Registration of changes in the nucleoide conformation based on the analysis of the time dependence of viscosity

The sensitivity of the method of anomalous time dependence of viscosity to changes in the conformation of DNA-protein complexes (such as nucleoide) by the action of (1) nalidixic acid on cells and (2) one- and two-charged ions Na+, Cl-, Cu2+, Ca2+, Mg2+, Zn2+, SO4(2-) on Escherichia coli cells has been studied. The data obtained suggest that the method anomalous time dependence of viscosity is highly sensitive to the conformation of high-molecular DNA-protein complexes. It was shown that the method anomalous time dependence of viscosity can be used to register cell sensitivity to changes in the concentration of one- and two-charged ions in suspension.     

Method of the anomalous time dependence of viscosity for registration of changes in conformational state of Escherichia coli cell genome

The sensitivity of the anomalous time dependence of viscosity to the concentration of the DNA-protein complexes (DNA + histone-like proteins of bacteria or, in other words, the genome) such as chromatin and the conformations of these complexes in lysates of E. coli AB1157 cells were studied. A linear region of the anomalous viscosity time dependence on the concentration of E. coli cells was found in which the interactions between single DNA-protein complexes can be neglected. The response of the genome of E. coli to ethidium bromide at concentrations of 0.0003-3 mg/ml was studied. Significant differences in the effect of ethidium bromide on E. coli cells in the stationary and logarithmic growth phases were found. The effect of heating cell lysates, the molar concentration of NaCl in lysates, and the addition of proteins into lysates on the parameters of the anomalous viscosity time dependence was studied. It was shown that proteins do not contribute significantly to the effect of anomalous viscosity time dependence. The results obtained confirm that the method is sensitive to changes in the conformational state of the genome of E. coli cells.     

Comparative analysis of mechanisms of the modification of microorganism viability under the effect of UHF heating and hyperthermia]

In experiments with models of isogenic Escherichia coli strains, a comparative study was made of the effect of SHF of electromagnetic field and hyperthermia. The survival rate of bacteria was determined and, simultaneously, injuries to genetic supramolecular structures were registered through measuring the anomalous time dependence of cell lysate viscosity. The combined effects of the mixture of these factors with H2O2 microconcentrations were studied. The differences observed in their realization were attributed to the different mechanisms of action of these factors on the repair enzyme systems of the studied cells. It has been found that the effect of microwaves on microorganisms causes much severer damages to DNA that hyperthermia does.     

The effect of ultrahigh frequency electromagnetic fields on the reduction of ferricyanide by human erythrocytes in the presence of methylene blue

Effect of microwave radiation with the frequency of 1000 +/- 10 MHz and specific absorption rate of 220-580 mV/g on the ferricyanide reduction by human red blood cells in the presence of methylene blue (carrier of oxidation-reduction equivalents through the membrane) was studied at different temperatures in the region of 23-34 degrees C. The temperature dependence of the ferricyanide reduction rate in Arrhenius plots shows two sharp "anomalous" sites with apparently negative activation energy at 26-27 and 29-30 degrees C. Broadness and expression of the "anomalous" sites increased with an increase of the blood storage time. The increase of the ferricyanide reduction rate under microwave irradiation was observed only in the temperature regions corresponding to the "anomalous" sites of the temperature dependence.     

Fifty hertz magnetic fields individually affect chromatin conformation in human lymphocytes: dependence on amplitude, temperature, and initial chromatin state

Effects of magnetic field (MF) at 50 Hz on chromatin conformation were studied by the method of anomalous viscosity time dependence (AVTD) in human lymphocytes from two healthy donors. MF within the peak amplitude range of 5-20 μT affected chromatin conformation. These MF effects differed significantly between studied donors, and depended on magnetic flux density and initial condensation of chromatin. While the initial state of chromatin was rather stable in one donor during one calendar year of measurements, the initial condensation varied significantly in cells from another donor. Both this variation and the MF effect depended on temperature during exposure. Despite these variations, the general rule was that MF condensed the relaxed chromatin and relaxed the condensed chromatin. Thus, in this study we show that individual effects of 50 Hz MF exposure at peak amplitudes within the range of 5-20 μT may be observed in human lymphocytes in dependence on the initial state of chromatin and temperature.     

Slowing of the time course of the excitation of squid giant axons in viscous solutions

Summary The time course of excitation of intracellularly perfused squid giant axons was slowed as the solution viscosity was raised by adding neutral molecules, i.e., glucose and glycerol. By twofold increase of the solution viscosity, the duration of action potential was prolonged to 2.7-fold and the maximum rate of rise decreased to one-half. At the same time, the membrane resistance at resting state increased by 60%. These effects were reversible. The time course of inward and outward currents was slowed also. When the solution viscosity increased to twofold, the time to peak inward current increased by 80%, and the amplitudes of peak inward and steady outward currents decreased by 60% and by 70%, respectively. These effects were not specific for the sodium or the potassium channel. Effects of solution viscosity occurred in both hypotonic and hypertonic solutions. Q₁₀ values of temperature dependence of the time course of the action potential were equal in any viscous solutions. These effects in viscous solutions were explained by the change in solution viscosity but not by the change in solution osmolarities, ionic activities, or solution resistivity.     

The effects of the microwaves on E. coli cells depend on oxygen concentration and static magnetic field

The effects of non-thermal microwaves (MW), 10(-4) and 10(-10) W/cm(2), on conformation of nucleoids in E. coli cells were analyzed by the method of anomalous viscosity time dependence (AVTD). MW exposure was performed at different values of static magnetic field and concentration of oxygen, 8-90 microT, and 2.3-7.8 mg/l, respectively. It was shown, that slight changes in both static magnetic field and oxygen concentration result in significant changes of MW effects up to their disappearance. It was established, that changes in static magnetic field affected significantly the time kinetics of the MW effects. The obtained data provide further evidence for strong dependence of the effects of non-thermal microwaves on physical parameters of exposure and physiological factors. These dependences should be taken into account in replication studies. The obtained results encourage further investigation of possible modulation of non-thermal MW effects by additional electromagnetic fields.     

Physio-chemical properties of polymyxan 88A–water system

The system exopolysaccharide polymyxan 88A–water was studied at several temperatures. The temperature dependence of viscosity at cooling and heating was obtained in order to estimate the phase separation temperature (T_s) and the gelation temperature (T_g). The experimental values of T_s and T_g were used to plot the phase diagram of the system under study at polymer concentrations below 1.5 wt%. Viscous flow in the system was examined by the cylinder–cylinder rotation method. It has been found that: (i) at shear rates within 1–100 s⁻¹ the dependence of viscosity on shear rate can be fairly expressed by the power low; (ii) the activation enthalpy of viscous flow practically does not depend on shear rate; and (iii) the activation entropy of viscous flow is negative, most likely due to an orienting action of mechanical field.     

Viscosity analysis of the temperature dependence of the solution conformation of ovalbumin

The viscosity of ovalbumin aqueous solutions was studied as a function of temperature and of protein concentration. Viscosity-temperature dependence was discussed on the basis of the modified Arrhenius formula at temperatures ranging from 5 to 55 degrees C. The activation energy of viscous flow for hydrated and unhydrated ovalbumin was calculated. Viscosity-concentration dependence, in turn, was discussed on the basis of Mooney equation. It has been shown that the shape parameter S decreases with increasing temperature, and self-crowding factor K does not depend on temperature. At low concentration limit the numerical values of the intrinsic viscosity and of Huggins coefficient were calculated. A master curve relating the specific viscosity etasp to the reduced concentration c[eta], over the whole range of temperature, was obtained and the three ranges of concentrations: diluted, semi-diluted and concentrated, are discussed. It has been proved that the Mark-Houvink-Kuhn-Sakurada (MHKS) exponent for ovalbumin does not depend on temperature.     

Possible effect of structural phase transition in the reactive center of Rhodobacter sphaeroides on the rate of dark adaptation of photooxidized bacteriochlorophyll from primary quinone

The dependence of the rate of dark recombination between the photooxidized primary donor--dimer bacteriochlorophyll molecule (P) and reduced primary quinone acceptor (QA), P+QA(-)-->PQA was studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides in the temperature range of 100-320 K. Control RC preparations, RC species with the removed H-subunit as well as RC samples with the hydrogen bonds network modified by isotopic D2O-H2O substitution were investigated. An anomalous temperature dependence of the recombination time (tau rec) of dark reaction P+QA(-)-->PQA was found for all RC samples. It was found that upon heating from 120 to 290 K tau rec increased 2.5 fold. However, upon further heating to 320 K, tau rec decreased again. The temperature dependences of the P+QA(-)-->PQA recombination time were compared with those of the thermodepolarization current of RC preparations in the same temperature range. The temperature curve of the thermodepolarization current was also nonmonotonous. The theoretical interpretation of the temperature dependence of tau rec as well as of the thermodepolarization current was made in the framework of the theory of structural phase transitions within the hydrogen bond network in the water-protein surrounding of the redox centers participating in the electron transfer reactions.     

Temperature dependence of the yield shear resultant and the plastic viscosity coefficient of erythrocyte membrane. Implications about molecular events during membrane failure.

Structural failure of the erythrocyte membrane in shear deformation occurs when the maximum shear resultant (force/length) exceeds a critical value, the yield shear resultant. When the yield shear resultant is exceeded, the membrane flows with a rate of deformation characterized by the plastic viscosity coefficient. The temperature dependence of the yield shear resultant and the plastic viscosity coefficient have been measured over the temperature range 10-40 degrees C. Over this range the yield shear resultant does not change significantly (+/- 15%), but the plastic viscosity coefficient changes exponentially from a value of 1.3 X 10(-2) surface poise (dyn s/cm) at 10 degrees C to a value of 6.2 X 10(-4) surface poise (SP) at 40 degrees C. The different temperature dependence of these two parameters is not surprising, inasmuch as they characterize different molecular events. The yield shear resultant depends on the number and strength of intermolecular connections within the membrane skeleton, whereas the plastic viscosity depends on the frictional interactions between molecular segments as they move past one another in the flowing surface. From the temperature dependence of the plastic viscosity, a temperature-viscosity coefficient, E, can be calculated: eta p = constant X exp(--E/RT). This quantity (E) is related to the probability that a molecular segment can "jump" to its next location in the flowing network. The temperature-viscosity coefficient for erythrocyte membrane above the elastic limit is calculated to be 18 kcal/mol, which is similar to coefficients for other polymeric materials.     

The decomposition of soluble collagen by γ-irradiation

1. The effect of gamma-irradiation in the range 1 krad-10 Mrads on freeze-dried acid-soluble collagen was studied. 2. The specific-rotation and reduced-viscosity recoveries after heating and cooling of the irradiated collagen in solution showed a high degree of dependence on irradiation dose, with reduced viscosity showing significantly less recovery than specific rotation on increasing the irradiation dose. 3. The dependence of reduced viscosity on concentration was greatly decreased with increased doses of gamma-irradiation. 4. The melting temperature measured by optical rotation also decreased as the irradiation dose was increased, and at low doses was distinctly biphasic. 5. Physical properties showed that the action of gamma-irradiation up to 10 Mrads occurred in two distinct phases, with the early changes being extremely sensitive to irradiation dose. 6. The action of the gamma-irradiation is discussed in terms of the structure of tropocollagen.     

Compensation of temperature and concanavalin A concentratration effects for glucose determination by the viscometric affinity assay

A viscometer suitable for rapid measurements in small volumes of highly viscous liquids is described. Using this device the viscometric affinity assay for glucose was studied under variable conditions in order to obtain basic information for the design of a viscometric glucose sensor. The viscosity of the dextran/Concanavalin A (ConA) solution is sensitive to glucose in a broad range of the shear stress. However, for measuring the glucose concentration with this sensitive liquid the strong dependence of the absolute viscosity on temperature and ConA concentration has to be taken into account. For the purpose of calibration a parameter more suitable than the absolute viscosity is the relative fluidity (F(r)) that is defined by the actual measured viscosity at a given glucose concentration, the reference viscosity at a standard glucose concentration, and a constant linearization coefficient. F(r) shows a linear dependence on the glucose concentration in the therapeutically interesting range up to 30 mM and is not significantly changed by moderate variations of the ConA concentration or temperature.     

Structural rearrangements of the chromatin during the adaptive response in two genetically close djungarian hamster fibroblast cell lines with different radiosensitivity

The capacity of cell for the adaptive response (AR) induction after gamma-irradiation using micronuclear test was investigated. Our model consists of the parental djungarian hamster embryonic fibroblast cell line DH-TK- and its radioresistant progeny (PIC-20). We demonstrated that AR for the more radiosensitive parental cell line was shifted to the lower adaptive and to the challenge doses. The maximal AR for DH-TK- cells was induced at 0.3 Gy adaptive dose and 1.5 Gy challenge dose (adaptive response coefficient (ARC) was 0.4+/- 0.1), whereas for PIC-20 cells these means were 0.5 Gy and 3.0 Gy correspondingly (ARC = 0.45+/-0.1). Using the method of anomalous viscosity time dependence (AVTD) we demonstrated the chromatin rearrangements in both cell lines during 3-5 h after adaptive dose application. The rearrangement degree evaluated by the relative maximal reduced viscosity was considerably higher in PIC-20 cell line than that in DH-TK cells (2.4+/-0.3 vs 1.4+/-0. 1). Interestingly, the time of chromatin rearrangement did not depend neither on the dose nor on the cell type and was similar in both cell lines after 5 h of adaptive dose application. It was also shown that during the AR chromatin relaxation was lower after exposure to both the adaptive and challenge doses than after challenge dose only. In contrast, in the degree of AR chromatin relaxation was higher for both cell lines.     

Acquired radioresistance of progeny of irradiated cells is accompanied by rearrangements in chromatin organization

gamma-Irradiation action within a dose range of 0-20 Gy on parental djungarian hamster fiborblasts, DH-TK- cell line, and the progenies of these irradiated cells, surviving acute exposure to 20 Gy irradiation, PIC-20 cell line, was examined. The PICs were 3 times more radioresistant than the parental cells as calculated from D0. Using a method of anomalous viscosity time dependence (AVTD) it was revealed that starting (initial) level (in untreated cells) of chromatin compactness in radioresistant progenies was more than 1.4 times as high as for parental cells. The analysis of dose dependence has shown that irradiation with a dose of 5 Gy resulted in complete chromatin loop relaxation in radiosensitive DH-TK- cells and partial one in radioresistant PIC-20 cells. Besides, the beginning of DNA-membrane complexes degradation following the irradiation with doses over 15 Gy in DH-TK- cells was observed. It was shown that the increased state of relative chromatin relaxation in PIC-20 cells determines an increasing in reparation effectiveness that resulted in lower percent of residual damages in these cells. Using the Nosern hybridization method the expression level of mts 1, tag 7 and vseap 1 genes was studied. It is revealed that tag 7 and vseap 1 gene expression in radioresistant cells were correspondingly 6 and 10 times higher than in radiosensitive parental cells and the level of mts 1 gene expression was not changed. So, based on the results obtained we suggest that acquired radioresistance in progenies of irradiated cells is determined by rearrangements in chromatin structure and accompanied constitutive changes of gene expression.     

Pairing mechanism for the high-TC superconductivity: symmetries and thermodynamic properties

The pairing mechanism for the high-Tc superconductors based on the electron-phonon (EPH) and electron-electron-phonon (EEPH) interactions has been presented. On the fold mean-field level, it has been proven, that the obtained s-wave model supplements the predictions based on the BCS van Hove scenario. In particular: (i) For strong EEPH coupling and T < T(C) the energy gap (Δtot) is very weak temperature dependent; up to the critical temperature Δtot extends into the anomalous normal state to the Nernst temperature. (ii) The model explains well the experimental dependence of the ratio R(1) ≡ 2Δ(tot)(0)/k(B)T(C) on doping for the reported superconductors in the terms of the few fundamental parameters. In the presented paper, the properties of the d-wave superconducting state in the two-dimensional system have been also studied. The obtained results, like for s-wave, have shown the energy gap amplitude crossover from the BCS to non-BCS behavior, as the value of the EEPH potential increases. However, for T > T(C) the energy gap amplitude extends into the anomalous normal state to the pseudogap temperature. Finally, it has been presented that the anisotropic model explains the dependence of the ratio R(1) on doping for the considered superconductors.     

Modification of the structure of plasmatic membranes of the liver by the action of alpha-tocopherol in vitro

The effect of the natural antioxidant alpha-tocopherol in a broad concentration range (10(-4) - 10(-25) M) on the viscosity characteristics and thermally induced structural transitions of a lipid bilayer of plasma membranes of murine hepatocytes in vitro has been studied. Changes in the rigidity of surface (approximately Abb) of the lipid bilayer were measured on a Bruker EMX EPR spectrometer (Germany) by the method of spin probes. Stable nitroxyl radicals of 5- and 16-doxylstearic acid, localized at different depth in the membrane served as spin probes. It was shown that the concentration dependence of the effect of alpha-tocopherol is linear and polymodal with three statistically significant increases in three ranges of its concentration: (1) in the range of traditional physiological concentrations 10(-4)-10(-9) M, (2) in the range of superlow doses 10(-9) - 10(-17) M, and (3) in the range of "imaginary" concentrations 10(-17) - 10(-25) M. The mechanisms of action of alpha-tocopherol in each of the three ranges are discussed. When studying the temperature dependences of viscous characteristics, a new thermally induced structural transition in the range of "physiological" temperatures 309-313 K for those alpha-tocopherol concentrations (including superlow ones) to which the maxima on the dose dependence curves at constant temperature of 293 K corresponded.     

The salting-out behavior of human plasma fibronectin and its possible correlation with heparin-induced cryoprecipitation of the protein

The solubility of human plasma fibronectin in concentrated ammonium sulfate solutions was measured at pH 7.0 and varying temperatures as well as at 25 degrees C and varying pHs. The salting-out parameters, KS and beta were found to increase linearly with temperature in the range 5 degrees-50 degrees C. KS-pH and beta-pH profiles were found to have maxima at pH 7.0. The dependence of both of the solubility parameters of plasma fibronectin on temperature and pH was thus found to be anomalous. The possibility of a correlation between the heparin-induced cryoprecipitation of fibronectin and the dependence of its solubility parameters on pH and temperature is considered. It is suggested that heparin-induced precipitation of human plasma fibronectin at low temperatures is caused by (i) a cold effect and (ii) conformational change in the protein due to heparin binding.     

Determination of Some Hydrodynamic Parameters of Ovine Serum Albumin Solutions Using Viscometric Measurements

The influence of protein concentration and temperature on the viscosity of ovine serum albumin (OSA) solutions was studied. The Mooney equation and a modified Arrhenius formula were used to described the viscosity-concentration and viscosity-temperature dependence of the solutions, respectively. The effective specific volume, the activation energy and entropy of viscous flow for hydrated OSA were calculated. The axial ratio and the dimensions of the main semi-axes of hydrated OSA were established. At low concentration limit, the temperature dependence of the intrinsic viscosity and Huggins coefficient is presented. Comparison of some hydrodynamic parameters obtained for different proteins has been made.     

Rheological characterization of the galactomannan from Leucaena leucocephala seed

A water soluble galactomannan isolated from Leucaena leucocephala seeds gave an intrinsic viscosity of 3.5dl/g and viscosity average molecular mass, M(v), of 6.98×10(5)g/mol. This was in reasonably good agreement with the value of the weight average molecular mass, M(w), of 5.44±0.20×10(5)g/mol determined by GPC-MALLS coupled to RI. The onset of polymer coil overlap occurred at c*[η] of 2.1, with slope of 3.0 above and 1.3 below the point of polymer coil overlap. The shear viscosity of the polysaccharide was temperature dependent and decreased with increasing temperature. The activation energy for viscous flow of 3.0% polysaccharide concentration obtained by Arrhenius plot of zero shear viscosity as a function of temperature was 26.4kJ/mol. Both the storage modulus (G') and loss modulus (G″) showed strong dependence on frequency indicating the presence of entangled coils. The Cox-Merz plot gave close superimposition of the complex and shear viscosities.