formed in the continuous lipid lamellae of the stratum corneum. Thus, peptide flux probably occurs through this region. ...
... Prausnitz et al. (1992) assert that the viable cells in the lower epidermis should be minimally affected; however, this has not been verified. Although pore formation in lipid lamellae is reversible, there is evidence that pores formed in cells may not be reversible (Chang, 1992). This possibility may affect the health of skin being electroporated. ...
Recently, a large number of new and potentially potent peptide and protein drugs have been developed. Their systemic delivery is difficult because they are rapidly cleared from the bloodstream, are of large molecular size, are vulnerable to proteolytic attack and tend to undergo aggregation, adsorption and denaturation. Controlled release strategies have many advantages over the current delivery method of injection or intervenous infusion. Controlled release allows for prolonged delivery while maintaining the drug concentration within therapeutic limits. It also improves and increases patient compliance by removing the discomfort of repeated applications. The transdermal route of delivery is particularly attractive because it avoids peptide and protein degradation via the gastrointestinal tract and the hepatic first-pass effect, and delivery can be interrupted by simply removing the device. Its primary problem is that the skin is an excellent barrier to large, hydrophilic, polar compounds. Recent work in the area of transdermal peptide and protein drug delivery is overviewed, including strategies such as prodrugs, chemical enhancement, iontophoresis, electroporation, and ultrasound, with focus on mass-transport mechanisms of different systems. Of the strategies studied, iontophoretic delivery appears the most promising. It is clear, however that more work remains to be done before transdermalpeptide and protein delivery devices come to market. Chemical engineers can contribute significantly to further research in this exciting area.
... The effects of PEF on biomembranes have been thoroughly studied since the use of PEF has attracted great interest in several scientific areas such as cell biology, biotechnology, medicine, or food technology (Zimmermann, 1986;Palaniappan and Sastry, 1990;Ho and Mittal, 1996;Prassanna and Panda, 1997). PEF can cause electrofusion or electroporation in cells (Chang, 1992). The electrofu- ...
... However, the exact mechanism of pore formation, expansion, and subsequent shrinkage and resealing or cell lysis remains controversial. Several theories (Zimmermann et al., 1974;Zimmermann, 1986;Tsong, 1991;Chang, 1992;Kinosita et al., 1992;Newman et al., 1992), based on the experiments carried out on model systems, such as liposomes or protoplasts, and on large eukaryote cells, such as erythrocytes, chloroplasts, green alga cells, sea urchin eggs, hamsters ovary cells, etc., have been proposed in order to explain the mechanism of the reversible electroporation and/or the electrical membrane breakdown. ...
Application of pulsed electric fields of high intensity and duration from microseconds to milliseconds may cause temporary
or permanent permeabilization of cell membranes. The generally accepted term describing this phenomenon is “electroporation”
or “electropermeabilization.” Permeabilization of cell membranes is the basis of different applications of PEF for the food
industry such as food pasteurization or extraction of intracellular components.
... 2) Molecular Dynamics Analysis: It is generally difficult to observe electropores, except perhaps in erythrocyte membranes in which large pores could be detected using electron microscopy [268]. However, MD simulations have proven to be effective in providing physical insights into both the dynamics of lipid membrane systems at the atomic level and allow for easy inclusion of the inherent many-body physics and details of a heterogenous atomic system [269], [270], [271], [272], [273], [274], [275], [276], [277], [278], [279], [280], [281]. ...
... 2) Molecular Dynamics Analysis: It is generally difficult to observe electropores, except perhaps in erythrocyte membranes in which large pores could be detected using electron microscopy [268]. However, MD simulations have proven to be effective in providing physical insights into both the dynamics of lipid membrane systems at the atomic level and allow for easy inclusion of the inherent many-body physics and details of a heterogenous atomic system [269], [270], [271], [272], [273], [274], [275], [276], [277], [278], [279], [280], [281]. ...
The field of bioelectrics lies at the intersection of biomedical engineering, biophysics, pulsed power technology, and bioscience, guided by the underlying theme of using electric pulses of varying waveforms and durations to drive and manipulate desired biological responses and outcomes in cells and tissues. The ever-growing list of applications includes tumor treatment, wound healing, neurostimulation, decontamination and inactivation of microorganisms and pathogens, cardiac ablation for the treatment of arrhythmias, and many more. Here, we present a broad review of the various advances in the field, the state of the hardware that drives the successful implementation of bioelectric systems, and some of the underlying biophysics and modeling efforts that help quantify and unravel the underlying mechanisms for eventual optimization of outcomes and their successful modulation. The primary focus is on the achievements over the past 50 years and the promise for future directions.
... Application of PEF treatments leads to the electroporation of the cell membranes, creating aqueous pathways (electropores) that increase the permeability of cell membranes to ions and other molecules [43]. In fact, the electroporation is the principal mechanisms involved in the microbial death. ...
... In erythrocyte membranes, large pores could be observed using electron microscopy (Chang 1992); but in general, direct observation of the formation of nano-sized pores is not possible with conventional techniques. Furthermore, due to the complexity and heterogeneity of cell membranes, it is difficult to describe and characterize their electroporation in terms of atomically resolved processes. ...
Externally applied nanosecond electric pulses are useful to trigger and tailor bioeffects in cells and tissues. However, the parameter space is large given the different types of cells, and the wide range of potential electrical parameters (such as pulse durations and waveforms, field intensities, and number of pulses) that are available for use. To maximize benefits, tailor a desired response, and devise an efficient system, it becomes necessary to first understand and quantify the biological behavior driven by the electrical input. Model development based on the inherent biophysical processes is an elegant and cost-effective option to help advance this technology. Given the merits and need for modeling then, this chapter focuses on the various schemes for analysis and simulations of the electrically driven bioeffects. Schemes ranging from molecular level descriptions to an averaged continuum analyses are presented and discussed in this chapter. Relevant examples and illustrative result are also given in this context of cellular bioelectrics.
... This physical delivery technique relies on the application of an electric pulse to the cells, which results in a transient rearrangement of the cell membrane, caused by a transient reorientation of bipolar lipid molecules under an intense electric field [34]. This rearrangement results in the formation of hydrophilic pores that last from several milliseconds to seconds and allow macromolecules to cross the cell membrane into the cytosol [35,36]. When it comes to antibodies, electroporation was shown to be more efficient compared to different profection reagents, such as ProteoJuice, Bioporter, ab-DeliverIN or Pulsin [37]. ...
Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells.
... Bu bağlamda, parçalanma ve gözenek oluşum süreci 3 farklı aşamadan oluşmaktadır: (1) gözeneklerin açılması ve oluşumu, (2) gözeneklerin büyümesi, (3) eğer tersinir ise tekrardan kapanması, tersinmez ise parçalanmanın meydana gelmesi şeklindedir. Gözenek oluşumu mikrosaniyeler içerisinde meydana gelirken, oluşan gözeneklerin kapanması yaklaşık 4 saniye sürmektedir (Chang, 1992). Bu teoriden yola çıkıldığında, elektroporasyonun veya gözenek oluşumunun meydana gelebilmesi için elektrik alan yoğunluğu kritik değeri aşmalıdır (Barbosa-Canovas ve Sepulveda, 2005). ...
In the food industry, using of new preservation and processing technologies, which may be an alternative to traditional methods, is becoming increasingly important. These novel and particularly non-thermal techniques have very important advantages such as enhancing food safety, reducing quality losses, and increasing production efficiency. One of these new technologies, Pulsed electric field (PEF) technique, stands out as a novel method that has been emphasized in recent years. It is used for different purposes in both liquid and solid foods, also various studies are carried out for the optimization. On the other hand, recently, there are several studies using PEF technique in meats and seafoods for the preservation purposes, as well as other positive effects (improving the functional properties of the product, accelerating processes such as drying, curing and freezing). In this regard, detailed information about the purposes for which PEF technique can be used in meats and seafoods has been tried to be given in this comprehensive review study.
... The application of PEF treatments leads to the electroporation of the cell membranes. The eventual result is the formation of aqueous pathways (or electropores), leading to an increase in cellular membrane permeability to ions and other molecules 5 . PEF is now recognized as a promising non-thermal technology for food preservation, since it maintains food quality while inactivating vegetative bacteria and yeast cells 6 . ...
The aim of this investigation was to lay the groundwork of the potential application of Pulsed Electric Fields (PEF) technology for accelerating the drying process of meat and meat products, and specifically in this work of Spanish dry-cured sausages “longaniza”. PEF treatments were applied to pork loin samples, and the influence of different PEF parameters on the process were evaluated. An optimal PEF treatment of 1 kV/cm, 200 μs of pulse width and 28 kJ/kg was determined as the most suitable to electroporate meat cells and to improve water transfer by achieving a water content reduction of 60.4% in treated-meat samples dried at 4 °C. The influence of PEF on meat drying rate was also studied on minced pork and the results showed that with a particle size of 4.0 mm higher drying rates were achieved. To validate the results, Spanish cured sausages were prepared from treated and untreated minced pork and stuffed into gauzes and natural pork casings at pilot plant scale. After the curing process, the application of PEF to sausages stuffed into gauze reduced the drying time from 17 to 9–10 days, a reduction of 41–47%, confirming the effects described at lab scale and the potential of PEF for accelerating the sausage-drying process.
... Electroporation refers to a process, by which the application of a strong external electrical field (measured as V cm-1 ) results in a transient permeabilization of the cell membrane ( Chang et al., 1992;Chen et al., 2006). Pores in the range of 1-100 nm (Chang, 1992) are created, allowing diffusion of small hydrophilic molecules into (and out of) the cell ( Tekle et al., 1994). ...
Photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of a range of bio-products and genetic engineering is an increasingly important strategy to improve the natural cellular features. For the vast majority of microalgal strains, however, genetic engineering is not yet possible and the establishment of efficient genetic tools for a broad range of microalgal species is an important task to reach biotechnological success. Stable DNA transformation is one of the crucial steps for most genetic engineering approaches. In this context, we report the first successful and stable nuclear genetic transformation of the biotechnologically promising oleaginous microalga M. neglectum. Transformation was achieved by electroporation and the efficiency could progressively be improved by implementation of an additional cell pretreatment step, aiming at weakening the rigid natural cell wall. Furthermore, a first reporter transgene was established for M. neglectum. As a result of this work, genetic engineering strategies now become accessible which are required to successfully establish M. neglectum as a novel host for biotechnological applications.
... Bacterial cells were transformed using electroporation as previously described [199,200,201]. Electro-competent E. coli cells were diluted 1:1 with 10% C.2 Tissue culture glycerol and 40µl of the prepared solution was added to a chilled eppindorf tube (1.5ml). ...
... Although the exact mechanism of electroporation is not well known, several theories such as electromechanical, electrohydrodynamic, electroosmotic, and the development of viscoelastic instabilities have been suggested. These mechanisms were proposed according to the studies carried out on different model systems such as liposomes, planar bilayers, and phospholipid vesicles (Chang, 1992;Neumann et al., 1992;Tsong, 1991;Zimmermann, 1986;Zimmermann et al., 1974). All of these theories have one common feature: the membrane plays an important role in amplifying the applied electric field (Weaver and Chizmadzhev, 1996). ...
... Indeed, we have already discussed that a cell exposed to an electric field exhibits an enhancement of its trans-membrane potential which is coupled with the formation of pores, something that has been known for several decades now (Cole, 1968;Neumann and Rosenheck, 1972). More recently, Chang (1992) has actually explained this induced transmembrane potential by the formula: V max = 1.5 r E cos θ, where r is the cell radius, E the electric field, and θ the angle between the normal vector of the membrane and the direction of the electric field applied. But we are still evoking the membrane and not the wall. ...
... The log-phase can be used for the determination of cell length alternating stress responses 240 referring to the close ranges of the reference data (Fig. 2) For treatments at 390 min the energy per pulse is more important due to the improved 276 membrane composure (Barsotti,1999). Thus more free charges have to be polarized by the 277 pulse to create a transmembrane potential of 1 V (Chang, 1992) that is necessary to form a 278 pore. During this trial just one treatment resulted in a cell length within the reference interval. ...
The aim of this work was to evaluate whether cell length alternations of Lactobacillus johnsonii NCC 533 can be used to monitor the response of this bacterium towards various stress factors during its growth. The selected stressors were heat shock (46 to 55 °C for 15, 25 min) and pulsed electric fields (PEF; at 15, 20 kJ kg− 1 with 7 to 20 kV cm− 1). These stress factors were applied either at the beginning or at the end of the log-phase in MRS broth. The cell length changes were also monitored during normal growth conditions. It was found, that the cell lengths of bacteria changed in the course of growth; with clear differences between lag, log and stationary growth phases. Heat shock led to significant cell elongation, whereas PEF-stress resulted in cell shrinkage. The stress treatments by heat and PEF were more influential on cell length changes at the beginning than at the end of the log-phase. The magnitude of cell length changes correlated with the intensity of the applied stresses.
Industrial relevance
Microbial fermentations require a reliable online monitoring, which rapidly gives information about occurring stresses, their extent and nature as well as the cell damage, the capability of cell recovery and the possibility of reaching the desired cell count. The present investigations and method developments could improve the understanding of these mechanisms to lead to better controlled industrial fermentations.
... Following ultrasonic treatment, the original permeability was eventually restored (26). Freeze-fracture electron microscopy has shown that, a few seconds after the electric pulse, the pores began to reseal; 5 sec post-electroporation, the majority of the pores became shallow and smaller, while at 10 sec the pore-like structures on the cell membrane had almost disappeared (32,41). Guzman et al reported similar results when cells were exposed to low frequency ultrasound (24 kHz) (37,43,44). ...
Staphylococcus aureus (S. aureus) is the principle causative agent of osteomyelitis, accounting for 80% of all human cases. S. aureus internalized in osteoblasts escapes immune response, including engulfment by phagocytes. It also escapes the action of a number of antibiotics. Ultrasound increases cell membrane permeability to a number of drugs. Following an internalization assay, we used low-frequency, low-power ultrasound combined with the antibiotic rifampin to target S. aureus internalized in human osteoblasts. Tryptic soy agar (TSA) was used to quantitate the antibacterial effect of rifampin combined with low-frequency ultrasound. A Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell viability following exposure to ultrasound. Our data revealed that rifampin successfully penetrates into osteoblasts and kills internalized S. aureus in osteoblasts, while low-frequency ultrasound promotes this process. Ultrasound had a negative impact on the cell viability of osteoblasts; however, this damage was slight and reversible. Ultrasound-enhanced antibiotic efficiency to bacteria internalized in the osteoblasts may contribute to the control of chronic infection to reduce recurrence.
... The predictions of the viscoelastic model and its generalizations [58] are in good agreement with data for cell membranes [39, 40] and bilayer lipid membranes [41]. The calculated concentration of pores is also close to that observed for erythrocyte membranes by Chang, which is 8/µm 2 [59]. It must be pointed out that models based on surface shape instabilities could describe only initial stages of pore formation. ...
... Alternative anticoagulation with hirudin for CPB in HIT II patients has been used successfully (2). However, in patients with renal insufficiency or failure bleeding complications after CPB occurred because of hirudin cumulation (2,3). In this case report, a high volume HD with a polysulfone high- flux-filter was instituted in the operating room after discontinuation of CPB in order to remove hirudin from plasma. ...
Heparin-induced thrombocytopenia type II (HIT II) requires alternative anticoagulation. Hirudin has been effectively used in patients with HIT II scheduled for cardiac surgery. However, bleeding complications were observed in patients with renal impairment. In vitro hemodialysis (HD) has been questioned over its efficacy in eliminating hirudin. Another approach to stop bleeding is the application of recombinant factor VIIa (rFVIIa). We report on a patient with HIT II and chronic renal failure who suffered from severe hirudin-induced bleeding after cardiac surgery who was safely treated with a combined approach of surgical hemostasis, substitution of blood products, HD, and rFVIIa to stop finally bleeding.
... The exact mechanisms of electroporation are not yet fully understood. Several theories [32,65,87,119,132,134] based on the experiments carried out on model systems such as liposomes, planar bilayers, and phospholipid vesicles have been proposed in order to explain the mechanism of the reversible electroporation and/or the electrical membrane breakdown. All of these theories have their relative advantages and disadvantages, but one common feature of great importance is the fact that the membrane plays a role in amplifying the applied electric field, as the conductivity of intact membrane is several orders of magnitude lower than the conductivities of extra cellular medium and cell cytoplasm [124]. ...
In the last decades, several non-thermal technologies have been proposed as alternative to the traditional ones to improve
the competitiveness of the food industry. The key to success was identified in offering to food industries the opportunity
to improve food quality, to introduce new foods in the market, and to optimize the processing procedures while reducing energy
costs. Pulsed electric fields (PEF) showed the potential to be one of the most promising novel technologies to reach these
objectives. The application of PEF as a pretreatment of permeabilization of vegetable and animal tissue to enhance the efficiency
of mass transfer of water or of valuable compounds from biological matrices demonstrated its efficiency in drying, extraction,
and diffusion processes. This article reviews the basic mechanisms of electroporation of plant tissues, discusses the methods
of detection of electrically induced cell damage, and analyses the influence of process parameters on the efficiency of the
treatment. Furthermore, this article focuses on the applications of PEF, its advantages, and energy costs in different fields
of food processing, such as juice expression, drying, and extraction, with special emphasis on the relevance of PEF to the
winemaking industry.
KeywordsPulsed electric field (PEF)-Cell permeabilization-Biological tissue-Complex impedance-Mass transfer
... The key features of electroporation are based on theories involving stochastic pore formation (Chen et al. 2006). In erythrocyte membranes, large pores could be observed using electron microscopy (Chang 1992); but in general, direct observation of the formation of nano-sized pores is not possible with conventional techniques. Furthermore, due to the complexity and heterogeneity of cell membranes, it is difficult to describe and characterize their electroporation in terms of atomically resolved processes. ...
The permeability of cell membranes can be transiently increased following the application of external electric fields. Theoretical approaches such as molecular modeling provide a significant insight into the processes affecting, at the molecular level, the integrity of lipid cell membranes when these are subject to voltage gradients under similar conditions as those used in experiments. This article reports on the progress made so far using such simulations to model membrane-lipid bilayer-electroporation. We first describe the methods devised to perform in silico experiments of membranes subject to nanosecond, megavolt-per-meter pulsed electric fields and of membranes subject to charge imbalance, mimicking therefore the application of low-voltage, long-duration pulses. We show then that, at the molecular level, the two types of pulses produce similar effects: provided the TM voltage these pulses create are higher than a certain threshold, hydrophilic pores stabilized by the membrane lipid headgroups form within the nanosecond time scale across the lipid core. Similarly, when the pulses are switched off, the pores collapse (close) within similar time scales. It is shown that for similar TM voltages applied, both methods induce similar electric field distributions within the membrane core. The cascade of events following the application of the pulses, and taking place at the membrane, is a direct consequence of such an electric field distribution.
... Erythrocytes were obtained from male albino Wistar rats' blood (160–190 g) after centrifugation by removing the buffy coat. The conductivity of cell suspensions was measured on the same day of blood collection after washing twice with isotonic low ionic strength solution (ILISS), composed of 13 mM NaCl, 150 mM sucrose, 14 mM NaOH, and pH 7.4 [2]. Spherical erythrocytes (spherocytes) were prepared in 67% of ILISS, σ o = 0.23 S/m at 23 @BULLET C (conductometer Metrohm mod. ...
An intense electric field can be applied to increase the membrane conductance G and consequently, the conductivity of cell suspension. This phenomenon is called electroporation. This mechanism is used in a wide range of medical applications, genetic engineering, and therapies. Conductivity measurements of cell suspensions were carried out during application of electric fields from 40 to 165 kV/m. Experimental results were analyzed with two electroporation models: the asymptotic electroporation model was used to estimate G<sub>m</sub> at the beginning and at the end of electric field pulse, and the extended Kinosita electroporation model to increase G<sub>m</sub> linearly in time. The maximum G was 1-7 × 10<sup>4</sup> S/m<sup>2</sup>, and the critical angle (when the G<sub>m</sub> is insignificant) was 50°-65°. In addition, the sensitivity of electroporated membrane conductance to extracellular and cytoplasmatic conductivity and cell radius has been studied. This study showed that external conductivity and cell radius are important parameters affecting the pore-opening phenomenon. However, if the cell radius is larger than 7 μm in low conductivity medium, the cell dimensions are not so important.
... The exact mechanism of electroporation is not yet fully understood. Several theories (Chang, 1992; Neumann et al., 1992; Zimmermann, 1986) based on the experiments carried out on model systems such as liposomes, planar bilayers, and phospholipid vescicles were proposed to explain the mechanism of the reversible electroporation and/or the electrical membrane breakdown. All of these theories in their differences are characterized by advantages and disadvantages, but they share a common feature: the cell membrane plays a significant role in amplifying the applied electric field, as the conductivity of intact membrane is several orders of magnitude lower than the conductivities of extra cellular medium and cell cytoplasm (Weaver and Chizmadzhev, 1996). ...
... EP transcription efficiency was also significantly higher than US, AAV and Lipo (p<0.02). The combination of high and low voltage pulses used for electroporation here is believed to create transient pores in both the cell and nuclear membranes enhancing DNA entry and subsequent nuclear localisation (Gothelf & Gehl ; Chang 1992). ...
... Erythrocytes were obtained from male albino Wistar rats' blood (160–190 g) after centrifugation by removing the buffy coat. The conductivity of cell suspensions was measured on the same day of blood collection after washing twice with isotonic low ionic strength solution (ILISS), composed of 13 mM NaCl, 150 mM sucrose, 14 mM NaOH, and pH 7.4 [2]. Spherical erythrocytes (spherocytes) were prepared in 67% of ILISS, σ o = 0.23 S/m at 23 @BULLET C (conductometer Metrohm mod. ...
Purpose: The characterization of electroporation in biological tissues is an important aspect to understand the
process, elaborate analytical or numerical models and prepare clinical protocols. A new technique is presented to
separate the effects of intense dielectric dispersion in the tissues from the results due to electroporation.
Methods: Rat liver samples are accommodated in a parallel plate system, their admittance is measured and the
electroporation protocol is applied.
Results: Experimental results of the intact tissue’s electrical properties are presented. The electroporation
threshold with a voltage ramp decreased from 385 to 309 V/cm when the rise time of the applied field was
reduced from 1000 μs to 200 μs. For a pulse train, the conductivity increased with each pulse and the memory
effect was significant during time intervals from 1 millisecond to 6 seconds.
Conclusion: The use of the Fourier series enabled the separation of the sample’s dispersive effects, allowing for the
evaluation of the electroporation effects and the determination of the electroporation threshold. The recovery
time of the electroporated tissue is similar to that obtained for isolated cells.
The techniques outlined in Chapter 4 (except for studies with the temperature-sensitive mutants) are geared toward temporarily changing the properties of the cells by altering their external environment. For example, suppose that you always culture your cells on a laminin-coated substratum (Timpl, 1989). Even though the cells are always cultured in this manner, the effect of the laminin is temporary because the cells will most likely behave differently in its absence. In displaying two distinct behaviors in culture (i.e., with or without laminin), the cell is said to express different phenotypes.
Olivier Sandre's PhD Thesis at UPMC Université Paris 6, under the supervision of Pr Françoise Brochard-Wyart, defended on September 27, 2000 (in French, only abstract in English).
Analysis of impedance spectrum of electroporated biological cell suspensions allows evaluating the state of permeabilization produced by the applied electric field. In this article we used a genetic algorithm to determine parameters of a proposed model for the impedance of suspension adjusting them numerically in order to minimize the error in relation to experimental spectra of Kluyveromyces marxianus CBS 6556 yeasts suspended in distilled water. It was possible to determine the variation of conductivity of suspension avoiding the influence of electrode polarization and dielectric dispersion due to interfacial and diffusion polarization on the cell surface. It was observed that the conductivity in the range of 1 to 100 KHz increases 2.5 times with pulses of 400 KV/m.
Electroporatlon Is a highly effective method to Increase permeability of cell membrane by using series of short intense electric pulses. Using this technique, we can Introduce small and large molecules Into cells. The electroporatlon of biological membranes has various applications In molecular biology and medicine. Despite the numerous applications of electroporatlon, the detailed effects of electric pulse on biological membranes as well as exact mechanisms of pore formation In lMng cells are not well understood. Several in-vitro and In-vlvo experimental studies have been conducted to determine the mechanisms of action of electroporatlon in various types of membranes. Because of the small spatial and fast temporal scales of this process, direct observation of electroporatlon is difficult, theoretical models and molecular dynamic simulations have been developed to facilitate the Interpretation of experimental data and the understanding of the mechanisms of action of electroporation. The role of phospholipids In respond to external electric fields, behavIor of water dipoles In the complex electric field landscape of the membrane Interface and reorganization of water dipoles In pore formation process have been proposed In these studies. Pore characteristics such as life time, ion selectlvlty size, kinetics of formation as well as number of pores are significant factors in the proposed mechanism of action. The present study reviews the different mechanisms of action of electroporatlon proposed by different experImental and modeling studies.
Gene therapy delivery using viral vectors has demonstrated efficient transfection but has safety issues. The need for safer yet effective delivery systems has led to the active development of non-viral techniques. In this chapter, we will discuss two evolving techniques - sonoporation with microbubble contrast agents and electroporation with focus on their basic principle, parameters affecting delivery efficiency, current evolving techniques and future prospects.
The effect of ``strong'' electromagnetic fields on cells and tissue can be dramatic but not necessarily harmful. The essentially universal biophysical phenomenon of ``electroporation'' occurs if an applied field causes the cell transmembrane voltage to reach about 0.5-1 V in a time of microseconds to milliseconds. Ordinarily the cell membrane is a formidable barrier to the transport of ions and charged molecules. However, electroporation results in a large increase in transmembrane conductance, which is believed to be caused by ion transport through temporary membrane openings (``pores''). This high-conductance state limits the transmembrane voltage and thereby protects the membrane. A large increase in molecular transport generally occurs for the same conditions and allows polar molecules to be introduced into cells. Similar enhanced molecular transport can be caused in living tissues. Not only cell membranes, but also cell layers or even the stratum corneum of human skin can be temporarily altered by the electrical creation of aqueous pathways. The mechanism of electroporation is partially understood, in that the electrical and mechanical behavior of artificial planar bilayer membranes can be described quantitatively by a theoretical model based on transient aqueous pores. More complex behavior in cell membranes may be due to both the complicated shapes of cell membranes and the additional participation of metastable pores and interactions with cell structures. In the case of tissues the situation is even more complex and has only recently begun to be studied but has the prospect of providing a new approach to transporting polar molecules across tissue barriers.
The psychrotrophic bacterium Bacillus psychrophilus was successfully transformed by electroporation with the cloning vector pC194 and the expression vector pPL708. Optimal electroporation parameters such as field strength, pulse length, and electroporation medium, as well as the influence of the growth phase of the bacterial cells, were determined. Maximum transformation efficiencies of 104 transformants/μg plasmid DNA were achieved with late logarithmic – early stationary phase cells grown in medium supplemented with 33.3 mM glycine and electroporated at high field strengths of 18–20 kV∙cm−1. By means of this procedure, a kanamycin-resistant gene was directly cloned into pPL708 and the recombinant plasmid electrotransformed into B. psychrophilus. In addition, the effect of electroporation on protein synthesis was analyzed by 2-dimensional polyacrylamide gel electrophoresis and computing scanning laser densitometry. At least eight proteins were induced by electroporation field strengths of 18 or 25 kV during the first 2 h immediately following electroporation. Conversely, the synthesis of at least five proteins was repressed by electroporation.Key words: electroporation, psychrotrophic bacterium.
Heart diseases are major causes of morbidity and mortality in Western society. Gene therapy approaches are becoming promising therapeutic modalities to improve underlying molecular processes affecting failing cardiomyocytes. Numerous cardiac clinical gene therapy trials have not yet demonstrated positive results. The success of gene therapy depends largely on the creation of a reliable and efficient delivery method. The establishment of such a system is determined by its ability to overcome the existing biological barriers including cellular uptake and intracellular trafficking as well as modulation of cellular permeability. In this chapter, we describe a variety of physical and mechanical methods based on the transient disruption of the cell membrane which are applied in non-viral gene transfer. In addition we focus on the use of different physiological techniques and devices and pharmacological agents to enhance endothelial permeability. Development of these methods will undoubtedly help to solve major problems facing gene therapy.
Conductivity measurements in suspensions of biological cells have been used since many years for electroporation effectiveness evaluation. However, conductivity modeling by means of instantaneous values of current and voltage during pulse application does not take into account the effects of the sample reactance and the dielectric dispersion of the medium. This can lead to misinterpretation in the electroporation analysis. The electrical modeling and characterization of electroporation using sinusoidal signal analysis at 10 kHz proposed in this paper allows us to avoid distortions due to reactive effects of the sample. A simple equation establishes the relation between suspension conductivity and membrane conductance. This model was used in experiments with suspensions of yeast cells and applied electric fields of up to 450 kV/m for 1 ms. The analysis using the proposed model resulted in membrane conductance values of up to 8000 S/m (2) and allowed estimating the distribution profile of conductance on the cell membrane.
Electroporation, the transient increase in the permeability of cell membranes when exposed to a high electric field, is an
established in vitro technique and is used to introduce DNA or other molecules into cells. When the trans-membrane voltage
induced by an external electric field exceeds a certain threshold (normally 0.2–1V), a rearrangement of the molecular structure
of the membrane occurs, leading to pore formation in the membrane and a considerable increase in the cell membrane permeability
to ions, molecules and even macromolecules. This phenomenon is, potentially, the basis for many in vivo applications such
as electrochemotherapy and gene therapy, but still lacks a comprehensive theoretical basis. This article reviews the state
of current electroporation theories and briefly considers current and potential applications in biology and medicine.
Electroporation is a process in which a controlled electrical pulse is applied to cells, inducing a transient destabilization
of the cell membrane. During this time, the cells are highly permeable to exogenous substances in the surrounding media. DNA,
proteins, and small molecules are all taken up by cells during electroporation; introduction of DNA into cells is the most
common application. Gene transfer by electroporation offers many advantages for analysis of gene expression. The technique
is simple, rapid, and reproducible. It is especially suited to suspension cultures and certain cell types that are poorly
trans-fected by other means. Because all cells are transfected instantaneously, and essentially simultaneously, it is particularly
suited to quantitation of gene transfer. Finally, single-copy, stable transfectants can often be isolated (1). Whereas the basic mechanisms of electroporation are still largely unknown, optimizing the conditions for electroporation
of any particular cell type is primarily empirical.
Gene transfer is an emerging therapeutic modality for a wide spectrum of diseases. Its clinical adoption is, however, limited
by the lack of safe and efficient gene delivery methods. Three classes of methods are currently under evaluation. The first
class consists of genetically modified viruses, which include retroviruses, adenoviruses, adeno-associated viruses, and several
others. These vectors are relatively efficient. However, their clinical application is associated with significant safety
concerns, such as oncogenesis and acute inflammatory response. The second class is nonviral vectors, which are composed of
synthetic components. These include complexes of DNA with lipids, polymers, or their combination. Many nonviral vector formulations,
which incorporate functional components to facilitate nuclease protection, cellular/tissue targeting, endosomal release, and
nuclear localization, have been investigated, mostly in vitro. These efforts have resulted in incremental advances in gene
transfer efficiency, requiring further improvements for clinical applications. The third class of methods is based on the
use of physical energy or force. Examples are gene gun, electroporation, and magnetofection. These methods are suitable for
locoregional gene delivery. In this chapter, we will provide an overview of the state-of-the-art gene transfer methods, their
strengths and weaknesses, and challenges and opportunities in this critical area of research, which will, to a large extent,
determine the future prospect of gene therapy in the clinic.
Key WordsGene therapy-gene delivery-viral vector-nonviral vector
Although technically established in mammalian species more than 15 year ago, the efficiency of embryo cloning by nuclear transfer in terms of offspring production has been invariably low. This article reviews a series of interrelated experiments carded out from 1992 to 1997 regarding the co-ordination of nuclear and cytoplasmic events after embryo reconstruction. The experiments were undertaken at the Istituto Zootecnico e Caseario per la Sardegna (IZCS) and the sheep used belonged to the Sardinian dairy breed. The use of enucleated, pre-activated recipient cytoplasts resulted in an increased frequency of development to blastocyst of embryos reconstructed with unsynchronized blastomere nuclei. This increase in development was due to the absence of chromatin damage and unbalanced ploidy in nuclei transferred after the decay of MPF (Maturation Promoting Factor). The combination of synchronous S-phase cytoplast-karyoplast nuclear transfer together with the reduction of embryo losses from the oviduct of temporary recipients allowed for the first time the production of large numbers of clones of genetically identical lambs, thus bringing nuclear transfer closer to practical application. Finally, the last series of experiments described deals with the establishment of an alternative protocol for embryonic nuclear transfer where the combined use of a chemical activating agent, ionomycin, with a protein kinase inhibitor, 6-Dimethylaminopurine, resulted in the highest development rates to blastocyst stage of metaphase II enucleated oocytes reconstructed with embryonic nuclei described so far.
Electroporation is a membrane phenomenon which involves fundamental behavior of cell and artificial bilayer membranes, and increasingly attracts consideration for applications in biology, biotechnology and medicine. Understanding of the basic mechanisms underlying electroporation is therefore important, and provides the motivation of this review of the essential features of theoretical models of electroporation. We particularly emphasize the ability of these models to describe experimental results. Here we discuss the theoretical models that have been proposed, their underlying assumptions, and their successes and failures. Most of our emphasis is on transient aqueous pore models, which can account for: (1) key features of mechanical instability (irreversible consequence of electroporation) in planar lipid bilayers at elevated voltages, (2) dramatic reversible electrical behavior of certain planar membranes and of cell membranes, and (3) some features of molecular transport. In contrast, theories which do not explicitly treat pores appear unable to account for key electroporation phenomena, and are only briefly discussed.
Externally applied voltages can create transient, non-selective pores in a cell's membrane, a phenomenon known as electroporation. Electroporation has reduced toxicity, is easy to perform, and does not induce the immune system. Therefore, the technique has a wide range of biological and medical applications. Previous experiments show that a two-pulse protocol, which consists of a fast, large-magnitude pulse and a slow, small-magnitude pulse, can increase the efficiency of drug delivery such as gene electrotransfer. In this work, we investigate the dynamics and control of the two-pulse protocol using a macroscopic model of electroporation. Numerical simulations show that there exists a range of pore radii that cannot be sustained using the conventional, open-loop, two-pulse protocol. As a result, one may need to use pores that are significantly larger than the sizes of the targeted molecules. Moreover, it is not possible to know the rate of delivery a priori. To ensure accurate drug delivery and avoid potential damage to the cell's membrane, we explore feedback mechanisms to eliminate the gap in sustainable pore radii and thus to precisely control the electroporation process. Numerical simulations show that a straightforward feedback algorithm can achieve robust control effects. Moreover, the control algorithm is effective without knowledge of the model and thus has the potential to be implemented in experiments.
The phenomenon of membrane electroporation (ME) methodologically comprises an electric technique to render lipid and lipid-protein membranes porous and permeable, transiently and reversibly, by electric voltage pulses. It is of great practical importance that the primary structural changes induced by ME, condition the electroporated membrane for a variety of secondary processes, such as, for instance, the permeation of otherwise impermeable substances.
We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.
The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, approximately 3 and 8%, respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including a single spectrin tetramer linking two junctional complexes, three-armed Y-shaped spectrin molecules linking three junctional complexes, three-armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, double spectrin filaments linking two junctional complexes, and four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetramers or higher order oligomer-like molecules, approximately 80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrinor ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.
A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.
A simple and reproducible procedure for the introduction of DNA into mammalian cells by electroporation is described. The
parameters involving the cells, the DNA, and the electric field are investigated. The procedure has been applied to a broad
range of animal cells. It is capable of transforming more than 1% of the viable cells to the stable expression of a selectable
marker.
Sea urchin eggs and embryos subjected to high-voltage electric discharge in a medium mimicking the intracellular milieu retain their structural integrity and remain permeable, permitting substrates to enter the cytoplasm and thus assay of enzyme activity. At saturating concentrations of substrates, five of six enzymes assayed for more active (three to fifteen times) in permeabilized embryos than in permeabilized eggs, but no fertilization-related differences are seen in homogenates prepared from these same permeabilized cells. Furthermore, enzyme activity in homogenates always exceeds that in the permeabilized cell suspensions. This difference in enzyme reaction rates between unfertilized eggs and fertilized eggs is not due to differences in the diffusibility of substrates into the permeabilized cells. The activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in permeabilized cells was studied in greater detail and has the following characteristics. (i) Regulation of activity persists during early development. (ii) This regulation is not mediated by diffusible allosteric agents. (iii) Stimulation at fertilization is initiated by a rise in intracellular calcium and is further promoted by cytoplasmic alkalinization. (iv) The microenvironment experienced by this enzyme intracellularly differs from that of the enzyme in homogenates as evidenced by markedly different pH vs. activity profiles. These results indicate that the regulatory status of enzymes is preserved in electrically permeabilized cells and suggest that this regulation depends on some cell structural feature(s) that is (are) destroyed upon homogenization.
This paper demonstrates the translational movement along the plane of the human erythrocyte ghost of the membrane particles exposed by freeze-fracture. The membrane particles can be aggregated by incubation of the ghosts in media with a pH in the vicinity of 5 5 or 3 5. The particles are disaggregated in neutral and alkaline media (pH 9 5) and also at pH 4.5 Aggregation of the particles at pH 5.5 is reversible, prevented by prefixation in glutaraldehyde and by media of high ionic strength. Particle aggregation occurs within 2-4 min. These results are consistent with the concept that the erythrocyte ghost membrane is a planar fluid domain formed by a bilayer membrane continuum which is interrupted by localized, yet mobile, proteic intercalations.
Summary Electric impulses were found to cause transient permeability changes in the membranes of vesicles storing biogenic amines. Release of catecholamines induced by electric fields (of the order of 20 kV/cm and decaying exponentially with a decay time of about 150 μsec) was studied, using the chromaffin granules of bovine adrenomedullary cells as a vesicular model system. Far-UV-absorption spectroscopy was applied to determine the amount of catecholamines released from suspended vesicles. A polarization mechanism is suggested for the induction of short-lived permeability changes caused by electric fields. Such transient changes in permeability may possibly represent a part of the sequence of events leading to stimulated neurohumoral secretion.
A new technique for assaying infectivity of adenovirus 5 DNA has been developed. Viral DNA was diluted in isotonic saline containing phosphate at a low concentration, and calcium chloride was added, resulting in the formation of a calcium phosphate precipitate. DNA coprecipitated with the calcium phosphate and, when the resulting suspension was added to human KB cell monolayers, became adsorbed to the cells. Following adsorption, uptake of DNA into the cells occurred during an incubation in liquid medium at 37 ° in the continued presence of extra calcium chloride.For adenovirus 5 DNA the assay resulted in up to 100-fold more plaques than could be obtained using DEAE-dextran. Furthermore a reproducible relationship between amounts of DNA inoculated per culture and numbers of plaques produced was demonstrated. The assay was most efficient at high DNA concentrations (10–30 μg/ml); below this range the addition of carrier DNA was necessary for optimum results.In addition to adenovirus 5 DNA, the technique has been used successfully to assay infectivity of DNA from adenovirus 1 and simian virus 40.
Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.
Fusion has been reported to occur in a variety of membrane systems in response to the application of certain electric currents to the medium (Zimmermann, U., 1982, Biochim. Biophys. Acta., 694:227-277). The application of a weak but continuous alternating current causes the membranes in suspension to become rearranged into the "pearl-chain" formation. Fusion can then be induced by one or more strong direct current pulses that cause pore formation. This results in the conversion of individual membranes in the "pearl-chain" formation to a single membrane with one or more hourglass constrictions that form lumens which connect the cytoplasmic compartments. As the diameter of the lumens increases, the overall membrane shape grows to one large sphere. To further characterize electric field-induced fusion, experiments were conducted using the erythrocyte ghost as a model membrane, and a new combination of electrical circuit and fusion chamber that is simpler and improved over previous systems. All odd-shaped ghosts (collapsed or partly collapsed spherical shapes, echinocytes, discocytes, and stomatocytes) in 30 mM phosphate buffer was first converted to spherocytes and then fused with increasing yields by increasing the number of pulses. After fusion, the lateral diffusion of a fluorescent lipid soluble label (Dil) from labeled to unlabeled membranes was observed to occur both with and without the appearance in phase-contrast optics of distinct communication (lumens) between cytoplasmic compartments of the fused membranes. Connections between cytoplasmic compartments, however, were unmistakable with the instant transfer of a fluorescent water-soluble label (fluorescein isothiocyanate-dextran) from labeled to unlabeled cytoplasmic compartments upon fusion. Although pulses still resulted in the lateral diffusion of Dil to unlabeled membranes, the presence of glycerol in the medium strongly reduced the yield of lumens observable by phase-contrast optics in fusion events. The presence of glycerol also inhibited the conversion of membranes to spherocytes, but did not inhibit the lateral diffusion of Dil from labeled to unlabeled membranes.
Structural changes underlying exocytosis evoked by the application of endotoxin to Limulus amebocytes were studied at the level of detail afforded by freeze-fracture and freeze-substitution techniques combined with the time resolution of direct rapid-freezing. The results with amebocytes prepared in this manner differed from those with other secretory cells prepared by conventional means. Exocytosis begins within seconds of endotoxin treatment when the plasmalemma invaginates to form pedestallike appositions with peripheral secretory granules. The juxtaposed membranes at these pedestal appositions form several punctate pentalaminar contacts, but examination of freeze-fractured pedestals failed to reveal any corresponding changes in the intramembrane particle distribution. Small secretory granule openings or pores, which are very infrequent, appear within the first 5 s after endotoxin treatment. These pores rapidly widen and this widening is immediately followed by the sequential dissolution of the granule contents, which then move into the surrounding extracellular space. Cytoplasmic filaments connecting the plasmalemma with the granule membrane are suitably deployed to be responsible for the plasmalemma invaginations. How pores begin is not certain, but the appearance of clear spaces between the granule core and the granule membrane at this point in exocytosis supports the possibility of a role of osmotic forces.
Cell fusion plays a very important role in modern biotechnology. For example, one key procedure in genetic engineering is the introduction of exogenous genetic material into a host cell. Such insertion of genes is accomplished by either permeabilizing the cell membrane to allow entry of genetic material, or fusing the host cell with a cell containing the desired genetic material. Furthermore, cell fusion is important in the production of monoclonal antibodies, which requires fusion of antibody-producing cells with continuously dividing cancer cells such as myeloma cells (Galfre et al., 1977; Lo et al., 1984). Also, cell fusion can be used as a microinjection technique to deliver drugs which normally cannot enter a cell. One can simply fuse the cell with liposomes or red blood cell ghosts that have been preloaded with specific drugs (Schlegel and Lieber, 1987).
External electric fields have traditionally been applied in physical chemistry and biophysics to probe the ionic—electric properties and reactivities of molecules and molecular organizations such as biological membranes (Eigen and DeMaeyer, 1963; Neumann, 1986a). In recent years, electric field pulse techniques have also gained increasing importance in cellular and molecular biology, in gene technology, and in medicine. In particular, the methods of electroporation (Neumann et al., 1982) and electrofusion (Senda et al., 1979; Neumann et al., 1980; Zimmermann and Scheurich, 1981; Weber et al., 1981) have become powerful tools for cell manipulations (for reviews see Zimmermann, 1986; Berg, 1987; Sowers, 1987) and for the physical chemical study of electrically induced structural rearrangements in membranes (for review see Neumann, 1986a).
Mouse thymocytes and erythrocytes are loaded electrically with drugs and dyes in isotonic solution. The loaded cells are used for targeting the drugs to specific sites in the organism in order to achieve a controlled drug release in time and space. Erythrocytes are directed to the liver by changing the volume and the shape. Using erythrocytes as drug carrier systems, methotrexate, the most widely studied agent in chemotherapeutic cancer treatment, could be directed exclusively to the liver.Directing to other organs is obtained by either using electrically fused cells or by loading cells with magnetic particles (about 10 nm in diameter) and the drug simultaneously and by guiding the cells to any selected site of the organism. The field technique used for the loading of the cells is based on the electrical breakdown of the cell membrane which is observed when cell suspensions are subjected to external field pulses of 2 to 20 kV/cm for short time intervals (ns to ms). When an apparent membrane potential of about 1 V (pulse length in the range of μs) is reached in response to the external field the membrane breaks down reversibly. The breakdown of the membrane is associated with a remarkable and reversible permeability increase of the cell membrane. The increase in permeability depends on the strength and the duration of the field pulse. The duration of the high conductance state of the cell membrane induced by the electric field is sufficiently long to entrap large quantities of drugs or dyes inside the cells.Electron-micrographs of thymocytes subjected to field pulses of various strengths and durations show that the pulse length is a critical factor for the reversibility of the field induced effects in the cell. When loading thymocytes the field pulse length has to be less than 1 μs to avoid irreversible changes in the ultrastructure of the cell. On the other hand, erythrocytes can be subjected to field pulses of 40 μs duration without any irreversible changes in the membrane structure.The life-span of the loaded erythrocytes in the blood circulation can be considerably prolonged if loss of haemoglobin and intracellular enzymes is avoided during field application and the resealing process. The loss of intracellular proteins can be minimized if the erythrocytes are immobilized in a polymeric network of calcium-alginate during the field application. The matrix is permeable to low molecular weight compounds but impermeable towards larger molecules such as proteins. Chelation of the Ca2+-ions in the alginate network by addition of sodium citrate leads to the release of the loaded erythrocytes from the calcium-alginate matrix.
Both irreversible mechanical breakdown (rupture) and reversible electrical breakdown can occur in bilayer membranes. A unified description of both phenomena is possible if it is hypothesized that a large number of transient aqueous pores are present because of thermal fluctuations. This hypothesis is in contrast to the usual picture of a bilayer membrane as an uninterrupted sheet. A statistical theory is presented which describes a pore population by providing a diffusion-convection equation for non-interacting pores. An approximate quasi-steady state solution to this equation describes the relative number of pores of different sizes. An expression for Dp, the effective lateral diffusion constant of a pore's radius, is also obtained. As found previously by others, rupture is identified with the occurrence of a single pore which expands beyond a critical radius, rc(U), where U is the transmembrane potential. The corresponding critical transmembrane potential for rupture, Uc, is a function of the total number of pores Ntot, and is also sensitive to the pore edge energy γ and the bilayer surface energy Γ.
Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either (i) never greater than 0.5 nm in radius, (ii) much smaller in number if they were greater than 0.5 nm in radius, or (iii) shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.
The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO22+-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO22+-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.
We describe a method of using flow cytometry for determining the distribution of electroporation effects within a statistically significant cell population. Here we illustrate basic aspects of the method by investigating the electroporation of red blood cells, which have been widely used by others in previous investigations of electroporation, including studies of reversible electrical breakdown, and molecular uptake or release associated with a transient high permeability state. We make two measurements on each cell in a population of 10,000 or more cells: (1) light scatter to indicate changes in cell morphology, and (2) fluorescence to determine the uptake of a fluorescence-labeled macromolecule (FITC-dextran; 70,000 dalton). Computer processing of the single cell data allows construction of statistical distributions which reveal how electroporation occurs within a large cell population. Using this method we find that after a single 50 μs square pulse of optimal magnitude about 30% have formed spherical ghosts because of electroporation, and that two distinct ghost subpopulations occur. One subpopulation (about 10% of analyzed cells) has negligible uptake, while the second subpopulation (about 20%) consists of ghosts which have taken up significant amounts of the test macromolecule. Two interesting findings are the high frequency-of-occurrence of electroporation due to a single, optimal pulse, and the implication, because of the two distinct ghost subpopulations, that there is a significant variation in pore sizes.
A simple physical chemical method of transferring genes into eucaryotic cells is described. Electric impulses in the intensity range of 510 kV/cm with a duration of 510 [mu]s were found to appreciably increase the uptake of DNA into cells. After electric field treatment, stable transformants were obtained in a system containing mouse cells deficient in thymidine kinase and a plasmid DNA harbouring the Herpes simplex thymidine kinase gene. The efficiency of transformation well compares with the results of biochemical methods of gene transfer. The electric field technique appears unique in its ease and simplicity.
The function and fate of antibodies introduced into living cells by red cell ghosts were studied using CRM 176 (a mutant diphtheria toxin having lower toxicity than the wild-type) and antibody against fragment A of diphtheria toxin. IgG labeled with iodine and FITC was found in the cytoplasm of the recipient cells. When about 1500 molecules of anti-fragment A antibody (rabbit IgG) were introduced into diphtheria toxin-sensitive Vero cells or FL cells, these cells became resistant to the toxin and formed normal colonies. It was calculated from the survival of cells without anti-fragment A IgG under these conditions that about 300 molecules of fragment A-176 were transferred to the cells. These results showed that the antigen-antibody reaction took place in living cells as effectively as in a cell-free system. The functional stability of antibody IgG in cells was examined by exposing Vero cells containing a subminimal amount of anti-fragment A IgG (about 1000 molecules) to the toxin for 2 hr at various times after the introduction of anti-fragment A IgG. More than 50% of the initial activity of the antibody to neutralize toxin still remained even after incubation of the cells at 37°C for 20 hr. The same degree of stability was also demonstrated using iodine-labeled specific anti-fragment A IgG. The IgG recovered from the recipient cells after various times of incubation at 37°C retained its full ability to bind to fragment A-conjugated Sepharose 4B, although the total amount of IgG associated with the cells decreased about 50% in 24 hr.
PROTEINS on the cytoplasmic surface of the erythrocyte membrane, including spectrin and actin, are postulated to comprise the red cell cytoskeleton1-3, but little is known about the role of actin or its association with the membrane. We have reported that monomeric (G) actin added to erythrocyte ghosts selectively associates with a component at the cytoplasmic surface of the membrane4,5. We now show that this component is unlikely to be spectrin and that actin binding occurs by stimulated actin polymerisation.
Isotonic suspensions of erythrocytes were exposed to intense electric fields for a duration in microseconds. Time-dependent increase in the conductivity of the suspension was observed under fields greater than a threshold of about 1.5 kV/cm. The threshold was independent of the ionic strength of the medium, and changed little with temperature or with the rise time of the applied field. Under fields greater than 3 kV/cm, the time course of the conductivity increase consisted of a rapid (approx. 1 μs) and a slow (approx. 100 μs) phases. The increase is attributed primarily to large membrane conductance induced by the applied field. The membrane conductance is in the order of in the rapid phase and in the slow phase. Comparison with previous results indicates that this induced membrane conductance corresponds to the formation of aqueous pores in the cell membrane. After the applied field was removed, the conductivity of the suspension returned nearly to its initial value, indicating that the induced membrane conductance is strongly dependent on the membrane potential. The conductivity then increased again in the time range of 10 s. This is attributed to the diffusional efflux of intracellular ions through the voltage-induced pores. From the rate of the efflux, number of the pores/cell is estimated to be in the order of 102. Final stage of the conductivity change was a slow decrease, corresponding to the colloid osmotic swelling of the perforated cells.
We have evaluated the cooling rates of specimens mounted in a variety of freeze-fracture holders when plunged into a series of liquid coolants. These rates were measured using miniature thermocouples placed within the mounted specimens. The most rapid cooling rates were obtained using propane at 83 K as the coolant. When mounted on a newly devised ‘copper sandwich’ holder, specimen cooling rates in excess of 4500 K/s have been recorded.
A simple guillotine-like device for quenching freeze-fracture specimens under reproducible conditions is presented.
APPLICATION of an electric pulse, at field intensities of a few kV cm-1 and of duration in the µs range, to an isotonic suspension of erythrocytes is known to cause haemolysis of the red cells1-4. Studies from different laboratories suggest that the haemolysis is due to the field-induced transmembrane potential1,3,4. Our recent experiments5 indicate that once the transmembrane potential reaches a threshold of approximately 1 V, which corresponds to an applied field of 2.2 kV cm-1, the erythrocyte membrane becomes leaky to normally impermeant ions or molecules. The permeation of solutes leads to the swelling and eventual lysis of the red cells. This type of haemolysis is known as colloid osmotic haemolysis6,7. The voltage-induced permeability change is consistent with the formation of pores in the membrane. We show here that the size of these pores can be varied in a controlled manner, and that the leaky membrane can be resealed while the haemolysis is prevented. Foreign molecules have successfully been incorporated into the resealed, but otherwise intact, erythrocytes.
In order to develop a safe and effective way to introduce exogenous genes into cells, we have experimented with a new method of electroporation which uses a radio-frequency (RF) electric field to permeabilize the cell membrane. This RF method has several advantages over the conventional electroporation method which uses a direct current (DC) field. We have shown that the RF electroporation method can be used to introduce marker genes into a wide variety of cell lines, including COS-M6, CV-1, CHO, 3T3 and hepatocytes, and is able to increase substantially the efficiency of gene transfection. (For example, the amount of DNA required for transfecting two million COS-M6 cells can be as low as 0.1 microgram). The transfection efficiency is shown to be affected by a number of factors, including cell type, field strength, pulse protocol and medium buffer. Because of its wide range of applications, high transfection efficiency and lack of harmful side-effect, the RF electroporation method would be particularly useful for introducing genes into human cells for gene therapy.
Cells from patients with the cancer-prone inherited disease, xeroderma pigmentosum (XP) are known to be defective in the endonuclease-mediated
incision step in excision repair of a number of different types of DNA adducts, but the molecular events responsible have
not been delineated. We have previously reported isolation of two DNA endonucleases, pI 4.6 and 7.6, from normal human chromatin
which recognize adducts produced by psoralen plus long wavelength ultraviolet radiation (UVA). These endonucleases are both
present in XP complementation group A (XPA) cells even though these cells are hypersensitive to this type of damage. We now
report that introduction by electroporation of either normal endonuclease into XPA cells restored their markedly deficient
DNA repair-related unscheduled DNA synthesis (UDS) to higher than normal levels following exposure to psoralen plus UVA. Introduction
of XPA endonucleases into similarly treated XPA cells had little or no restorative effect on UDS. However, both normal and
XPA endonucleases increased UDS in normal cells to higher than normal levels. These results indicate that XPA cells have endonucleases
which can repair these adducts but which cannot function in intact cells unless a factor(s), which they lack is provided by
normal cells.
Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.
Chinese hamster ovary cells (CHO) grown in monolayers were permeabilized to molecules with molecular weight up to 1000 by high intensity 100 mus square wave electric field pulses. This permeability was transient and the cell viability was not affected. It was not possible for molecules with molecular weight larger than 1500 to penetrate inside the cytoplasm if lytic pulsing conditions were not used. In order to investigate the ultrastructural changes associated with this transient and limited permeabilization, cells were chemically fixed a few seconds after their pulsation and observed by electron microscopy. By scanning electron microscopy, numerous microvilli and blebs were observed almost immediately after application of the field. No other membrane changes were observed. Permeabilization of the membrane was visualized at the electron microscopic level by penetration of Ruthenium red. The appearance of osmotic pressure-dependent 'blebs' was indicative of local weakening of the plasma membrane. Most of these effects were fully reversible and disappeared within 30 min at 37 degrees C with the formation of huge polykaryons when cells were in contact before pulsing.
The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.
To examine the mechanism by which polymorphonuclear leukocytes (PMNs) move, phalloidin labelled with fluorescein isothiocyanate was introduced into freshly sampled cells by use of an electric-cell fusion system. The best conditions for treatment were three pulses of direct current at 100 V for a pulse duration of 3 microseconds. The treated cells retained their usual motility when observed under a microscope, so the method was suitable for the analysis of motile living cells. We used the method to study PMNs during locomotion, spreading and phagocytosis. In locomotion, fluorescence first appeared at the head of the cell and shifted gradually along the cell margin from head to tail. In spreading, diffuse fluorescence around the marginal part of the cytoplasm was strongest near both the attachment sites and the perinuclear area of the cell and spots of fluorescence appeared in the cytoplasm. In phagocytosis, fluorescence developed from the attachment sites, spread to the entire phagocytizing area of the cytoplasm and disappeared when phagocytosis ended. Cells treated with cytochalasin B were randomly spotted with fluorescence. Freshly sampled cells had diffuse and scattered fluorescence, without the lines observed in fixed cells.
Restriction endonucleases that make either blunt- or cohesive-end DNA double-strand breaks can induce chromosome aberrations. We have used cell electroporation with great success to permeabilize Chinese hamster ovary cells for the introduction of restriction enzymes. The introduction of restriction enzymes by this method resulted in extremely high frequencies (greater than 90%) of aberrant metaphase cells and also a dramatic decrease in cell survival, as measured by subsequent colony formation. Cell electroporation by itself caused no increase in aberrant chromosomes and had only a slight effect on cell survival.
It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.
Il apparait que l'electroporation (qui permet de realiser des trous dans des membranes cellulaires grâce a un choc electrique de haut voltage) est une methode simple et efficace pour introduire des gene dans des cellules animales, vegetales, des organismes unicellulaires et des microorganismes. Cette methode permet egalement un traitement des maladies genetiques chez l'homme
Controlled permeability can be conferred to cell membranes by exposing cells to a microsecond electric pulse of sufficient intensity (electroporation). By constructing a fluorescence microimaging system with a submicrosecond time resolution we have been able to resolve temporally and spatially the events in a single cell under a microsecond electric pulse. An enormous membrane conductance, corresponding to a loss of 0.01-0.1% of the membrane area, was observed in those membrane regions where the transmembrane potential induced by the electric pulse exceeded a critical value. The conductance decreased to a low level in a submillisecond after the pulse, leaving a moderately electroporated cell.
The considerable amount of activity in the field of electrofusion and electropermeabilization is very promising from the point of view of new insights into biomembranes and new technologies in the future for the production of new compounds and modification of cell systems for nutrition, energy production and the removal of waste products. It is particularly gratifying to see how basic science has provided the foundation for a useful technology, although in some cases the time needed to develop an application is very long. In other cases, it is necessary to overcome the difficulties posed by existing schools of thought which have been shown to be wrong. It is fascinating to observe the many developments and discoveries in the areas of physics, material science, space technology and electronics which are just waiting to be applied to biological systems. An increased interdisciplinary collaboration between physicists and biologists could provide considerable impetus to biology and its application in technology. However, this can only be achieved if basic research into biological membranes is accelerated. The techniques for electrical breakdown, electropermeabilization and electrofusion could be an important tool in this process, since we cannot rule out the possibility that the high electrical fields occurring naturally in the membrane play an important role in the selective transport of substances across the membrane as well as in natural regulatory processes.
The sequence of events during the electrically induced fusion of human erythrocytes was studied by rapid quench freeze-fracture electron microscopy. A single electric field pulse was used to induce fusion of human erythrocytes treated with pronase and closely positioned by dielectrophoresis. The electronic circuit was coupled to a rapid freezing mechanism so that ultrastructural changes of the membrane could be preserved at given time points. Pronase treatment enabled adjacent cells to approach each other within 15 nm during dielectrophoresis. The pulse caused a brief disruption of the aqueous boundaries which separated the cells. Within 100 msec following pulse application, the fracture faces exhibited discontinuous areas which were predominantly free of intramembranous particles. At 2 sec after the pulse, transient point defects attributed to intercellular contact appeared in the same membrane areas and replaced the discontinuous areas as the predominant membrane perturbation. At 10 sec after the pulse, the majority of the discontinuous areas and point defects disappeared as the intercellular distance returned to approximately 15 to 25 nm, except at sites of cytoplasmic bridge formation. Intramembranous particle clearing was observed at 60 sec following pulse application in discrete zones of membrane fusion.
A simple method, employing high-voltage electric discharge (electroporation), was developed to introduce phosphorylated nucleosides into the cytoplasm of viable cells. HL-60 leukemia cells permeabilized by this technique remained viable and incorporated deoxyribonucleoside triphosphates into nuclear DNA. Furthermore, DNA synthesis was depressed for at least 24 h in HL-60 cells made permeable to 1-beta-D-arabinosylcytosine 5'-triphosphate by this methodology. Electroporation was found to be applicable to the permeabilization of a wide variety of cell lines in culture to nucleotides, suggesting that this methodology may be useful for the introduction into intact cells of a wide variety of molecules that are not normally transported effectively.
An infectious retrovirus vector has been used to transfer a bacterial gene encoding resistance to the neomycin analogue G418 into pluripotent haematopoietic stem cells present in explanted murine bone marrow tissue. Subsequent transplantation of the cells into lethally irradiated mice results in engraftment of the animals with donor haematopoietic tissue containing the bacterial gene. This approach affords an efficient and rapid means of re-introducing genetically modified tissue into intact organisms and provides a system whereby the expression and regulation of cloned genes can be followed within the context of a well characterized developmental programme.
This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.
By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, τ=200 μsec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these ‘equivalent pores’ remain patent for up to 1 hr. The formation and stability of these ‘pores’ is not affected by the Ca content of the bathing solution. The ‘pores’ permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water.
Cells rendered ‘leaky’ in K glutamate medium containing 5mm Mg-ATP and EGTA to give an ionized Ca close to 10−8m release less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10−5m Ca. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 μm Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions.
Calcium-evoked release of catecholamine is associated with the release of dopamine-β-hydroxylase (DβH) but not lactate dehydrogenase. The ratio DβH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DβH and catecholamine is identical. Transmission electron microscopy of ‘leaky’ cells exposed to 10−8m Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of ‘leaky’ cells exposed to 10−5m Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles.
Ca-dependent release of both catecholamine and DβH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca.
Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DβH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate−>acetate−>Cl−>Br−>SCN−.
Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in ‘leaky’ cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic pressure, (iii) lowering the temperature below 20°C, and (iv) N-ethyl maleimide.
The resealing process of lipid bilayer membranes after reversible electrical breakdown was investigated using two voltage pulses switched on together. Electrical breakdown of the membranes was induced with a voltage pulse of high intensity and short duration. The time course of the change in membrane conductance after the application of the high (short) voltage pulse was measured with a longer voltage pulse of low amplitude. The decrease in membrane conductance during the resealing process could be fitted to a single exponential curve with a time constant of 10--2 micros in the temperature range between 2 and 20 degrees C. The activation energy for this exponential decay process was found to be about 50 kJ/mol, which might indicate a diffusion process. Above 25 degrees C the resealing process is controlled by two exponential processes. The data obtained for the time course of the resealing process can be explained in terms of pore formation in the membrane in response to the high electrical field strength. A radius of about 4 nm is calculated for the initial pore size. From the assumed exponential change of the pore area with progressive resealing time a diffusion constant of 10(-8) cm2/s for lateral lipid diffusion can be estimated.
Amphotericin B is a polyene that binds to sterols and perforates cell membranes. An antileukemic drug such as daunomycin added exogenously is impermeable to the red cell membrane. However, when the cells are incubated with a low concentration of amphotericin B, daunomycin is entrapped in the red cells without hemolysis or alteration in the chemical parameter of the erythrocytes. The erythrocyte has been used as a carrier vehicle to enhance the cytotoxic activity of daunomycin against L1210 leukemic cells. In comparison to control preparations, the greatest increase in survival was obtained in vivo when the erythrocytes with entrapped daunomycin were given to C57BL X DBA/2 F1 mice bearing L1210 cells.