Changes of intracellular Na+ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intracellular Na⁺ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na concentrations and the intensities of PEF does not follow linear or exponen-tial behavior. As the intensities increase, the intracellular Na⁺concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na⁺ concentration decreases. Ouabain can in-hibit the decrease of intracellular Na concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na⁺ , K⁺ -ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell perme-abilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell mem-branes are discussed.     

Changes of intracellular Na+ concentration in erythrocytes caused by pulsed electrical field

Changes of sodium ionic concentration of human erythrocytes applied to pulsed electrical field (PEF) were studied by using shift reagent and NMR spectroscopy. The results show that the concentration of intraceUular Na⁺ increases with the increasing intensity of PEF when the erythrocytes are applied to PEF with higher intensities. The relationship between intracellular Na⁺ concentrations and the intensities of PEF does not follow linear or exponential behavior. As the intensities increase, the intracellular Na⁺ concentrations increase even faster by an exponential curve. However under effects of PEF at lower intensities, intracellular Na⁺ concentration decreases. Ouabain can inhibit the decrease of intracellular Na⁺ concentration, and the inhibition increases with the increasing concentration of ouabain, suggesting that Na⁺, K⁺-ATPase on cell membrane can be activated by PEF at lower intensities. Direct measurement of activities of the enzyme by using Malachite green method has confirmed this observation. Cell permeabilities to ions, activation of enzymes by electrical fields and transmission of physical signals like PEF across cell membranes are discussed.     

Influence of pulsed electromagnetic and pulsed vector magnetic potential field on the growth of tumor cells

Aims and Background: Tumor diseases cause 20% of deaths in Europe and they are the second most common cause of death and morbidity after cardiovascular diseases. Thus, tumor cells are target of many therapeutic strategies and tumor research is focused on searching more efficient and specific drugs as well as new therapeutic approaches. One of the areas of tumor research is an issue of external fields. In our work, we tested influence of a pulsed electromagnetic field (PEMF) and a hypothetic field of the pulsed vector magnetic potential (PVMP) on the growth of tumor cells; and further the possible growth inhibition effect of the PVMP. Methods: Both unipolar and bipolar PEMF fields of 5?mT and PVMP fields of 0?mT at frequencies of 15?Hz, 125?Hz and 625?Hz were tested on cancer cell lines derived from various types of tumors: CEM/C2 (acute lymphoblastic leukemia), SU-DHL-4 (B-cell lymphoma), COLO-320DM (colorectal adenocarcinoma), MDA-BM-468 (breast adenocarcinoma), and ZR-75-1 (ductal carcinoma). Cell morphology was observed, proliferation activity using WST assay was measured and simultaneous proportion of live, early apoptotic and dead cells was detected using flow cytometry. Results: A PEMF of 125?Hz and 625?Hz for 24?h–48?h increased proliferation activity in the 2 types of cancer cell lines used, i.e. COLO-320DM and ZR-75-1. In contrast, any of employed methods did not confirm a significant inhibitory effect of hypothetic PVMP field on tumor cells.     

Alterations of intracellular calcium concentration in mice neuroblastoma cells by electrical field and UVA

By using a FURA2 ratio imaging method, the intracellular free calcium concentration was investigated in cultured mice neuroblastoma cells under the influence of an amplitude-modulated (AM) field (5 kHz sine wave AM 16 Hz sinusoidal 800 V/m and 80 V/m), as well as of electric field pulses (300-ms unipolar pulses of 1000 V/m and 800 V/m, 5 pulses during 10 s and 50 pulses during 100 s). An increase in free intracellular calcium was found in about 50% of cells after field application, whereas in control experiments only about 20% of the cells showed similar increases. However, this effect depended on the amount of UV irradiation used for excitation of FURA2 fluorescence. Experiments with 1/30 to former total illumination no longer demonstrated an increase in control cells or in cells treated with AM fields. The number of cells showing calcium increase after the application of pulsed fields was reduced significantly. Therefore, the UV light itself, applied as double flashes for the fluorescence measurement, activates the cellular calcium regulation. These findings offer a possible explanation for the low reproducibility of field effects found in different laboratories, in which investigations were performed with different equipment using different intensities of UV excitation. Bioelectromagnetics 18:595–597, 1997. © 1997 Wiley-Liss, Inc.     

Changes in biosynthesis of exopolysaccharide in Lactococcus lactis subspecies cremoris treated by moderate pulsed electric field treatment

Metabolome analysis and physicochemical analyses were executed with cell extracts of a Lactococcus lactis subspecies cremoris strain treated by moderate pulsed electric field (PEF) to elucidate the mechanism of enhanced production of exopolysaccharide (EPS) by the treatment. Metabolome analysis by capillary electrophoresis time of flight mass spectrometry annotated 224 metabolites from the cytoplasmic extract of the strain, which, however, showed no significant changes in metabolites related to the EPS production. Electron microscopic observation and chemical analysis of undecaprenoids as carrier of EPS biosynthetic intermediates suggested that PEF treatment dissociated immature EPSs from the intermediates due to the focal electro-condensation of hydrogen ions at the cell surface. Thus, liberated undecaprenyl phosphates were recycled efficiently, which resulted in mass increase of EPS with smaller molecular weight. The study suggested the feasibility of moderate PEF treatment as a food processing technique and revealed the mechanism of enhanced production of EPS by the treatment.     

Experimental studies on ultralow frequency pulsed gradient magnetic field inducing apoptosis of cancer cell and inhibiting growth of cancer cell

The morphology characteristics of cell apoptosis of the malignant tumour cells in magnetic field-treated mouse was observed for the first time. The apoptotic cancer cell contracted, became rounder and divorced from adjacent cells; the heterochromatin condensed and coagulated together along the inner side of the nuclear membrane; the endoplasmic reticulums (ER) expanded and fused with the cellular membrane; many apoptotic bodies which were packed by the cellular membrane appeared and were devoured by some lymphocytes and plasma. Apoptosis of cancer cells was detected by terminal deoxynucleotidyl transferase mediated in situ nick end labeling (TUNEL). It was found that the number of apoptosis cancer cells of the sample treated by the magnetic field is more than that of the control sample. The growth of malignant tumour in mice was inhibited and the ability of immune cell to dissolve cancer cells was improved by ultralow frequency (ULF) pulsed gradient magnetic field; the nuclei DNA contents decreased, indi     

Effects of pulsed magnetic field on the formation of magnetosomes in the Magnetospirillum sp. strain AMB‐1

Magnetotactic bacteria are a diverse group of microorganisms which possess one or more chains of magnetosomes and are endowed with the ability to use geomagnetic fields for direction sensing, thus providing a simple and excellent model for the study of magnetite‐based magnetoreception. In this study, a 50 Hz, 2 mT pulsed magnetic field (PMF) was applied to study the effects on the formation of magnetosomes in Magnetospirillum sp. strain AMB‐1. The results showed that the cellular magnetism (R_mag) of AMB‐1 culture significantly increased while the growth of cells remained unaffected after exposure. The number of magnetic particles per cell was enhanced by about 15% and slightly increased ratios of magnetic particles of superparamagnetic property (size <20 nm) and mature magnetosomes (size >50 nm) were observed after exposure to PMF. In addition, the intracellular iron accumulation slightly increased after PMF exposure. Therefore, it was concluded that 50 Hz, 2 mT PMF enhances the formation of magnetosomes in Magnetospirillum sp. strain AMB‐1. Our results suggested that lower strength of PMF has no significant effects on the bacterial cell morphologies but could affect crystallization process of magnetosomes to some extent. Bioelectromagnetics 31:246–251, 2010. © 2009 Wiley‐Liss, Inc.     

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 μmol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [¹⁴C]pyruvate to haemoglobin and the rate of efflux of [¹⁴C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent K_m and V_max for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.     

~(12)C~(6 )注入小麦种子胚乳引起M_1代的变化

通过控制单核能大小 ,将 1 2 C离子注入小麦种子胚乳 ,结果引起 M1 代植株的较大变化。这些变化包括 :生物学性状的变异 ;根尖细胞染色体畸变率和微核率的显著提高 ;POD酶活性显著提高和 CAT酶活性、MDA含量和蛋白质含量的较大程度的下降等。本文也讨论了诱变胚乳对胚作用的可能机理     

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.     

Changes in streptonigrin lethality during adaptation of Escherichia coli to picolinic acid correlation with intracellular picolinate and iron uptake

Uptake studies with [¹⁴C]picolinate and ⁵⁵Fe³⁺ have provided an explanation for the change in streptonigrin killing on adaptation of Escherichia coli to picolinate, in terms of the available iron within the cell. When picolinic acid is added to a growing culture of E. coli an interval of bacteriostasis ensues; this adaptation period is followed by resumption of exponential growth. Addition of picolinate (4 mM) to a log phase culture of strain W3110 gave protection from the lethal action of streptonigrin (30 μM) when the two agents were added simultaneously. In contrast streptonigrin killed cells that had adapted to picolinate; however, a preincubation of adapted W3110 with phenethyl alcohol protected the cells from streptonigrin lethality. [¹⁴C]Picolinate uptake studies showed that initially picolinate entered the cells, but that it was excluded from adapted cells; addition of phenethyl alcohol permitted the entry of picolinate into adapted W3110. The changes in streptonigrin killing parallel the changes in concentration of intracellular picolinate, which can chelate the iron required by streptonigrin for its bactericidal action. ⁵⁵Fe³⁺ uptake studies showed that initially picolinate prevented iron accumulation by strain W3110, whereas adapted cells did take up iron in the presence of picolinate. Addition of phenethyl alcohol prevented any observed uptake of iron by adapted W3110. This modulation of iron transport by picolinate also affects streptonigrin lethality. Experiments with iron transport mutants showed that picolinate acted on both the enterochelin and citrate routes of uptake. Therefore picolinate affects the concentration of available iron within the cell both by (a) its intracellular presence resulting in chelation of iron and (b) its action on iron uptake; these effects explain the change in streptonigrin killing on adaptation of E. coli to picolinate.     

Absorbance changes of carotenoids and bacteriochlorophylls responding to the change of local electrical field induced by hydrophobic anion,tetraphenylborate in membranes of photosynthetic bacteria

Large blue-shifts of carotenoid absorption bands were induced by dark addition of a hydrophobic anion, tetraphenylborate, in chromatophores and cell membranes of photosynthetic bacteria, Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata. Tetraphenylborate also induced a red-shift of the 850 nm absorption band and a blue-shift and broadening of the 800 nm band of bacteriochlorophyll. From the analysis of the relation between the magnitude and isosbestic wavelength of the absorbance changes the tetraphenylborate-induced carotenoid band shift were assumed to reflect the change of local electrical field close to each carotenoid molecule which exists as a minor pool on the light-harvesting pigment-protein complex II (LHC II). Absorbance changes of carotenoid and chlorophylls were also induced by tetraphenylborate in membranes of spinach chloroplasts.     

Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions

Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl₃ was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 μM; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 μM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 μm-1 mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 μM to 100 μM.     

The effect of lead ions on the energy metabolism of human erythrocytes in vitro

The aim of this work was to evaluate the influence of chronic exposure to lead ions on the parameters of energetic status of human erythrocytes in vitro. Umbilical cord erythrocytes were incubated with lead acetate at final lead ion concentrations ranging from 10 to 200 microg/dl. ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine, IMP, inosine, hypoxanthine, NAD and NADP concentrations in erythrocytes were determined using HPLC. Scanning electron micrographs of erythrocytes were taken. The mean concentrations of ATP, GTP, NAD and NADP, and mean values of adenylate energy charge (AEC) and GEC in cells incubated at the presence of lead ions were significantly lower after 20 h of incubation. Concentrations of purine degradation products (Ado, Guo, Ino) and Hyp were significantly higher. It is suggested that lead ions affect the energy metabolism of erythrocytes. Morphological changes in erythrocytes correspond to the increase of lead ions in the incubation mixture and to the decrease of ATP concentration in erythrocytes. A decrease in NAD and ATP concentration in erythrocytes could be a sensitive indicator of energy process disturbance, useful in monitoring in case of chronic lead exposure.     

Pertussis toxin inhibits the rise in the intracellular concentration of free calcium that is induced by chemotactic factors in rabbit neutrophils: possible role of the "G proteins" in calcium mobilization

The addition of pertussis toxin to rabbit neutrophils inhibits the rise in the intracellular concentration of free calcium induced by the chemotactic factors fMet-Leu-Phe and leukotriene B4. At high concentrations of fMet-Leu-Phe, the inhibitory effect of the toxin is more on the stimulus-induced increase in membrane permeability to calcium than on calcium mobilization from internal stores. These results suggest that the "G protein" system either directly or indirectly is involved in the regulation of the stimulus-induced changes in the calcium mobilization and/or gating systems.     

Location of the extrinsic subunit PsbP in photosystem II studied by pulsed electron-electron double resonance

The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (Y_D) in PsbD, the D2 subunit of plant photosystem II, were 50.8?±?3.5?Å, 54.9?±?4.0?Å, 57.8?±?4.9?Å, and 58.4?±?14.1?Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of Y_D relative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed Y_D coordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.