Kinetics of the enzyme titration process by reversible modifiers.

The effect of reversible modifiers on the initial rate of enzyme catalysed reactions has been investigated in a quasi-equilibrium approximation using the general modifier mechanism of Botts and Morales. It has been shown that, when investigating the dependence of the initial rate on the modifier concentration at a fixed substrate concentration, the kinetics of enzyme titration by reversible modifiers can generally be described using two kinetic constants. Just as the dependence of the initial rate on the substrate concentration (at a fixed modifier concentration) is described using two kinetic constants: the Michaelis constant Km and the limiting rate Vm. Only one constant M50 is needed to describe the kinetics of linear inhibition, and in the case of nonlinear inhibition and activation, along with M50 the constant QM is also needed. Knowing the values of the constants M50 and QM, it is possible to unambiguously determine the modification efficiency, that is, to calculate how many times the initial rate of the enzyme catalysed reaction will change when a certain modifier concentration is added to the incubation medium. The properties of these fundamental constants have been analysed in detail and the dependence of these constants on other parameters of the Botts-Morales model have been shown. Equations describing the dependence of relative reaction rates on the modifier concentration using these kinetic constants are presented. Various ways of linearising these equations for calculating the kinetic constants M50 and QM from experimental data are also presented.

Analysis of decay kinetics of the cytosolic calcium transient induced by oxytocin in rat myometrium smooth muscle cells.

The method of kinetic analysis of the relaxation phase of the mechanical response of the smooth muscle previously proposed by Burdyga and Kosterin was applied to study the dynamics of the decay of oxytocin-induced calcium transients in cytosol of the rat myometrium smooth muscle cell detected by a fluorescence signal generated by a calcium-sensitive probe fluo-4 using a laser scanning confocal microscope. The experimental data were well linearized in the coordinates ln [(Fm - F)/F] vs lnt (F and Fm are the current fluorescence intensity of the calcium probe and the fluorescence intensity at the maximum of the calcium transient, respectively, while t is the time). The empirical parameters n and τ were determined by which the maximal normalized relaxation rate Vn was calculated for five different ROIs (regions of interest) in the myocyte cytosol. It proved to be almost the same for all ROIs. The maximal normalized relaxation rate calculated from the fluorescence intensity was always lower than that calculated from the corresponding calcium concentration, i.e. the cytosolic Ca2+ concentration in the relaxation phase decreases faster than the corresponding fluorescence intensity. The value of the maximal normalized relaxation rate calculated both from the fluorescence intensity and from the force of oxytocin-induced contractions of isolated rat uterus longitudinal smooth muscles (according to Tsymbalyuk and Kosterin) was exactly the same. This indicates that in the relaxation phase, the decreasing curves of both the fluorescence intensity and the contraction forces coincide.

The effect of 2D tungsten disulfide nanoparticles on Lewis lung carcinoma cells in vitro.

The unique physicochemical properties of modern two-dimensional (2D) nanomaterials with graphene-like structures make them promising candidates for biology and medicine purposes. In this article, we investigate the influence of the two-dimensional tungsten disulfide (2D WS2) water suspension nanoparticles obtained by an improved mechanochemical method from powdered WS2 on morphological and structural characteristics of Lewis lung carcinoma cells using FT-IR, Raman spectroscopy, and confocal microscopy. The characterization of the 2D WS2 nanoparticles by different physical methods is given also. We have highlighted that 2D WS2 does not exert cytotoxic activity in the case of 1 day incubation with tumor cells. Prolongation of the incubation period up to 2 days has caused a statistically significant (p < 0.05) concentration-dependent decrease of the number of viable cells by more than 30% with the maximum cytotoxic effect at concentrations of 2D WS2 close to 2 µg ml-1. In the Raman spectra of 2D WS2 treated cells the bands centered at 354 cm-1 and 419 cm-1, which are assigned to characteristics and modes of WS2 nanoparticles were observed. The obtained data indicate, that the cytotoxic effect of 2D WS2 on tumor cells in the case of long-term incubation is realized particularly through the ability of 2D WS2 to enter tumor cells and/or accumulate on their surface, which gives a rationale to conduct further studies of their antitumor efficacy in vitro and in vivo when combined with chemotherapeutic drugs.

Kinetics simulation of transmembrane transport of ions and molecules through a semipermeable membrane.

We have developed a model to study the kinetics of the redistribution of ions and molecules through a semipermeable membrane in complex mixtures of substances penetrating and nonpenetrating through a membrane. It takes into account the degree of dissociation of these substances, their initial concentrations in solutions separated by a membrane, and volumes of these solutions. The model is based on the assumption that only uncharged particles (molecules or ion pairs) diffuse through a membrane (and not ions as in the Donnan model). The developed model makes it possible to calculate the temporal dependencies of concentrations for all processing ions and molecules at system transition from the initial state to equilibrium. Under equilibrium conditions, the ratio of ion concentrations in solutions separated by a membrane obeys the Donnan distribution. The Donnan effect is the result of three factors: equality of equilibrium concentrations of penetrating molecules on each side of a membrane, dissociation of molecules into ions, and Le Chatelier's principle. It is shown that the Donnan distribution (irregularity of ion distribution) and accordingly absolute value of the Donnan membrane potential increases if: (i) the nonpenetrating salt concentration (in one of the solutions) and its dissociation constant increases, (ii) the total penetrating salt concentration and its dissociation constant decreases, and (iii) the volumes ratio increases (between solutions with and without a nonpenetrating substance). It is shown also that only a slight difference between the degrees of dissociation of two substances can be used for their membrane separation.

Vibrational spectra of DNA in the confined interglobular volume of photonic crystal.

The impact of confinement of DNA molecules in a limited volume of the cavity of photonic crystals (PC) on the vibrational properties of the DNA molecule and its conformation is studied. According to our preliminary study, the aqueous shell is removed when the DNA molecules are infiltrated into the PC cavities. Raman scattering (RS) DNA marker lines showed a dramatic conformational change of DNA in the PC cavities and the appearance of new unknown conformational states. We observed the enhancement of vibrational modes of DNA in the PC in comparison with free DNA of about tenfold and the absence of vibrational modes in DNA bases in a region of 1450-1700 cm-1. The observed features in the RS spectra of DNA are explained by the impact of confined interglobular volume and strong localization of the electromagnetic field. Namely, FDTD simulations in linear regime demonstrate the localization of light in cavities of PC with an approximately ninefold enhancement of the electric field within the photonic stop-band, which is the main reason for RS amplification.

2D-BN nanoparticles as a spectroscopic marker and drug delivery system with protection properties.

An application of 2D-BN nanoparticles as a spectroscopic marker, weak luminescent marker and anticancer drug (doxorubicin, DOX) delivery system with protection properties was studied for the LNCaP strains of cancer cells using FTIR and Raman spectroscopy for analysing the cancer cells, cells with BN, the cancer cells with DOX, and the cancer cells with BN nanoparticles loaded by DOX. Study of IR absorption and Raman spectra of the LNCaP strains of cancer cells incubated with 2D-BN nanoparticles for 1 hour showed that the 2D-BN nanoparticles could pass through the cell membrane and localize inside the membrane or close to the membrane in the cytoplasm of the cells. We registered the spectra of the disturbed lipids during the DOX-2D-BN passing through the membrane. After incubation for 2 hours and more, spectral changes in other structural components of the cell (nuclei, cytoplasm, mitochondria) can be registered. Confocal microscopy showed that a gold nanostructured support enhances the fluorescence of the cancer cells with 2D-BN as well as that with DOX, however the double action of 2D-BN and DOX on the cancer cells aggravates the emission property of the studied system. An MTT test showed that the toxicity of DOX on the 2D-BN nanoparticles is less than that on the reference cells, and at the same time the efficiency of the DOX action on the cancer cells does not change.

CALMODULIN ANTAGONISTS EFFECT ON Ca2+ LEVEL IN THE MITOCHONDRIA AND CYTOPLASM OF MYOMETRIUM CELLS.

It is known that Ca(2+)-dependent regulation of this cation exchange in mitochondria is carried out with participation of calmodulin. We had shown in a previous work using two experimental models: isolated mitochondria and intact myometrium cells, that calmodulin antagonists reduce the level of mitochondrial membrane polarization. The aim of this work was to investigate the influence of calmodulin antagonists on the level of ionized Ca in mitochondria and cytoplasm of uterine smooth muscle cells using spectrofluorometry and confocal microscopy. It was shown that myometrium mitochondria, in the presence of ATP and MgCl2 in the incubation medium, accumulate Ca ions in the matrix. Incubation of mitochondria in the presence of CCCP inhibited cation accumulation, but did not cease it. Calmodulin antagonist such as trifluoperazine (100 µm) considerably increased the level of ionized Ca in the mitochondrial matrix. Preliminary incubation of mitochondria with 100 µM Ca2+, before adding trifluoperazine to the incubation medium, partly prevented influence of the latter on the cation level in the matrix. Incubation of myometrium cells (primary culture) with another calmodulin antagonist calmidazolium (10 µM was accompanied by depolarization of mitochondrial membrane and an increase in the concentration of ionized Ca in cytoplasm. Thus, using two models, namely, isolated mitochondria and intact myometrium cells, it has been shown that calmodulin antagonists cause depolarization of mitochondrial membranes and an increase of the ionized Ca concentration in both the mitochondrial matrix and the cell cytoplasm.

[Modulation of myometrium mitochondrial membrane potential by calmodulin antagonists].

Influence of calmodulin antagonists on mitochondrial membrane potential was investigated using a flow cytometry method, confocal microscopy and fluorescent potential-sensitive probes TMRM and MTG. Influence of different concentrations of calmodulin antagonists on mitochondrial membrane potential was studied using flow cytometry method and a fraction of myometrium mitochondria of unpregnant rats. It was shown that 1-10 microM calmidazolium gradually reduced mitochondria membrane potential. At the same time 10-100 microM trifluoperazine influenced as follows: 10 microM--increased polarization, while 100 microM--caused almost complete depolarization of mitochondrial membranes. In experiments which were conducted with the use of confocal microscopy method and myometrium cells it was shown, that MTG addition to the incubation medium led to the appearance of fluorescence signal in a green range. Addition of the second probe (TMRM) resulted in the appearance of fluorescent signal in a red range. Mitochondrial membrane depolarization by 1 microM CCCP or 10 mM NaN3 was accompanied by the decline of "red" fluorescence intensity, "green" fluorescence was kept. The 10-15 minute incubation of myometrium cells in the presence 10 microM calmidazolium or 100 microM trifluoperazine was accompanied by almost complete decrease of the TMRM fluorescent signal. Thus, with the use of potential-sensitive fluorescent probes TMRM and MTG it was shown, that calmodulin antagonists modulate mitochondrial membrane potential of myometrium cells.

[Investigation of nitrosactive compounds influence on polarization of the mitochondrial inner membrane in the rat uterus myocytes using potential sensitive fluorescent probe DiOC6(3)].

The effect of nitrosactive compounds (sodium nitroprusside and sodium nitrite) on the polarization level of the uterus myocytes inner mitochondrial membrane using the confocal laser microscopy and fluorescent probe potentialsensitive DiOC6(3) (3,3'-dihexyloxacarbocyanine) was ivestigated. Colocalisation of mitochondrial membranes specific fluorescent probes (MitoTracker Orange CM-H2TMRos, 10 - nonyl acridine orange and DiOC6(3)) was demonstrated. It was shown that sodium nitroprusside at 0.1 mM concentration caused a moderate decrease in mitochondrial transmembrane potential. That observation was confirmed by flow cytometry. Action efficiency of sodium nitrite in a similar concentration was significantly lower than that of sodium nitroprusside. It is shown that it was sodium nitroprusside which caused a slight swelling of the mitochondria. A possible protecting role of nitric oxide as to mitochondria was discussed.

[Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

[Fluorescent derivatives of diphtheria toxin subunit B and their interaction with Vero cells].

Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell.

[The action of nitrite-anions and hydrogen peroxide on surface properties of the smooth muscle plasmatic membrane].

In the presence of vesicular preparations of sarcolemma (near 70% reverse cytoplasmic sideways inward) fluorescence of ANS--1-(fenilamino)-8-naftylamine--rises more than 10 times. In the conditions of increase of concentration of sodium nitrite and hydrogen peroxide from 1 to 5 microM the probe fluorescence diminishes. Extinguishing of ANS fluorescence under the action of nitrite anions can be explained by chemical modification of the positively charged superficial groups (amino- and sulfhydrile). It will result in the decrease of amounts of local "+"-charges on a membrane, proper decrease of fastening ANS with a membrane and fluorescence extinguishing. H2O2 is able to oxidize superficial -SH-groups which carry a partial positive charge. Consequently a decrease of general amount of fixed "+" charges on a membrane, diminishing of ANS fastening with sarcolemma and the proper fluorescence extinguishing takes place. Probed and calculated by the method "ion beats" superficial closeness of charges of vesicular preparations makes 2.3 +/- 0.1 mC/m2. At the action of 50 microM sodium nitrite and hydrogen peroxide it diminishes to 2.0 +/- 0.1 mC/m2 and 1.7 +/- 0.07 mC/m2 accordingly. The less effective action of NaNO2 can be determined by the influence of Na+ on the superficial field of membrane. With the purpose to confirm the previous suppositions we used the method of laser-correlation spectroscopy, that registered the mean value of hydrodynamic diameter (GD) of vesicules and their division by sizes. Middle GD makes in control 327 +/- 16 nm, and GD which is most often met in population--291 +/- 11 nm. Adding of nitrite anions to the system in a growing concentration results in the substantial decline of GD of both all the membrane population and of plasma vesicules. Analogous results are obtained in the case of H2O2. In the presence of specific modifiers of sulfhydrile- and aminogroup of membrane surface (DTT and TNBS) the decline of GD under the action of nitrite anions or hydrogen peroxide is not observed, or diminishes substantially. Consequently, the diminishing of GD in our system under the action of the probed compounds can be related to chemical modification of amino or/and sulfhydrile groups of membrane surface which complements and confirms experiments with ANS. Diminishing of density of superficial positive charges is possible under the condition of the action of nitrogen oxides and hydrogen peroxide on the membrane can result in strengthening of cations interaction with a membrane and, in principle, will influence their reception and transport.

[Identification of mechanism of enzyme/transport process in the system of "enzyme/ transporter--substrate--effector" using the "ratio" method].

Using equilibrium assumption the analysis of kinetics of enzyme/transport process has been developed. In the course of this process the enzyme can interact simultaneously with substrate S and effector E and as a result two products are generated: P1 (from substrate) and P2 (from effector) after catalytic effect of enzyme or transportation across the biological membrane. It has been demonstrated that the ratio (R = V0,1/V0,2) of initial rates of formation of reaction products P, (V0,1) and P2 (V0,2) represented in linearized form as a dependence on concentration of both substrate S0 and effector E0 allows to identify a specific mechanism of the present process. An algorithm of such identification has been developed.