Dielectric properties of mouse lymphocytes and erythrocytes

In order to study the effect of the nucleus on dielectric behavior of the whole cell, permittivity (dielectric constant) and conductivity of mouse lymphocytes and erythrocytes were measured over a frequency range from 0.1 to 250 MHz. Erythrocytes (spherocytes) showed a single dielectric dispersion, which was explained by a single-shell model that is a conducting sphere covered with a thin insulating shell. On the other hand, lymphocytes showed a broad dielectric dispersion curve which was composed of two subdispersions. The high-frequency subdispersion, which was not found for erythrocytes, was assigned to the Maxwell-Wagner dispersion of the nucleus occupying about 65% of the total cell volume. Analysis of the lymphocyte dispersion was carried out by a double-shell model, in which a shelled sphere, i.e., nucleus, is incorporated into the single-shell model. The following electrical parameters were consequently estimated; the capacitance of the plasma membrane, 0.86 microF.cm-2; the conductivity of the cytoplasm, 3.2 mS.cm-1; the capacitance and conductance of the nuclear envelope are, respectively, 0.62 microF.cm-2 and 15 S.cm-2, and the permittivity and conductivity of the nucleoplasm are 52 and 13.5 mS.cm-1.     

Transient Features in Nanosecond Pulsed Electric Fields Differentially Modulate Mitochondria and Viability

It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0–80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.     

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells

Nanosecond, high‐voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP‐induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators—rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt‐quenched calcein—we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO‐PRO‐1 influx) was detected in addition to mitochondrial membrane permeabilization. Bioelectromagnetics 33:257–264, 2012. © 2011 Wiley Periodicals, Inc.     

Fine structural observations on nuclear maturation during spermiogenesis in Hydra littoralis

Cytodifferentiation during spermiogenesis in Hydra littoralis was studied at the fine structural level. Concentration of nuclear material as well as specific orientation of granular and filamentous nuclear elements are apparent in two regions of the early spermatid: where the nuclear envelope is in contact with mitochondrial membranes at one pole of the cell and at an opposite region where the nucleus is closely apposed to the plasma membrane. Ultimately the mass of condensed nuclear material becomes concentrated at the mitochondrial pole of the cell. Additional electron-dense material is extruded from the nucleus into a large vacuole which is in continuity with the nuclear membrane as well as associated with Golgi lamellae and vesicles. Eventually all residual cytoplasm is sloughed, leaving the nucleus, mitochondria, and flagellum. These observations are suggestive of nucleocytoplasmic interactions during development, especially influences of mitochondria and plasma membranes on chromatin condensation.     

Monitoring the disruption of nuclear envelopes in interphase cells with GFP-beta-galactosidase.

The nuclear envelope of eukaryotic cells provides a barrier separating nucleus from cytoplasm, thereby regulating the exchange of macromolecules between both compartments. However, in cells exposed to severe forms of stress this barrier may break down, resulting in the mixing of nuclear and cytoplasmic contents. We show here that the fusion protein GFP-beta-galactosidase can be used to evaluate the intactness of nuclear envelopes in HeLa cells that have been exposed to heat and oxidative stress. GFP-beta-galactosidase is restricted to the cytoplasm of interphase cells, but enters the nucleus when nuclear membranes are disrupted. For comparison, we have analyzed the barrier function of nuclear membranes with antibodies against lamin B. Treatment of fixed cells with digitonin permeabilizes the plasma membrane, but leaves nuclear envelopes intact. Consequently, after digitonin incubation antibodies to lamin B can bind their antigen only if nuclear membranes are damaged. For various heat and oxidative stress conditions, we have compared the distribution of GFP-beta-galactosidase with the accessibility of lamin B to antibodies. Our results demonstrate that nuclear envelopes are permeable to antibodies whenever GFP-beta-galactosidase enters the nucleus. GFP-beta-galactosidase is therefore a useful tool for evaluating the disintegration of the nuclear envelope and identifying cells in which a mixing of nuclear and cytoplasmic material takes place.     

Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.     

Protein translocation in apoptosis.

In programmed cell death (apoptosis), receptor-generated or other signals are transmitted to all cellular compartments, resulting in an apoptotic cell with extensive cytoplasmic and nuclear alterations. Protein translocation is now recognized as being crucial in the induction, amplification and regulation of this process. Diverse mechanisms trigger protein translocation to and from the plasma membrane, mitochondrion and nucleus during apoptosis. This review discusses where, why and how the various protein-translocation events take place and highlights their importance in the execution and regulation of apoptosis.     

Nonlinear Dispersive Model of Electroporation for Irregular Nucleated Cells

In this work, the electroporation phenomenon induced by pulsed electric field on different nucleated biological cells is studied. A nonlinear, non‐local, dispersive, and space–time multiphysics model based on Maxwell’s and asymptotic Smoluchowski’s equations has been developed to calculate the transmembrane voltage and pore density on both plasma and nuclear membrane perimeters. The irregular cell shape has been modeled by incorporating in the numerical algorithm the analytical functions pertaining to Gielis curves. The dielectric dispersion of the cell media has been modeled considering the multi‐relaxation Debye‐based relationship. Two different irregular nucleated cells have been investigated and their response has been studied applying both the dispersive and non‐dispersive models. By a comparison of the obtained results, differences can be highlighted confirming the need to make use of the dispersive model to effectively investigate the cell response in terms of transmembrane voltages, pore densities, and electroporation opening angle, especially when irregular cell shapes and short electric pulses are considered. Bioelectromagnetics. 2019;40:331–342. © 2019 Wiley Periodicals, Inc.     

DNA damage targets PKCη to the nuclear membrane via its C1b domain

Translocation to cellular membranes is one of the hallmarks of PKC activation, occurring as a result of the generation of lipid secondary messengers in target membrane compartments. The activation-induced translocation of PKCs and binding to membranes is largely directed by their regulatory domains. We have previously reported that PKCη, a member of the novel subfamily and an epithelial specific isoform, is localized at the cytoplasm and ER/Golgi and is translocated to the plasma membrane and the nuclear envelope upon short-term activation by PMA. Here we show that PKCη is shuttling between the cytoplasm and the nucleus and that upon etoposide induced DNA damage is tethered at the nuclear envelope. Although PKCη expression and its phosphorylation on the hydrophobic motif (Ser675) are increased by etoposide, this phosphorylation is not required for its accumulation at the nuclear envelope. Moreover, we demonstrate that the C1b domain is sufficient for translocation to the nuclear envelope. We further show that, similar to full-length PKCη, the C1b domain could also confer protection against etoposide-induced cell death. Our studies demonstrate translocation of PKCη to the nuclear envelope, and suggest that its spatial regulation could be important for its cellular functions including effects on cell death.     

Theoretical evaluation of voltage inducement on internal membranes of biological cells exposed to electric fields

Several reports have recently been published on effects of very short and intense electric pulses on cellular organelles; in a number of cases, the cell plasma membrane appeared to be affected less than certain organelle membranes, whereas with longer and less intense pulses the opposite is the case. The effects are the consequence of the voltages induced on the membranes, and in this article we investigate the conditions under which the induced voltage on an organelle membrane could exceed its counterpart on the cell membrane. This would provide a possible explanation of the observed effects of very short pulses. Frequency-domain analysis yields an insight into the dependence of the voltage inducement on the electric and geometric parameters characterizing the cell and its vicinity. We show that at sufficiently high field frequencies, for a range of parameter values the voltage induced on the organelle membrane can indeed exceed the voltage induced on the cell membrane. Particularly, this can occur if the organelle interior is electrically more conductive than the cytosol, or if the organelle membrane has a lower dielectric permittivity than the cell membrane, and we discuss the plausibility of these conditions. Time-domain analysis is then used to determine the courses of the voltage induced on the membranes by pulses with risetimes and durations in the nanosecond range. The particularly high resting voltage in mitochondria, to which the induced voltage superimposes, could contribute to the explanation why these organelles are the primary target of many observed effects.     

Nanosecond Electric Pulse Effects on Gene Expression

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 microns2, of which 34.5 microns2 was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 micron2 of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 microns2 (6.2%). Mitochondria occupied 7.3 microns2 (3.3%) and the Golgi network 5.49 microns2 (2.5%). Decidual secretory bodies occupied 0.91 micron2 (0.42%) and cytoplasmic processes 1.89 micron2 (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 microns of plasma membrane. The mean nuclear membrane measured 28.3 microns (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 microns (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 microns (400% pm, 52.6% cm); Golgi membrane measured 30.77 microns (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 microns (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 microns or 2.9% cm). A mean of seven such processes was observed per 87.8 microns of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

Leukemic cell intracellular responses to nanosecond electric fields

Intense, nanosecond (ns) pulsed electric fields (PEFs) are known to affect the intracellular structures of cells. The probability of preferentially inducing subcellular effects increases with decreasing pulse length while effects on the plasma membrane are diminished. This has been demonstrated by applying electrical pulses of 60 and 10 ns duration with electric field intensities of up to 6.5 MV/m to HL-60 cells. Using confocal microscopy, PEF-induced changes in the integrity of the plasma membrane and nucleus were measured by recording fluorescence changes with propidium iodide (PI) and acridine orange (AO), respectively. Results suggest that high voltage, nsPEFs target the nucleus and modify cellular functions while plasma membrane effects are delayed and become smaller as pulse duration is shortened. Cell viability was not affected by these pulses. In spite of the high pulsed electric fields, thermal effects can be neglected because of the ultrashort pulse duration. The results suggest application of this ultrashort pulse technology to modulate nuclear structure and function for potential therapeutic benefit.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE : III. Continuity between Plasma Membrane and Nuclear Envelope in Androgonial Cells of Blasia

An ultrastructural study of late-stage androgonial cells of Blasia pusilla, a thallose liverwort, showed the nearly spherical nuclei often lying close or appressed to the cell walls. In some cells the two membranes comprising the nuclear envelope separated, the inner membrane continuing intact as a limiting boundary of the nucleus and the membrane on the outer, cytoplasmic side recurving away from the nucleus to continue without evident interruption around the periphery of the cell as the plasma membrane. It is believed that Blasia offers the first completely convincing demonstration of the heretofore problematic continuity of cytoplasmic membranes. A possible sequence of events leading to this unusual relationship between nucleus and cytoplasm is suggested. The sequence includes blebbing of the outer membrane of the nuclear envelope and subsequent membrane proliferation, apparent isolation of cytoplasmic ground substance, fusion of internal membrane with the ectoplast, and migration that finally brings the nucleus into flat contact with the wall. While this manifestation of membrane continuity may be anomalous, it is not presently considered the result of cell injury.     

Ultrastructural studies on early events in fertilization of the bivalveLaternula limicola

Summary Ultrastructural studies on sperm-egg interaction at the time of fertilization inLaternula limicola were performed. The temporary-acrosome did not change morphologically while the sperm passed through the egg investments. At the onset of sperm entrance into the egg, however, the temporary-acrosome and mitochondria were eliminated from the sperm. Afterwards the sperm was engulfed by the egg surface without membrane fusion of the gametes. After entry the sperm nucleus was surrounded by four membranes: the plasma membranes of the egg and of the sperm, and the membranes of the sperm nuclear envelope. As the sperm nucleus differentiated into the male pronucleus, the plasma membranes of both the sperm and egg were initially vesiculated, then dispersed into the egg cytoplasm. Finally, the sperm nuclear envelope changed into the male pronuclear membrane accompanying sperm chromatin dispersion.     

Luteinizing Hormone Receptors are Confined in Mesoscale Plasma Membrane Microdomains Throughout Recovery from Receptor Desensitization

We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane. After brief exposure to human chorionic gonadotropin (hCG), LH receptors remained for several hours desensitized to hCG challenge. Throughout this period, significantly increased numbers of LH receptors were confined within smaller diameter (<120 nm) membrane compartments and associated with DRM fragments of characteristically low density. By 5 h, when cells again produced cAMP in response to hCG, unoccupied LH receptors were found in larger 169 ± 22 nm diameter cell-surface membrane compartments and >90 % of LH receptors were again found in high-density membrane fragments characteristic of bulk plasma membrane. Taken together, these results suggest that, during recovery from LH receptor desensitization, LH receptors are both located with DRM lipid environments and confined within small, mesoscale (80–160 nm) cell-surface compartments. This may reflect hormone-driven translocation of receptors into DRM and formation there of protein aggregates too large or too rigid to permit effective signaling. Once bound hormone is removed, receptor structures would have to dissociate before receptors can again signal effectively in response to hormone challenge. Moreover, such larger protein complexes would be more easily constrained laterally by membrane structural elements and so appear resident in smaller cell-surface compartments.     

Measurement of Dipole Potential in Bilayer Lipid Membranes by Dielectric Spectroscopy

Planar bilayer lipid membranes formed from egg phosphatidylcholine in aqueous media containing the lipophilic anion, dipicrylamine (DPA), were studied by dielectric spectroscopy over a frequency range of 10 Hz–10 MHz. The membranes showed dielectric relaxation due to the translocation of DPA between the membrane interfaces. Incorporating either cholesterol or 6-ketocholestanol into the membranes increased the characteristic frequency of the relaxation, which is proportional to the translocation rate constant of DPA. The results suggested that the sterol dipoles induced positive potential changes within the membrane interior. The changes of the dipole potential were 70 mV for cholesterol and 150 mV for 6-ketocholestanol when the sterol mole fraction was 0.67. The opposite effect was caused by phloretin added to the aqueous media, and the maximum dipole potential change was ?90 mV at 100 μM.     

Effects of Heracleum persicum ethyl acetate extract on the growth, hyphal ultrastructure and aflatoxin biosynthesis in Aspergillus parasiticus

The ethyl acetate extract of leaves, seeds and flowers of Heracleum persicum, a medicinal plant of Iran (family Apiaceae) inhibited growth and aflatoxin (AF) production of Aspergillus parasiticus. On the basis of total dry weight growth inhibition by the leaf extract ranged from 17.1 to 36.9 %, by the flower extract from 32.2 to 75.6 %, and by the seed extract from 27.5 to 74.9 %. Production of AFB₁ and AFG₁ was inhibited in a dose-dependent manner, with a reduction of 88.5–100 % at the highest concentration of 8,000 μg/ml tested. The flower extract decreased ergosterol content of hyphae most significantly. Electron microscopy further revealed structural defects in the treated A. parasiticus including disruption of cytoplasmic membranous compartments, detachment of plasma membrane from the cell wall, and disorganization of hyphal compartments. Collapsed hyphae without conidiation, shorter branches and undifferentiated hyphal tips were also evident. The results indicate that H. persicum extract exerts antifungal and anti-AF activities by disrupting plasma membrane integrity and permeability mainly through interference with ergosterol biosynthesis. These results show that H. persicum can serve as a potent and safe alternative for inhibiting toxigenic aspergilli growth and thus preventing AF contamination of foods and feeds.