Theoretical and experimental study of the effect of membrane pores on spectra of amplitudes of polarization of a cell

Spectra of amplitudes of polarization of erythrocytes, yeast cells, and latex particles in the range of 1-10 Hz were investigated by the method of dielectrophoresis. Positive dielectrophoresis of erythrocytes and yeast cells the frequency range of 60 - 100 Hz was revealed. The theoretically positive dielectrophoresis was evidenced by the occurrence of channels across the cell membrane and bacterial cell wall.     

Sensing Biophysical Alterations of Human Lung Epithelial Cells (A549) in the Context of Toxicity Effects of Diesel Exhaust Particles

Diesel exhaust particles (DEP) in urban air are associated with numerous respiratory diseases. The role of underlying biomechanics in cytotoxicity of individual lung cells relating to DEP exposure is unclear. In this study, atomic force microscopy (AFM), confocal Raman microspectroscopy (RM), and fluorescence (FL) microscopy were used to monitor alterations of single A549 cells exposed to DEP. Results revealed a significant decrease in membrane surface adhesion force and a significant change in cell elasticity as a function of DEP–cell interaction time, and the dynamic changes in cellular biocomponents which were reflected by changes of characteristic Raman bands: 726 cm^?1 (adenine), 782 cm^?1 (uracil, cytosine, thymine), 788 cm^?1 (O–P–O), 1006 cm^?1 (phenylalanine), and 1320 cm^?1 (guanine) after DEP exposure. These findings suggest that the combination of multi-instruments (e.g., AFM/FL) may offer an exciting platform for investigating the roles of biophysical and biochemical responses to particulate matter-induced cell toxicity.     

Participation of protein phosphatases in regulation of sodium transport across the erythrocyte membrane of the lamprey Lampetra fluviatilis

Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with ²²Na to determine the unidirectional Na⁺ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na⁺ transport into erythrocytes. The stimulation of Na⁺ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na⁺ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na⁺ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na⁺ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (～12%), but statistically significant decrease of intracellular Na⁺ content. A small decrease of intracellular K⁺ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na⁺ transport across the lamprey erythrocyte membrane in both directions.     

Impact of thermohaemolysis-associated membrane alteration on the passive ion permeability and life-span of erythrocytes

Suspending erythrocytes in medium containing sucrose prevented heat-induced lysis at its early stage. This allowed determination of the thermohaemolysis-related ion permeability by measuring the initial rate of the stipulated shrinkage of erythrocytes. Thus, correspondingly, the coefficient P of the ion permeability was calculated for heated human erythrocytes using ouabain-pretreated cells in 37–45°C range and intact cells in 50–58°C range. The values obtained for P obeyed a straight line Arrhenius plot over the entire 37–58°C range suggesting that the ion permeability was activated by a single mechanism earlier identified as relevant to thermohaemolysis. At the 37–58°C range, the activation energy of the P was 250±15 kJ/mol which was markedly different from the value of 56 kJ/mol known for the 10–37°C range. For erythrocytes from five mammals, similar temperature dependencies of the P were obtained over 45–60°C range. For erythrocytes from all species, excluding horse, the P, extrapolated at 37°C, had a value comparable with the known coefficient of the passive, ouabain-insensitive cation permeability at 37°C. For ouabain-treated human erythrocytes at 37°C, the period of thermohaemolysis-related shrinkage in sucrose containing media was found to be about six times shorter than the life-span of intact cells which substantiated the role of the active transport in balancing the thermohaemolysis-related diffusion of ions at 37°C. Consequently, the thermal resistance of erythrocytes, which was earlier related to their sphingomyelin content, was now found also to be in good correlation with their life-span in the circulation of 11 mammals.     

Ultra-wide band electromagnetic radiation does not affect UV-induced recombination and mutagenesis in yeast

Cell samples of the yeast Saccharomyces cerevisiae were exposed to 100 J/m² of 254 nm ultraviolet (UV) radiation followed by a 30 min treatment with ultra-wide band (UWB) electromagnetic pulses. The UWB pulses (101–104 kV/m, 1.0 ns width, 165 ps rise time) were applied at the repetition rates of 0 Hz (sham), 16 Hz, or 600 Hz. The effect of exposures was evaluated from the colony-forming ability of the cells on complete and selective media and the number of aberrant colonies. The experiments established no effect of UWB exposure on the UV-induced reciprocal and non-reciprocal recombination, mutagenesis, or cell survival. Bioelectromagnetics 19: 128–130, 1998. © 1998 Wiley-Liss, Inc.     

Interaction of diesel exhaust particles with human, rat and mouse erythrocytes in vitro

Inhaled ultrafine (nano) particles can translocate into the bloodstream and interact with circulatory cells causing systemic and cardiovascular events. To gain more insight into this potential mechanism, we studied the interaction of diesel exhaust particles (DEP) with human, rat and mouse erythrocytes in vitro. Incubation of erythrocytes with DEP (1, 10 or 100 μg/ml) for 30 min caused the highest hemolytic effect (up to 38%) in rats, compared to small but significant hemolysis in mice (up to 2.5%) and humans (up to 0.7%). Transmission electron microscopy of erythrocytes revealed the presence of variable degrees of ultrafine (nano)-sized aggregates of DEP either internalized and/or adsorbed onto the erythrocytes in the three species. A significant amount of DEP was found in rat and mouse (but not human) erythrocytes. Lipid erythrocyte susceptibility to in vitro peroxidation measured by malondialdehyde showed a significant and dose-dependent increase in erythrocytes of rats, but not humans or mice. Unlike in human erythrocytes, total antioxidant status (TAS) and superoxide dismutase (SOD) activity in rats were significantly and dose- dependently decreased. In mouse erythrocytes, DEP caused a decreased in SOD (at 10 μg/ml) and TAS (at 100 μg/ml) activities. In conclusion, DEP caused species-dependent erythrocyte hemolysis and oxidative stress, and were either taken up and/or adsorbed onto the red blood cells. Rat (and to a lesser degree mouse) erythrocytes were susceptible to DEP. Human erythrocytes showed the highest resistance to the observed effects. These species difference should be noted when using rats and mice blood as models for humans.     

Electric field distribution in biological systems

Electric field distribution in biological systems was investigated. In the analysis both the conductive and the dielectric properties of biological systems were considered. Making use of the complex dielectric coefficient, equations which describe the electric field behavior in such media, were formulated. These equations were solved numerically for a few biological systems. The solutions show that the macroscopic field distribution, for example, the refraction of the ECG wave upon passing from one tissue into another, is mainly determined by the tissue's conductive properties (in the frequency range of 0–10⁸ Hz). However, the microscopic field distribution around the individual cells is determined by the conductive, the dielectric or both properties, depending on the frequency. At frequencies below 10⁴ Hz the field configuration is determined largely by the system's conductive properties. At frequencies above 10⁷ – 10⁸ Hz, by the dielectric properties and in the range of 10⁴ – 10⁶ Hz both properties affect the field distribution. In this range the field direction may be shifted by as much as 90° by relatively small frequency changes.     

Specific inactivation of glucose metabolism from eucaryotic cells by pentalenolactone

Pentalenolactone, an antibiotic related to the class of the sesquiterpene-lactones and produced by the strain Streptomyces arenae Tü-469, inhibits specifically the glucose metabolism by inactivation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD⁺ oxidoreductase (phosphorylating) EC 1.2.1.12). The sensitivity of several eucaryotic cell-systems for pentalenolactone was shown under in vivo conditions. The glycolytic as well as the gluconeogenetic pathway of mammalian cells can be completely inhibited with low concentration of the antibiotic. In all cases, the minimum inhibitory concentration is dependent on cell density. The inhibitory effect in vivo and in vitro does not seem to be species-specific. In erythrocytes from rats, in Ehrlich-ascites tumor cells and in Plasmodium vinckei infected erythrocytes from mice glycolysis can be inhibited with concentrations of 18–90 μM pentalenolactone. In hepatocytes, glycolysis as well as gluconeogenesis is prevented by the same concentrations. In contract to these results, in yeast the inhibition depends on growth conditions. The inhibition in glucose medium is cancelled by precultivation on acetate-containing medium.     

Role of phospholipids in the DDT-induced efflux of potassium in human erythrocytes

Incubation of human erythrocytes for 1–2 h at 37°C in a suspension of dipalmitoylphosphatidylcholine (DPPC) liposomes results in a phospholipid enrichment of erythrocyte membranes by 45–55% and a depletion of cholesterol by 19–24%. The enrichment by DPPC was time and concentration dependent. By contrast, dioleoylphosphatidylcholine (DOPC) liposomes were less effective in enriching the membranes with phospholipid and in depleting the membranes of cholesterol. Concomitantly, the DDT-induced efflux of K⁺ was reduced in the case of DPPC-enriched erythrocytes but enhanced in DOPC-enriched erythrocytes. These results suggest that DDT partitions more readily into the unsaturated than the saturated phospholipids of the erythrocyte membrane. It is concluded that the extent to which DDT affects the flux of K⁺ across the membrane is dependent on the fluidity of the lipid phase. We also report here a rapid method for cholesterol depletion of red blood cells in comparison to previously reported methods.     

The taurine content of avian erythrocytes and its role in osmoregulation

• 1.1. The taurine content of erythrocytes from 15 avian species contained levels of taurine in the range of 20–70 mmol/kg of hemoglobin, about 100-fold that of mammalian red blood cells.
• 2.2. This high taurine content did not appear to be related to the nucleation of these cells as nucleated amphibian erythrocytes and human reticulocytes contained low levels.
• 3.3. The erythrocytes lacked cysteine sulfinic acid decarboxylase, a key enzyme in the synthesis of taurine from cysteine, indicating a probable lack of synthetic capabilities.
• 4.4. The cells were able to accumulate labeled taurine against a concentration gradient. This uptake was inhibited by β-alanine and was Na⁺-dependent.
• 5.5. When incubated in hypotonic medium, the cell volume of pigeon erythrocytes rapidly increased and was followed by a much slower return to normal size. The cell volume reduction was accompanied by a slow efflux of taurine into the medium.
• 6.6. These data suggest that taurine plays a role in cell volume maintenance and osmotic regulation in avian erythrocytes.

     

Modeling the influence of slurry concentration on Saccharomyces cerevisiae cake porosity and resistance during microfiltration

Filtration of an isotonic suspension of baker's yeast through a 0.45‐μm membrane was studied at two different pressures, 40 and 80 kPa, for yeast concentrations ranging from 0.14 to 51 kg/m³ (dry weight). For a yeast volume fraction above 0.06 (～21.8 kg/m³), the porosity of the yeast cake is less dependent on the suspension concentration. For highly diluted suspensions, the specific cake resistance approaches a minimum that depends on the filtration pressure. Correlation functions of cake porosity and specific cake resistance were obtained for the concentration range investigated showing that the Kozeny–Carman coefficient increases when the applied pressure increases. Both filtration pressure and slurry concentration can be process controlled. In the range of moderate yeast concentration, the filtrate flux may be increased by manipulating the filtration pressure and the slurry concentration, thereby improving the overall process efficiency. The complex behavior of yeast cakes at high slurry concentration can be described by a conventional model as long as part of yeast cells are assumed to form aggregates, which behave as single bigger particles. The aggregation effect may be accounted for using a binary mixture model. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Synchronization of pacemaker cell firing by weak ELF fields: Simulation by a circuit model

Entrainment of output action potentials from repetitively firing pacemaker cells, brought about by regularly spaced excitatory or inhibitory postsynaptic inputs, is a well-known phenomenon. Synchronization of neural firing patterns by extremely low frequency (ELF) external electric fields has also been observed. Whereas current densities of ≈10 A-m⁻² are required for direct excitation of otherwise quiescent neural tissue, much lower peak current densities (≈10⁻² A-m²) have been reported to entrain spontaneously firing molluscan pacemaker cells. We have developed a neural spike generator circuit model that simulates repetitive spike generation by a space clamped patch (area ≈ 10⁻⁷ m²) of excitable membrane subjected to depolarizing current. Picoampere (pA) range variation of DC depolarizing current causes a corresponding smooth variation of neural spike frequency, producing a physiologically realistic stimulus-response (S-R) characteristic. When lower pA range 60 Hz AC current is superposed upon the DC depolarizing current, smooth variation of the S-R characteristic is distorted by subharmonic locking of the spike generator at 30, 20, 15, 12, 10 Hz, and higher order subharmonic frequencies. Although the additional superposition of a physiologically realistic level of “white” current noise, covering the bandwidth 4–200 Hz, suffices to obscure higher order subharmonic locking, locking at 30, 20, and 15 Hz is still clearly evident in the presence of noise. Subharmonic locking is observed at a root mean square AC simulated tissue current density of ≈10⁻⁵ A-m⁻². Bioelectromagnetics 19:92–97, 1998. © 1998 Wiley-Liss, Inc.     

NMR field-cycling relaxation spectroscopy of bovine serum albumin,muscle tissue,micrococcus luteus and yeast: 14N1H-quadrupole dips

The frequency dependence of the proton spin lattice relaxation time of bovine serum albumin, muscle tissue, Micrococcus luteus and yeast has been measured by the aid of the field-cycling technique. In all systems ¹⁴N¹H-quadrupole dips have been observed. The conclusion is that amide groups are the dominating relaxation centers up to approx. 10⁷Hz. This finding can be understood by the fact that protein backbone fluctuations and, if possible, tumbling of the whole molecule rather than side group motions are the relevant mechanisms in this frequency range. A proton relaxation scheme for cells and tissue is presented.     

Effect of static magnetic fields on the budding of yeast cells

The effect of static magnetic fields on the budding of single yeast cells was investigated using a magnetic circuit that was capable of generating a strong magnetic field (2.93 T) and gradient (6100 T² m^?1). Saccharomyces cerevisiae yeast cells were grown in an aqueous YPD agar in a silica capillary under either a homogeneous or inhomogeneous static magnetic field. Although the size of budding yeast cells was only slightly affected by the magnetic fields after 4 h, the budding angle was clearly affected by the direction of the homogeneous and inhomogeneous magnetic fields. In the homogeneous magnetic field, the budding direction of daughter yeast cells was mainly oriented in the direction of magnetic field B. However, when subjected to the inhomogeneous magnetic field, the daughter yeast cells tended to bud along the axis of capillary flow in regions where the magnetic gradient, estimated by B(dB/dx), were high. Based on the present experimental results, the possible mechanism for the magnetic effect on the budding direction of daughter yeast cells is theoretically discussed. Bioelectromagnetics 31:622–629, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of manganese on potassium outflow from erythrocytes and on respiration of rat liver mitochondria

In this work, effects of manganese on respiration of rat liver mitochondria and the rate of K⁺ outflow from rat erythrocytes are studied in a broad range of concentrations. It is shown that manganese ions at low concentrations (1 × 10^–7–3 × 10^–5 М) inhibit K⁺ outflow from rat erythrocytes; this can be used to prevent their lysis. At high concentrations (1 × 10^–4–1 × 10^–3 M), manganese activates K⁺ outflow from the erythrocytes but inhibits the valinomycin-induced outflow of the ion from the erythrocytes. This fact is an indication of manganese influence on physicochemical properties of membranes. At low concentrations manganese does not affect parameters of respiration and oxidative phosphorylation of rat liver mitochondria, while at high concentrations it exerts acceleration of the mitochondrial respiration, i.e., uncouples respiration from phosphorilation and, hence, inhibits ATP synthesis.     

Kinetic properties of sodium transport pathways in the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> erythrocytes

To activate Na⁺/H⁺ exchange, intracellular pH (pH_i) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na⁺/H⁺ exchanger activity was estimated from the values of amiloride-sensitive components of Na⁺ (²²Na) inflow or of H⁺ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na⁺ and H⁺ transport on pH_i value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H⁺ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H⁺ outcome from acidified erythrocytes (pH_i 5.9) with increase of H⁺ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na⁺ for the semimaximal stimulation of H⁺ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H⁺ from the cytoplasm side and the presence of positive cooperativity in Na⁺-binding from the extracellular side of the Na⁺/H⁺ exchanger. Na⁺ efflux from cells in the Na⁺-free medium did not change at a 10-fold increase of H⁺ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na⁺/H⁺ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na⁺ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na⁺ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Li+ effect on the cell wall of the yeast Saccharomyces cerevisiae as probed by FT-IR spectroscopy

The effect of Li⁺ ions as a transformation inducing agent on the yeast cell wall has been studied. Two Saccharomyces cerevisiae strains, p63-DC5 with a native cell wall, and strain XCY42-30D(mnn1) which contains structural changes in the mannan-protein complex, were used. Fourier transform infrared (FT-IR) spectroscopy has been used for the characterization of the yeast strains and for determination of the effect of lithium cations on the cell wall. A comparison of the carbohydrate absorption band positions in the 970–1185 cm^?1 range, of Na⁺ and Li⁺ treated yeast cells has been estimated. Absorption band positions of the cell wall carbohydrates of p63-DC5 were not influenced by the studied ions. On the contrary, the treatment of XCY42-30D(mnn1) cells with Li⁺ ions shifted glucan band positions, implying that the cell wall structure of strain XCY42-30D(mnn1) is more sensitive to Li⁺ ion treatment.     

Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2)

The mechanism by which H⁺ alters the kinetics of the H⁺-coupled peptide transporters PEPT 1 and PEPT 2 was investigated in two different cell lines which differentially express these transporters, namely Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). The effects of H⁺ on the affinity and the maximal velocity of Gly-Sar uptake were analyzed in these cells under identical conditions. In both cells, H⁺ influenced only the maximal velocity of uptake and not the apparent affinity. The effects of H⁺ on the IC₅₀ values (i.e., concentration necessary to cause 50% inhibition) of the cationic dipeptide Ala-Lys and the anionic dipeptide Ala-Asp for inhibition of Gly-Sar uptake were also investigated. H⁺ did not change the IC₅₀ value for Ala-Lys but did decrease the IC₅₀ value for Ala-Asp considerably. The influence of diethylpyrocarbonate (DEP) on the kinetic parameters of PEPT 1 and PEPT 2 was then studied. Histidyl residues are the most likely amino acid residues involved in H⁺ binding and translocation in H⁺-coupled transport systems and DEP is known to chemically modify histidyl residues and block their function. DEP treatment altered the maximal velocity of Gly-Sar uptake but had no effect on its K_t (Michaelis-Menten constant) or the IC₅₀ values of Ala-Lys or Ala-Asp for the inhibition of Gly-Sar uptake. It is concluded that H⁺ stimulates PEPT 1 and PEPT 2 primarily by increasing the maximal velocity of the transporters with no detectable influence on the substrate affinity.     

Spinning response of yeast cells to rotating electric fields

The frequency-dependent rotation or spinning motion of yeast cells subjected to a fourpole rotating electric field was examined over a very wide frequency range (500 Hz to 500 MHz). In the lower frequency range (500 Hz – 700 KHz) the yeast cells were observed to spin in a direction counter to the applied field, with a small peak at about 600 Hz and a more pronounced one at 20 KHz. For frequencies above 700 KHz the spinning of the cells switched direction from counter-field to co-field, with a maximum in the rotation rate at about 70 MHz and a subpeak at 20 MHz. The rate was also observed to exhibit a square dependence on the magnitude of the applied rotating field.     

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance.