Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissues.     

Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

Previous studies have shown greater fluorophore uptake during electroporation on the anode-facing side of the cell than on the cathode-facing side. Based on these observations, we hypothesized that hyperpolarizing a cell before electroporation would decrease the requisite pulsed electric field intensity for electroporation outcomes, thereby yielding a higher probability of reversible electroporation at lower electric field strengths and a higher probability of irreversible electroporation (IRE) at higher electric field strengths. In this study, we tested this hypothesis by hyperpolarizing HL-60 cells using ionomycin before electroporation. These cells were then electroporated in a solution containing propidium iodide, a membrane integrity indicator. After 20 min, we added trypan blue to identify IRE cells. Our results showed that hyperpolarizing cells before electroporation alters the pulsed electric field intensity thresholds for reversible electroporation and IRE, allowing for greater control and selectivity of electroporation outcomes.     

Electroporation: parameters affecting transfer of DNA into mammalian cells

Electroporation, the reversible breakdown of cell membranes caused by a high-voltage discharge, is a rapid, simple, and efficient method for introducing DNA into mammalian cells. An instrument for electroporation which permits the high-voltage discharge waveform to be varied with respect to rise time, peak voltage, and fall time is described. The uptake and expression of SV40 DNA following electroporation of two cell types, a human carcinoma-derived cell line, HEp-2, and a human lymphoblastoid cell line, 721, depended on the peak voltage and the fall time of the voltage discharge. The electronic parameters which produced optimum DNA transfer, however, differed for the two cell types. DNA as large as 150 kb was introduced into cells by electroporation. Cells can be electroporated in either phosphate-buffered saline or culture medium containing fetal bovine serum, and the efficiency of DNA transfer does not vary with cell densities from 10(6) to 2 X 10(7)/0.5 ml. Exposing the cells to multiple voltage discharges did not improve DNA transfer. DNA has been introduced by electroporation into all cell types tested, including human carcinoma-derived cell lines, human lymphoblastoid cell lines, human fibroblast strains, and primary human lymphocytes. To obtain maximal DNA transfer by this method, however, one must optimize the peak voltage and fall time of the discharge waveform for each cell type.     

A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.     

Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii.

Azotobacter vinelandii cells were transformed via high-voltage electroporation, with the broad host-range plasmid pRK2501. The number of transformants was dependent on the applied voltage, capacitance, and recovery procedure after electroporation. For example, Log, 4.44 transformants microgram-1 DNA were recovered in the A. vinelandii cell suspension electroporated at 1500 V and 25 microF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 microgram/ml-1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance). Electroporation at 2500 V and capacitance settings of 25 and 3 microF did not produce any transformants. Cell survival was also poor at high voltages. A. vinelandii transformants were not recovered on N-free agar medium. In addition, no viable cells were recovered on N-free agar after electroporation at 2500 V, 25 microF; 2500 V, 3 microF; and 1500 V, 25 microF. Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.     

Electroporation of cells

By measuring uptake of the membrane impermeable dye. phenosafranine, it can be shown that the plasma membrane of intact cells within cell aggregates can be reversibly permeabilized by electroporation. However, the plant cell wall is a barrier to DNA uptake by intact cells, although under certain circumstances expression of DNA, electroporated into intact cells, can be demonstrated. The level of expression is about 20–50 times lower than that obtained by electroporation of protoplasts, and depends on cell wall properties and pretreatments of cell aggregates. In contrast, efficient transformation of whole cells of bacteria and yeasts can be achieved by electroporation. Factors which influence DNA transfer into whole plant cells and the possibility of stable transformation are discussed.     

Effect of Pulsed Electric Field Treatments on Permeabilization and Extraction of Pigments from Chlorella vulgaris

The effect of pulsed electric field (PEF) treatments of different intensities on the electroporation of the cytoplasmatic membrane of Chlorella vulgaris, and on the extraction of carotenoids and chlorophylls were investigated. Staining the cells with propidium iodide before and after the PEF treatment revealed the existence of reversible and irreversible electroporation. Application of PEF treatments in the range of 20–25 kV cm^?1 caused most of the population of C. vulgaris to be irreversibly electroporated even at short treatment times (5 pulses of 3 µs). However, at lower electric field strengths (10 kV cm^?1), cells that were reversibly electroporated were observed even after 50 pulses of 3 µs. The electroporation of C. vulgaris cells by PEF higher than 15 kV cm^?1 and duration is higher than 15 µs increased significantly the extraction yield of intracellular components of C. vulgaris. The application of a 20 kV cm^?1 for 75 μs increased the extraction yield just after the PEF treatment of the carotenoids, and chlorophylls a and b 0.5, 0.7, and 0.8 times, respectively. However, further increments in electric field strength and treatment time did not cause significant increments in the extraction yield. The extraction of carotenoids from PEF-treated C. vulgaris cells after 1 h of the application of the treatment significantly increased the extraction yield in comparison to the yield obtained from the cells extracted just after the PEF treatment. After PEF treatment at 20 kV cm^?1 for 75 µs, extraction yield for carotenoids, and chlorophylls a and b increased 1.2, 1.6, and 2.1 times, respectively. A high correlation was observed between irreversible electroporation and percentage of yield increase when the extraction was conducted after 1 h of the application of PEF treatment (R: 0.93), but not when the extraction was conducted just after PEF treatment (R: 0.67).     

Transient gene expression in electroporated protoplasts and intact cells of sugar beet

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.     

High-efficiency gene transfection by in situ electroporation of cultured cells

It is demonstrated in this study that high-efficiency gene transfection can be obtained by directly electroporating cultured mammalian cells in their attached state using a pulsed radio-frequency (RF) electric field. A plasmid DNA containing the reporter gene beta-gal was introduced into COS-M6 cells and CV-1 cells using this in situ electroporation method. At the optimal electric field strength (1.2 kV/cm), we found that over 80% of the M6 cells took up and expressed the beta-gal gene with a cell survival rate of about 50%. In contrast, the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension. It was shown that CV-1 cells could also be electroporated highly efficiently using the in situ method. Furthermore, we have measured the time required to express the beta-gal gene after the plasmid DNA was introduced. We found that the percentage of cells expressing beta-gal reached a peak value about 10 h after electroporation. This time-course was the same for both attached and suspended cells, suggesting that the observed difference in transfection efficiency was mainly the result of effects of the detachment treatment on the electroporation process rather than on the gene expression.     

Cell membrane electroporator with digital generation of random shaped pulses

A Digital Poration System (DPS), a versatile device for electrotreatment of biological objects by electric field pulses; was designed, constructed, and implemented. A feature distinguishing DPS from the currently available electroporators based on capacitor discharge through the load is the use of a digital-to-analog converter card as a generator of pulses applied for electroporation of biological membranes, with further amplification of the pulse by both voltage and current. The shape of pulses, including bipolar pulses, is arbitrarily programmable in DPS unlike other electroporators providing exponentially decaying and square-wave pulses only. Thus, the application area of DPS is substantially extended. In DPS, many of the drawbacks inherent in capacitor electroporators are removed, including the need for an additional external pulse analyzer monitoring and logging the electroporation processes, the necessity to recharge the capacitor before any new pulse, a poor precision of setting and measuring the pulse parameters, the need for an additional generator of long-lasting low-voltage signals for electrophoresis of ions into the porated object, the need for additional AC generators for the alignment of cells before, after, and during electroporation, and the need for an additional microprocessor to control multi-pulse and/or repetitive protocols. DPS provides a slew rate of about 1 V/1 ns required for the electroporation of most mammalian somatic cells, with +/- 250 V output voltage and 500 Ohm load resistance. The application area of DPS is much wider than for the available porators. It includes electrochemotherapy, cell electrofusion, oocyte activation by mimicking calcium waves (the latter two are the crucial components of mammalian organism cloning technology), dielectrophoretic bunching and orientation ordering of cells, sorting of cells, and electrophoresis of charged species into the cells.     

Cell membrane fluidity related to electroporation and resealing

In this paper, we report the results of a systematic attempt to relate the intrinsic plasma membrane fluidity of three different cell lines to their electroporation behaviour, which consists of reversible and irreversible electroporation. Apart from electroporation behaviour of given cell lines the time course required for membrane resealing was determined in order to distinguish the effect of resealing time from the cell’s ability to survive given electric pulse parameters. Reversible, irreversible electroporation and membrane resealing were then related to cell membrane fluidity as determined by electron paramagnetic resonance spectroscopy and computer characterization of membrane domains. We found that cell membrane fluidity does not have significant effect on reversible electroporation although there is a tendency for the voltage required for reversible electroporation to increase with increased membrane fluidity. Cell membrane fluidity, however, may affect irreversible electroporation. Nevertheless, this effect, if present, is masked with different time courses of membrane resealing found for the different cell lines studied. The time course of cell membrane resealing itself could be related to the cell’s ability to survive.     

Addition of serum to electroporated cells enhances survival and transfection efficiency

Optimal electroporation efficiency of many cell types is associated with poor survival. We show that serum rapidly reseals the membranes of electroporated cells and that timely addition of serum following electroporation can improve cell survival and transfection efficiency.     

The effects of Tubulin denaturation on the characterization of its polymerization behavior

We report here upon a simulation study examining the effect of a dynamic mode of tubulin denaturation upon the kinetic and thermodynamic characterisation of the polymer formed for two idealized models of a tubulin polymerization reaction: (i) an irreversibly polymerizing system; and (ii) a reversibly polymerizing system. The effects of each denaturation mode upon the two model systems behavior are highlighted by interpretation of the data in terms of the classical Oosawa reversible condensation polymerization model. We reveal here findings which suggest that the measurement strategy in concert with Tubulin's instability over the time course of the experiment may bias the results obtained so as to make an irreversible system's behavior conform to the equilibrium model or alternatively distort the results obtained from a truly reversible system to produce values of the critical concentration quite seriously in error. It was also found that Tubulin denaturation may seriously distort parameter estimates gained from a kinetic characterization of the system (e.g. nucleus size and growth rate constant).     

The invasion of HeLa cells by Salmonella typhimurium: reversible and irreversible bacterial attachment and the role of bacterial motility

The interactions which brought about the invasion of HeLa cells by Salmonella typhimurium consisted of a sequence of three phases. Initially, the motility of the bacteria facilitated their contact with the HeLa cells whereupon the bacteria became attached in a reversible manner (i.e. the bacteria could be removed readily by washing the HeLa cell monolayers with Hanks' Balanced Salt solution). The binding forces responsible for reversible attachment were probably the weak long-range forces of the secondary minimum level of attractive interactions between the bacterium and the HeLa cell. Reversible attachment was a necessary interlude before the bacteria became irreversibly attached to the surfaces of the HeLa cells (i.e. the bacteria were no longer removed by the washing procedure that removed the reversibly attached salmonellae). Irreversible attachment was prevented in solutions of low ionic strength; the forces responsible were probably those of the primary minimum generated between the HeLa cell and a bacterial adhesion which was capable of acting over only short distances between the reversibly attached bacterium and the HeLa cell (i.e. probably less than 15 nm). Only irreversibly attached bacteria proceeded to the third phase and were internalized by the HeLa cells.     

Voltages up to 600V did not affect cryopreserved bovine spermatozoa on capillary-type electroporation

Physical methods such as electroporation have been used to improve the DNA uptake efficiency of sperm cells. This study aims to develop an efficient capillary-type electroporation method for incorporation of exogenous DNA into bovine cryopreserved sperm cells with minimal detrimental effects for later use in SMGT. Electroporation of the samples was performed in 2 different groups (with 1?μg of DNA and without DNA transfection) and under five different voltages: 500?V, 600?V, 700?V, 800?V and 900?V. Non-electroporated sperm cells (with and without DNA) were used as control. Kinetics parameters were determined using computer assisted semen analyses, whereas membrane integrity, fluidity, mitochondrial function and DNA uptake were evaluated by flow cytometry. Results revealed that all tested voltages reduced electroporated sperm motility (P?<?0.05) when compared to the control (non-electroporated cells). Mitochondrial function results showed no statistical difference among groups. Similarly, groups electroporated with lower (500?V, 600?V and 700?V) voltages showed no difference in cell membrane integrity and fluidity. Groups electroporated at higher voltages (800?V and 900?V) demonstrated negative effects in cells membrane integrity when compared to other groups and control. Also, all electroporated groups demonstrated significant higher percentages of transfected sperm cells when compared to the control group (P?<?0.05). Under the recommendation of using voltages up to 600?V, this method represents a safe and efficient alternative for electroporation of bovine spermatozoa.     

A role for clathrin in reassembly of the Golgi apparatus

The Golgi apparatus is a highly dynamic organelle whose organization is maintained by a proteinaceous matrix, cytoskeletal components, and inositol phospholipids. In mammalian cells, disassembly of the organelle occurs reversibly at the onset of mitosis and irreversibly during apoptosis. Several pharmacological agents including nocodazole, brefeldin A (BFA), and primary alcohols (1-butanol) induce reversible fragmentation of the Golgi apparatus. To dissect the mechanism of Golgi reassembly, rat NRK and GH3 cells were treated with 1-butanol, BFA, or nocodazole. During washout of 1-butanol, clathrin, a ubiquitous coat protein implicated in vesicle traffic at the trans-Golgi network and plasma membrane, and abundant clathrin coated vesicles were recruited to the region of nascent Golgi cisternae. Knockdown of endogenous clathrin heavy chain showed that the Golgi apparatus failed to reform efficiently after BFA or 1-butanol removal. Instead, upon 1-butanol washout, it maintained a compact, tight morphology. Our results suggest that clathrin is required to reassemble fragmented Golgi elements. In addition, we show that after butanol treatment the Golgi apparatus reforms via an initial compact intermediate structure that is subsequently remodeled into the characteristic interphase lace-like morphology and that reassembly requires clathrin.     

Carbohydrate control over carotenoid build‐up is conditional on fruit ontogeny in clementine fruits

The final contents of primary and secondary metabolites of the ripe fruit depend on metabolic processes that are tightly regulated during fruit ontogeny. Carbohydrate supply during fruit development is known to influence these processes but, with respect to secondary metabolites, we do not really know whether this influence is direct or indirect. Here, we hypothesized that the sensitivity of clementine fruit metabolism to carbohydrate supply was conditional on fruit developmental stage. We applied treatments increasing fruit load reversibly or irreversibly at three key stages of clementine (Citrus clementina Hort. ex Tan.) fruit development: early after cell division, at the onset of fruit coloration (color break) and near maturity. The highest fruit load obtained by early defoliation (irreversible) had the highest impact on fruit growth, maturity and metabolism, followed by the highest fruit load obtained by early shading (reversible). Final fruit size decreased by 21 and 18% in these early irreversible and reversible treatments, respectively. Soluble sugars decreased by 18% in the early irreversible treatment, whereas organic acids increased by 46 and 29% in these early irreversible and reversible treatments, respectively. Interestingly, total carotenoids increased by 50 and 18%, respectively. Changes in leaf starch content and photosynthesis supported that these early treatments triggered a carbon starvation in the young fruits, with irreversible effects. Furthermore, our observations on the early treatments challenge the common view that carbohydrate supply influences positively carotenoid accumulation in fruits. We propose that early carbon starvation irreversibly promotes carotenoid accumulation.     

高压电场不可逆电穿孔诱发A549肺癌细胞凋亡的实验研究

目的：不可逆电穿孔是治疗肿瘤的新兴技术,本文探讨高压电场引起的不可逆电穿孔诱发A549肺癌细胞凋亡的特点。方法：选择处于生长周期的A549细胞,共分为A—G7个组进行研究,其中A组为不施加电场的空白对照组,B-G组为实验组,B组施加500V／cm强度高压电场,G组施加1750V／cm的高压电场,BG组之间各组的高压电场强度间隔为250V／cm。采取细胞抑制实验、不可逆电穿孔示踪实验、细胞凋亡实验,检验A549细胞细胞凋亡与电场强度的关系。结果：①各实验组与对照组、各实验组之间的细胞抑制率,均存在显著性差异（P〈0．05）;②电场强度≥1000V／cm时,细胞不可逆电穿孔率明显增加,有统计学意义（P〈0．05）;电场强度≥1500V／cm时,细胞不可逆电穿孔率增加不明显,无统计学意义（P〉0．05）;③电场强度≥1250V／cm时,细胞早期凋亡率明显增加,有统计学意义（P〈0．05）。结论：高压电场不可逆电穿孔诱发A549肺癌细胞发生早期凋亡的强度为1250V／cm,发生晚期凋亡的强度为1500V／cm,且凋亡率随着电场强度的增加持续升高。这对于高压电场不可逆电穿孔效应引起的肿瘤细胞凋亡机制的研究具有重要意义。     

Irreversible inactivation of magnesium-dependent neutral sphingomyelinase 1 (NSM1) by peroxynitrite,a nitric oxide-derived oxidant

Previous results have indicated that the generation of ceramide by hydrolysis of sphingomyelin by magnesium-dependent neutral sphingomyelinase 1 (NSM1) is reversibly inhibited by hydrogen peroxide (H2O2) and oxidized glutathione (GSSG). This redox-dependent reversible regulation of NSM1 activity has been shown to involve the reversible formation and breakage of disulfide bonds. In this paper, we show that peroxynitrite, a nitric oxide-derived oxidant generated by SIN1, inactivates dose-dependently the NSM1 activity in an irreversible manner. In addition, we show that, in contrast to the reversible inhibition of NSM1 by H2O2 or GSSG which involves the formation of disulfide bonds, irreversible inactivation of this enzyme by peroxynitrite generated from SIN1 is likely due to definitive oxidative thiol modification. These results suggest that depending on the nature of the oxidative stress, the enzymatic activity of NSM1 could be reversibly or irreversibly inactivated.