Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA

Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form “pearl chains” which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.     

Effect of Twisted Fiber Anisotropy in Cardiac Tissue on Ablation with Pulsed Electric Fields

BackgroundAblation of cardiac tissue with pulsed electric fields is a promising alternative to current thermal ablation methods, and it critically depends on the electric field distribution in the heart.MethodsWe developed a model that incorporates the twisted anisotropy of cardiac tissue and computed the electric field distribution in the tissue. We also performed experiments in rabbit ventricles to validate our model. We find that the model agrees well with the experimentally determined ablation volume if we assume that all tissue that is exposed to a field greater than 3 kV/cm is ablated. In our numerical analysis, we considered how tissue thickness, degree of anisotropy, and electrode configuration affect the geometry of the ablated volume. We considered two electrode configurations: two parallel needles inserted into the myocardium (“penetrating needles” configuration) and one circular electrode each on epi- and endocardium, opposing each other (“epi-endo” configuration).ResultsFor thick tissues (10 mm) and moderate anisotropy ratio (a = 2), we find that the geometry of the ablated volume is almost unaffected by twisted anisotropy, i.e. it is approximately translationally symmetric from epi- to endocardium, for both electrode configurations. Higher anisotropy ratio (a = 10) leads to substantial variation in ablation width across the wall; these variations were more pronounced for the penetrating needle configuration than for the epi-endo configuration.For thinner tissues (4 mm, typical for human atria) and higher anisotropy ratio (a = 10), the epi-endo configuration yielded approximately translationally symmetric ablation volumes, while the penetrating electrodes configuration was much more sensitive to fiber twist.ConclusionsThese results suggest that the epi-endo configuration will be reliable for ablation of atrial fibrillation, independently of fiber orientation, while the penetrating electrode configuration may experience problems when the fiber orientation is not consistent across the atrial wall.     

Structural transition produced by electric fields in aqueous sodium deoxyribonucleate

It was found that the birefringence of aqueous solutions of sodium DNA is anomalous when electric fields of high intensity (≥10⁴ v/cm) are applied. The magnitude of the birefringence first rose upon application of the orienting pulse, then fell as the field was sustained above a critical value. The occurrence of the effect depended upon macromolecular and electrolyte concentrations. Upon removal of the field, the birefringence was rapidly restored and then it decayed with an increase of the reorientational relaxation times, relative to those observed below the critical field. It is proposed that the electric field may cause aggregation of the macromolecules and then produce a structural transition concomitant with the electric field orientation effect. This transition may correspond to the “B” “A” structures identified in x-ray studies, or to a “B” “V” structure change, where “V” is a postulated new helical form stabilized by cooperative interactions of base and dipoles in the electric field. Field induced transitions of this type would be of interest in connection with molecular mechanisms of transport through membranes, nerve impulse transmission, or information storage.     

Electroendocytosis Is Driven by the Binding of Electrochemically Produced Protons to the Cell’s Surface

Electroendocytosis involves the exposure of cells to pulsed low electric field and is emerging as a complementary method to electroporation for the incorporation of macromolecules into cells. The present study explores the underlying mechanism of electroendocytosis and its dependence on electrochemical byproducts formed at the electrode interface. Cell suspensions were exposed to pulsed low electric field in a partitioned device where cells are spatially restricted relative to the electrodes. The cellular uptake of dextran-FITC was analyzed by flow cytometery and visualized by confocal microscopy. We first show that uptake occurs only in cells adjacent to the anode. The enhanced uptake near the anode is found to depend on electric current density rather than on electric field strength, in the range of 5 to 65 V/cm. Electrochemically produced oxidative species that impose intracellular oxidative stress, do not play any role in the stimulated uptake. An inverse dependence is found between electrically induced uptake and the solution’s buffer capacity. Electroendocytosis can be mimicked by chemically acidifying the extracellular solution which promotes the enhanced uptake of dextran polymers and the uptake of plasmid DNA. Electrochemical production of protons at the anode interface is responsible for inducing uptake of macromolecules into cells exposed to a pulsed low electric field. Expanding the understanding of the mechanism involved in electric fields induced drug-delivery into cells, is expected to contribute to clinical therapy applications in the future.     

Kinetic Theory Model for Ion Movement through Biological Membranes: II. Interionic Selectivity

The equation presented in the previous paper for steady-state membrane ionic current as a function of externally applied electric field strength is numerically analyzed to determine the influence of ionic and membrane molecule parameters on current densities. The model displays selectivity between different ions. A selectivity coefficient S_i, defined as the ratio of current carried by an ionic species i at a given field strength to the current carried by a reference species at the same field strength, has the following properties: (a) S_i is a function of electric field strength except for ion-membrane molecule interactions yielding velocity independent collision frequencies; (b) for ion-membrane molecule interactions characterized by a collision frequency that is a decreasing (increasing) function of increasing ionic velocity, ions whose S_i > 1 (<1) at zero field strength will show maxima (minima) (minima[maxima]) in their S_i vs. electric field strength curves.     

Neural crest cell galvanotaxis: new data and a novel approach to the analysis of both galvanotaxis and chemotaxis

The galvanotaxis response of neural crest cells that had migrated out of the neural tube of a 56-hr-old quail embryo onto glass coverslips was observed using time-lapse video microscopy. These cells exhibit a track velocity of about 7 microns/min and actively translocate toward the negative pole of an imposed DC electric field. This nonrandom migration could be detected for fields as low as 7 mV/mm (0.4 mV/cell length). We find that this directional migration is independent of the speed of migration and have generated a rather simple mathematical equation that fits these data. We find that the number of cells that translocate at a given angle, phi, with respect to the field is given by the equation N(phi) = exp(a0 + a1cos phi), where a1 is linearly proportional to the electric field strength for fields less than 390 mV/mm with a constant of proportionality equal to KG, the galvanotaxis constant. We show that KG = (150 mV/mm)-1, and at this field strength the cellular response is approximately half maximal. This approach to cellular translocation data analysis is generalizable to other directed movements such as chemotaxis and allows the direct comparison of different types of directed movements This analysis requires that the response of every cell, rather than averages of cellular responses, is reported. Once an equation for N(phi) is derived, several characteristics of the cellular response can be determined. Specifically, we describe 1) the critical field strength (390 mV/mm) below which the cellular response exhibits a simple, linear dependence on field strength (for larger field strengths, an inhibitory constant can be used to fit the data, suggesting that larger field strengths influence a second cellular target that inhibits the first); and 2) the amount of information the cell must obtain in order to generate the observed asymmetry in the translocation distribution (for a field strength of 100 mV/mm, 0.3 bits of information is required).     

Differential Epigenetic Effects of Atmospheric Cold Plasma on MCF-7 and MDA-MB-231 Breast Cancer Cells

Cold atmospheric plasma (plasma) has emerged as a novel tool for a cancer treatment option, having been successfully applied to a few types of cancer cells, as well as tissues. However, to date, no studies have been performed to examine the effect of plasma on epigenetic alterations, including CpG methylation. In this study, the effects of plasma on DNA methylation changes in breast cancer cells were examined by treating cultured MCF-7 and MDA-MB-231 cells, representing estrogen-positive and estrogen-negative cancer cells, respectively, with plasma. A pyrosequencing analysis of Alu indicated that a specific CpG site was induced to be hypomethylated from 23.4 to 20.3% (p < 0.05) by plasma treatment in the estrogen-negative MDA-MB-231 cells only. A genome-wide methylation analysis identified “cellular movement, connective tissue development and function, tissue development” and “cell-to-cell signaling and interaction, cell death and survival, cellular development” as the top networks. Of the two cell types, the MDA-MB-231 cells underwent a higher rate of apoptosis and a decreased proliferation rate upon plasma treatment. Taken together, these results indicate that plasma induces epigenetic and cellular changes in a cell type-specific manner, suggesting that a careful screening of target cells and tissues is necessary for the potential application of plasma as a cancer treatment option.     

Differential Cellular Effects of Electroporation and Electrochemotherapy in Monolayers of Human Microvascular Endothelial Cells

In vivo electroporation of tumours shows disruption of blood flow and creates a vascular effect with an initial rapid and transient vasoconstriction phase and a much longer lasting phase with changed microvascular endothelium. These changes are not well understood but are presumed to involve the cytoskeleton. The paper presents for the first time differential in vitro effects describing cytoskeleton changes and monolayer integrity changes by both electroporation and electrochemotherapy of monolayers of human microvascular endothelial cells (HMEC-1). After the application of electric field pulses, the morphology of cells, and both the F-actin and Beta-tubulin cytoskeleton proteins were affected. During both electroporation and electrochemotherapy, the initial phase of cellular damage was noticed at 10 min as swollen cells and honeycomb-like actin bundles. The electroporation-induced cellular effects, observed from electric pulses >150 V, were voltage-dependent and within 24 hrs partly recoverable. The electrochemotherapy-induced cellular effects developed at 2 hrs in spindle-like cells, and more densely packed F-actin and Beta-tubulin were observed, which were dependent on the amount of bleomycin and the voltages applied (>50 V). In addition, for electrochemotherapy with electric pulses >150 V cellular changes were not recoverable within 24 hrs. The effects on monolayer integrity were reflected in the enhanced monolayer permeability, with the electrochemotherapy showing an earlier onset and synergy. We conclude that electrochemotherapy as compared to electroporation leads within 24 hrs to a quicker and more pronounced monolayer integrity damage and endothelial cell death, which together provide further insight into the cellular changes of the vascular disruption of electrochemotherapy.     

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation.     

Effect of Direct-Current Electric Field on Enzymatic Activity and the Concentration of Laccase

This work investigates the effect of direct-current electric field on the extracellular enzymatic activity, concentration and other experimental parameters of laccase from Trametes versicolor. The results showed that laccase could significantly contribute to the change of pH at the end of graphite electrode. In addition, it increased the electrical conductivity of the water. In the experiment, the optimum pH and catalytic pH range for laccase activity were 3.0 and pH 2.5–4.0. The application of 6 V direct current showed significant effects on the laccase enzyme activity. The activity of laccase was enhanced in the anodic region, but at the same time was strongly inhibited at the cathode. The electric charge characteristics of laccase were changed when exposed to electric field, and some laccases molecules moved to the anode, which produced a slight migration phenomenon. This study is the basis of combination of laccase and electrical technology, at the same time, providing a new direction of enhancing laccase activity. Compared to immobilization, using electric field is simple, no chemical additives, and great potential.     

Isolation and Validation of an Endogenous Fluorescent Nucleoid Reporter in Salmonella Typhimurium

In this study we adapted a Mud-based delivery system to construct a random yfp reporter gene (encoding the yellow fluorescent protein) insertion library in the genome of Salmonella Typhimurium LT2, and used fluorescence activated cell sorting and fluorescence microscopy to screen for translational fusions that were able to clearly and specifically label the bacterial nucleoid. Two such fusions were obtained, corresponding to a translational yfp insertion in iscR and iolR, respectively. Both fusions were further validated, and the IscR::YFP fluorescent nucleoid reporter together with time-lapse fluorescence microscopy was subsequently used to monitor nucleoid dynamics in response to the filamentation imposed by growth of LT2 at high hydrostatic pressure (40–45 MPa). As such, we were able to reveal that upon decompression the apparently entangled LT2 chromosomes in filamentous cells rapidly and efficiently segregate, after which septation of the filament occurs. In the course of the latter process, however, cells with a “trilobed” nucleoid were regularly observed, indicative for an imbalance between septum formation and chromosome segregation.     

Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10⁶ V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase.     

Biophysical aspects of dielectrophoresis

Dielectrophoresis, the translational motion produced by the action of a nonuniform electric field upon neutral polarizable bodies, is proving useful in handling biological systems. It is especially useful in studies of suspensions of cells. Normal and abnormal cells respond differently, and may be separated on the basis of their differing polarizational response. Because the phenomenon depends upon the polarization and not upon the charge of the particles, subtle new differences are available for exploitation by dielectrophoresis. Operation can be carried out in a high frequency electric field. The frequency spectrum of cellular response to dielectrophoresis varies with the species, and with the physiological state of the individual cell. Cells may be studied individually or in large numbers at a time. Cells may be gathered and formed into fleshlike aggregates using dielectrophoresis.Extension of the techniques to the study of the fundamental nature of the polarizabilities of cells and subcellular particles should provide useful insight into the roles of electronic, atomic dipole orientational, and nomadic polarization, as well as that due to interfacial (Maxwell-Wagner) polarization in biological systems.     

Electric Imaging through Evolution,a Modeling Study of Commonalities and Differences

Modeling the electric field and images in electric fish contributes to a better understanding of the pre-receptor conditioning of electric images. Although the boundary element method has been very successful for calculating images and fields, complex electric organ discharges pose a challenge for active electroreception modeling. We have previously developed a direct method for calculating electric images which takes into account the structure and physiology of the electric organ as well as the geometry and resistivity of fish tissues. The present article reports a general application of our simulator for studying electric images in electric fish with heterogeneous, extended electric organs. We studied three species of Gymnotiformes, including both wave-type (Apteronotus albifrons) and pulse-type (Gymnotus obscurus and Gymnotus coropinae) fish, with electric organs of different complexity. The results are compared with the African (Gnathonemus petersii) and American (Gymnotus omarorum) electric fish studied previously. We address the following issues: 1) how to calculate equivalent source distributions based on experimental measurements, 2) how the complexity of the electric organ discharge determines the features of the electric field and 3) how the basal field determines the characteristics of electric images. Our findings allow us to generalize the hypothesis (previously posed for G. omarorum) in which the perioral region and the rest of the body play different sensory roles. While the “electrosensory fovea” appears suitable for exploring objects in detail, the rest of the body is likened to a “peripheral retina” for detecting the presence and movement of surrounding objects. We discuss the commonalities and differences between species. Compared to African species, American electric fish show a weaker field. This feature, derived from the complexity of distributed electric organs, may endow Gymnotiformes with the ability to emit site-specific signals to be detected in the short range by a conspecific and the possibility to evolve predator avoidance strategies.     

Cellular spin resonance: Theory and experiment

Living and dead cells, as well as certain inanimate particles, can be made to spin in a rotating electric field, such as that produced by a four-pole electrode system. The theory for the effects and the experimental results for a number of cells are given. Yeast cells (Saccharomyces cerevisiae, dead, and as protoplasts;Schizosaccharomyces octospora); an alga (Chlorella pyrenoidosa); mouse myeloma cells; human Hela cells; and several model particles were studied. Their spectra of spin response are shown to agree with the theory presented. Interestingly, live cells spin in a contrafield direction, while dead ones spin co-field in the low-frequency range. The result indicate that this new technique of cellular spin resonance (CSR) will be useful in determining the effective dielectric constant of individual cells, for the direction and magnitude of the CSR is directly proportional to the effective dielectric constant, and the spin rate can readily and directly be determined over a wide frequency range.     

A Targeted Mutation within the Feline Leukemia Virus (FeLV) Envelope Protein Immunosuppressive Domain To Improve a Canarypox Virus-Vectored FeLV Vaccine

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the “mechanical” function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be “switched off” by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

Existence of abnormal protein aggregates in healthy Escherichia coli cells

Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and “healthy” Escherichia coli cells. We observed overrepresentation of “aberrant proteins,” as well as substrates of the major conserved chaperone DnaK (Hsp70) and the protease ClpXP (a serine protease), in the aggregates. In addition, the protein aggregates appeared to interact with chaperones known to be involved in the aggregate repair pathway, including ClpB, GroEL, GroES, and DnaK. Finally, we showed that the levels of reactive oxygen species and unfolded or misfolded proteins determine the levels of protein aggregates. Our results led us to speculate that protein aggregates may function as a temporary “trash organelle” for cellular detoxification.     

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO₄) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.