Erythrocyte acetylcholinesterase (AChE) is bound to the membrane by a complex glycosylphosphatidylinositol anchor, so the effect of alcohol on AChE activity may reflect direct and/or membrane-mediated effects. The indication of a direct interaction between n-butanol and AChE molecules is the activation/inhibition of AChE by occupation of the enzyme’s active and/or regulatory sites by alcohol. The activation of AChE can occur only at low concentrations of alcohols, while at high concentrations AChE is inhibited. In this work the mechanism of inhibition of erythrocyte AChE by n-butanol at high concentrations was studied. The values of activity, calculated assuming parabolic competitive inhibition, which implies that one or two molecules of inhibitor bind to the enzyme, fit well to the experimental values. From the values of the inhibition constants it was concluded that at high n-butanol concentrations two alcohol molecules usually interact with AChE.

Production of paclitaxel and other taxanes by cell suspension cultures of Taxus species is an alternative to their extraction from yew tree bark or shoots. Little is known about the mechanism of taxane transport in Taxus cells, in either cell cultures or plants. In our study we measured the influence of jasmonic acid (JA), an elicitor of taxane production, on plasma membrane permeability in cell suspensions of Taxus x media. TEMPOL spin probe was added to the suspension of cells grown in the presence of JA and to control cells. Reduction of TEMPOL in the cell interior was measured by electron paramagnetic resonance (EPR). Plasma membrane permeability was derived from the kinetics of reduction. JA was shown to decrease the membrane permeability of Taxus x media cells. The observed inhibition of the spin probe permeability by JA can be ascribed to modification of the membrane structure and lipid composition or, at least, to changes in the lipid lateral domain structures, thus affecting the transport of TEMPOL. The changed permeability to small uncharged molecules could influence the primary cellular metabolism and possibly also the transport of taxanes.

The perturbation of the physical state of cell membranes triggered by an external oxidative stimulus has been studied with sperm cells which were chosen as a model system. Electron paramagnetic resonance (EPR) spectroscopy was applied and three different nitroxides bearing a paramagnetic group on the 5th, 7th and 13th carbon of the stearic acid acyl chain were used to probe different regions of the membrane. The theoretical simulations of the experimental spectra indicate distinct domains in the intact cell plasma membrane where local steric constraints impose different degrees of motional averaging experienced by the reporter group. Upon the external oxidative stimulus the spectral changes were proportional to the accumulated oxidation products and were detected only for the reporter group residing close to the lipid-water interface. The EPR spectra reveal that the motion of the reporter group has slowed down in the oxidized cells and that the oxidatively modified membrane shows up as a more rigid structure as compared with the native state. The results also indicate that the oxidation-induced spectral changes are supressed in the presence of gangliosides.

An electron paramagnetic resonance (EPR)-based membrane surface characterization method is presented to detect the properties of the carbohydrate-rich part of membrane surfaces as well as carbohydrate interaction with other membrane constituents and water-soluble molecules. The proposed method relies on the spin-labeling and spectral decomposition based on spectral simulation and optimization with EPRSIM software. In order to increase the sensitivity of characterization to the carbohydrate-rich part of the membrane surface, the sucrose-contrasting approach is introduced. With this method, which was established on model membranes with glycolipids and tested on erythrocyte membrane, we were able to characterize the surface and lipid bilayer lateral heterogeneity. Additionally, some properties of the interaction between glycocalyx and lipid bilayer as well as between glycocalyx and sucrose molecules were determined. The experiments also provided some information about the anchoring and aggregation of the glycosylated molecules. According to the results, some functions of the glycosylated surface are discussed.

The stability of torocyte red blood cell daughter endovesicles induced by octaethylene-glycol dodecylether (C12E8) was studied theoretically. In addition, the effects of C12E8 and tetraethylene-glycol dodecylether (C12E4) on physical properties of the red blood cell membrane were studied experimentally, using the electron spin resonance (ESR) technique. In the theoretical part, it was assumed that the stable vesicle shape corresponds to the minimum of its membrane free energy, which is the sum of the membrane bending energy and the contribution of the C12E8-induced membrane inclusions. We found that the torocytic vesicle shape may be stable due to quadrupolar ordering of the C12E8 anisotropic inclusions that are embedded in the vesicle membrane. It was also shown how a preference of the membrane inclusions for a specific membrane curvature might lead to their non-homogeneous lateral distribution. In the experimental part, it was shown that C12E4 drastically changes the proportions of the membrane lipid domains (characterized by different ‘fluidity’), while C12E8 induces much smaller changes in the proportions of the domains. A possible relation between the difference in the effects of C12E8 and C12E4 on the membrane lipid domains, and their distribution between the membrane leaflets, is discussed.

Using EPR spectroscopy a typical lateral domain structure was detected in the membranes of spin-labeled bovine erythrocyte ghosts. The spectral parameters were determined by decomposing the EPR spectrum into three spectral components and tuned by a hybrid-evolutionary-optimization method. In our experiments the lateral domain structure and its properties were influenced by the variation in the temperature and by the addition of n-butanol. The specific responses of the particular domain types were detected. For the most-ordered domain type a break was seen in the temperature dependence of its order parameter, while the order parameters of the two less-ordered domain types exhibited a continuous decrease. Below the break-point temperature the alcohol-induced membrane fluidity variation is mainly a consequence of the change in the proportions of the least- and the most-ordered domain type and not the change of the domain-type ordering or dynamics (with n-butanol concentration). On the other hand, the fluidity variation above the break-point temperature arises from both types of changes. Interestingly, the proportion of the domain type that has its order parameter between that of the least- and the most-ordered domain type remains almost constant with concentration as well as with temperature, which implies its stability. Such characterization of the lateral membrane domain structure could be beneficial when considering the lipid–protein interactions, because it can be assumed that the activity of the membrane-bound enzyme depends on the properties of the particular domain type.

The action of mercuric chloride and methyl mercuric chloride on the membrane lateral domain organization of bovine, equine, and canine erythrocytes was studied. Electron paramagnetic resonance (EPR) spectra of spin-labeled erythrocytes were analyzed with respect to their lateral domain structure. Continuous alteration of the membrane domain populations revealed that mercuric compounds affect the membrane via the evolution of toxic events in the cells.

Two main high-density lipoprotein subfractions, HDL2 and HDL3, were spin labelled with TEMPO which partitions both in aqueous and lipid phase. The dynamics of the lipid phase was monitored via the reduction of incorporated TEMPO with ascorbic acid. The reduction of the paramagnetic nitroxide into the nonparamagnetic hydroxilamine form decreases the ESR signal with time. The reduction curves show complex behaviour while the partition coefficients of TEMPO remain unchanged during the reaction. The reduction process in the samples containing HDL particles proceeds faster than in the aqueous solution of pure reactants, e.g. spin label and ascorbic acid. In order to explain experimental data the model for reduction of TEMPO by ascorbic acid is proposed. It assumes that the processes taking place in these heterogeneous systems are determined by the overall reaction rate, which depends on the local concentrations of the reactants, as well as on their transport properties in the particular phases.

The antioxidative properties of pig urinary bladder mucosa were compared with those of gastric and intestinal mucosa using nitroxide radicals. Electron paramagnetic resonance (EPR) method was used to monitor the metabolic processes of nitroxides in mucosae. The reduction of nitroxides was measured on intact luminal surfaces of gastric, intestinal, and urinary bladder mucosa, as well as in homogenates of mucosa surface layer. Furthermore, N-ethylmaleimide and ascorbate oxidase have been used to characterize the reducing agents in urinary bladder mucosa homogenates. The nitroxide concentration decrease on intact mucosa of the urinary bladder was significantly different from those of the gastric and the intestinal mucosa. The concentration decrease was the largest for intestinal mucosa and the smallest for bladder mucosa. On the other hand, homogenates exhibit the largest nitroxide reduction rates for the bladder mucosa and the smallest for the gastric mucosa. In the bladder surface layer homogenates ascorbate and thiol-containing reducing agents were found and their coupled action in the nitroxide reduction process was established. The mucosa of urinary bladder is protected against nitroxide free radicals by a relatively low permeability and very active endogenous reducing agents. The gastric and intestinal mucosa are more permeable and/or have greater antioxidant activity on their surface. The reduction of nitroxides in the urinary bladder mucosa occurs via the ascorbate-thiol coupled reducing system.

A novel thiol-specific spin labeling procedure for the protein component (apoprotein B, apoB) of low density lipoproteins (LDLs) is presented. A methanethiosulfonate spin label was used to probe the free cysteine residues of apoB with electron paramagnetic resonance (EPR) spectroscopy. The results indicated that the spin labeled sites are predominantly buried in the LDL particle in two distinct environments that differ in their mobility restrictions. The suitability of thiol-specific labeling for the study of the stability and conformation of apoB was demonstrated in experiments with denaturing agents. The results presented in this work offer a new approach for the matching of EPR data with the primary structure of apoB.

A method by which it is possible to characterize the membranes of biological samples on the basis of the EPR spectral lineshape simulation of membrane-dissolved nitroxide spin probes is described. The presented simulation procedure allows the determination of the heterogeneous structure of biological membranes and fluidity characteristics of individual membrane domains. The method can deal with isotropic and anisotropic orientations of nitroxides introduced into the biological samples described by restricted fast motion with a correlation time between 0.01 and 10 ns. The linewidths of the Lorentzian lineshapes are calculated in a restricted fast-motion approximation. In the special case of samples with high concentrations of nitroxides or in the presence of paramagnetic ions, the lineshapes are calculated directly from the exchange-coupled Bloch equations. The parameters describing ordering, relaxation, polarity, and the portions of the individual spectral components are extracted by optimizing the simulated spectra to the experimental spectrum with either a Simplex or a Monte Carlo algorithm. To improve the algorithm's efficiency, a new way of characterizing the goodness of fits is introduced. The new criterion is based on the standard least-squares function, but with special weighting of the partial sums. Its benefits are confirmed with membrane spectral simulation. Two classes of examples—simulation and optimizations of synthetic spectra to evaluate the accuracy of the optimization algorithms and simulation and optimization of EPR spectra of nitroxides in liposome suspensions in the presence of a broadening agent and in human leukocytes are shown.

In this work, the interaction of different isomers of lower aliphatic alcohols with LDL representing a complex macromolecular assembly is investigated in vitro. Emphasis is given to the comparison of the impact of molecular architecture of methanol, ethanol, propanol (n-, iso-) and butanol (n-, iso-, sec-, tert-) in perturbing the lipid–protein assembly. The geometrical characteristics as well as the lipophilicity of the respective alcohol are considered. The EPR method combined with the spin labeling of both the apoB and the lipid monolayer allowed parallel detection of changes provoked in both phases. In addition to the change in protein environment, the spectral decomposition of the experimental data revealed a decrease in lipid ordering with the increasing concentration of the alcohols. This phenomenon for aliphatic alcohols is linearly correlated with the equal volume occupation (EVO) of alcohol in LDL. The results support the molecular mechanism of alcohol action through its interference with the lipid–protein interactions in LDL, which could be applicable to the molecular mechanism of alcohol interaction with integral membrane proteins.

The reaction kinetics, measured by electron paramagnetic resonance (EPR) was used to study the involvement of uric acid in the reactions of nitroxide radical (TEMPONE) scavenging by ascorbate (PBS, pH 7.4, and T = 37 degrees C). In absence of iron ions, the oxygen reoxidation of hydroxylamine was slow and the addition of uric acid did not change the observed kinetics. On the other hand, in presence of iron ions the reoxidation starts to recover the nitroxide radical. This reaction rate is strongly enhanced by uric acid. In fact there are two hypotheses, the first ascribes more efficient iron complexes with uric acid, by which the reoxidation rate increases. Especially the competition between the phosphate and uric acid ligands might be important. However, uric acid could be directly involved in scavenging the hydroxyl radicals, and herewith influence the reaction rate. No scavenging activity of uric acid in UV irradiated aqueous solutions was found. The trapped adducts of the hydroxyl and the superoxide radicals show the same concentration in presence and absence of uric acid.

Lateral heterogeneity in terms of co‐existing domains with a distinct molecular organization is an area of increasing interest in membrane biology. The structural and dynamic aspects of the in‐plane domain organization of lipids are becoming well documented, especially for model membrane systems. Potato ( Solanum tuberosum L. cv. Desirée) callus cells and roots of plantlets from stem node culture were doped with a spin‐labeled analog of the methyl ester of palmitic acid bearing the paramagnetic nitroxide group at position C—5 of the acyl chain, which serves as a monitor of membrane fluidity of the region close to the polar phospholipid head groups of the bilayer. Model reconstruction of the line‐shapes of the experimental spectra revealed the co‐existence of two types of membrane domains with different ordering and dynamics of lipids in the membranes of both callus and root cells. With changes in temperature, relatively small differences were detected in either type of domain in the lipid ordering of the bilayer as characterized by order parameter S . However, the relative population of domains in the bilayer exhibited stronger temperature dependence. Typically, the relative proportion of disordered domains with less molecular order (smaller S ) was larger in the membranes of callus cells compared to those of root cells, indicating higher fluidity throughout the measured temperature range (5–35°C). The Arrhenius activation energies for rearrangement of lipid molecules within the bilayer were found to be higher for root tissue membranes, indicating the ability of root cells to oppose actively any drastic changes of membrane structuring under temperature stress. The distinctions in organization of lateral domains between the callus and root cell membranes may be correlated with differences in growth rate and metabolic activity between these two types of tissue.

Antioxidant properties have been attributed to melatonin; it seemed therefore worthwhile to determine its effects in relation to the prooxidant action of adriamycin, which contributes to its toxic and therapeutic effects. Melatonin effectively acts as a direct free radical scavenger in the concentration range of 20-100 microM as determined in vitro, using Fenton reaction as a source of free radicals that were determined by EPR using spin trapping method. Following the administration of a single i.v. dose of 28 mg/Kg or of 3 repeated i. p. doses of 5 mg/Kg adriamycin to CBA mice, glutathione levels in the liver cells were significantly reduced. When the treatment with adriamycin was preceded by the s.c. administration of 2 mg/Kg melatonin, the decrease in total and reduced glutathione concentrations was significantly prevented. A significant increase in lipid peroxidation was observed in liver cells after a single administration of adriamycin which was not attenuated by pretreatment with melatonin. These results indicate that further examination of the possible protective action of melatonin on the toxic effects of prooxidant antitumor drugs on normal and neoplastic tissues would be of interest also in relation to their chronotoxicological properties.

Membrane fluidity of non-cultured lung cancer tissue was studied by electron paramagnetic resonance (EPR). EPR spectra of a lipophilic spin probe in a tissue of resected tumor samples from 51 patients were compared with computer simulated spectra, which were superimpositions of spectra characterizing membrane domains with different fluidity. The membranes of tumor tissues were more fluid, than those of normal lungs; the most fluid domains were enlarged and their order parameter decreased in comparison to normal tissue. An empirical fluidity parameter (H13) was defined as the criterion to correlate EPR and clinical data. The histology of tumor, the quantitative presence of different tumor and non-tumor cells and the pathohisthological stage of the disease had no significant influence on fluidity.

Clinical procedures, such as acid etching and reshaping of the teeth supporting removable partial dentures by grinding off some enamel surface, increase the permeability of dental enamel. Teeth take several months in vivo to partially recover from such damage. In the meantime, the tooth is more susceptible to carious decay. To prevent this the ground or etched enamel should be effectively protected. Using electron paramagnetic resonance (EPR) and a two-chamber diffusion cell the authors studied the influence of adhesive resin applied to the ground and acid-etched enamel surfaces on the diffusion of spin label TMAPO (2,2,6-6 tetramethyl-4-acetamido-piperidine-1-oxyl) molecules through the enamel. The enamel permeability was measured in samples exposed to 1-min etching with 37% phosphoric acid, in samples etched for 5 min, and in samples ground with a diamond bur. Next, all the treated enamel surfaces were coated with Scotchbond Multi-Purpose Plus(R) dental adhesive system and the permeability measurements repeated. Scanning electron microscopy (SEM) was used to study the porosity of enamel surfaces. The adhesive resin film covering the etched or ground enamel surfaces was found to decrease significantly the diffusion through dental enamel. This finding confirms the clinical value of dental adhesives used to protect ground or accidentally acid-etched enamel surfaces. SEM analysis showed that adhesive resin covers the porous surface of the acid-etched and ground enamel tightly.

The structure and permeability of cementum are changed during the course of periodontal disease. In this study, the transport of water-soluble, spin-labelled molecules through cementum was studied by electron paramagnetic resonance (EPR). Cementum samples cut from different parts of the root were classified into four different groups: (A) samples exposed to the oral environment, (B) samples exposed to the periodontal-pocket environment; (C) samples cut from periodontally involved teeth but not exposed to saliva or periodontal pocket and (D) samples from sound young teeth extracted for orthodontic reasons. In order to obtain undamaged cementum, a dentine layer was left on each sample. Two methods were used to measure the diffusion coefficients of spin-labelled molecules in cementum dentine samples. First, the method of one-dimensional EPR imaging (EPRI) was used to evaluate the penetration of spin-labelled molecules into the cementum/dentine structure. Second, the diaphragm-cell method was used to determine the diffusion coefficients of the labelled molecules through the cementum under steady-state conditions. The results indicate that the interface between cementum and dentine is a barrier to diffusion. A set of diffusion (D) and partition (K) coefficients to describe the molecular transport in cementum, barrier and dentine was generated from the experimental data of both methods. For cementum (c), the barrier (b) and dentine (d) these coefficients were: Dc= 10(-8)cm2/s, Db= 10(-10)cm2/s, Dd= 10(-6)cm2/s and K=0.1. For the particular periodontally involved and uninvolved teeth the value of the rate-limiting barrier was DbA= 0.3 +/- 0.03 x 10(-10)cm2/s, DbB= 1 +/-0.3 x 10(-10)cm2/s, DbC= 0.3 +/- 0.03 x 10(-10)cm2/s, DbD= 0.4 +/- 0.05 x 10(-10)cm2/s. The largest diffusion flux across the dental hard tissue was found in the samples that had been exposed to the pocket environment (3.1 +/- 0.2) x 10(-9)cm2/s (p < 0.01), which coincided with the permeability calculated from the data evaluated by EPRI. The transport of the labelled molecules into and through the cementum dentine samples depends on the structure of the dental hard tissues, which changes during the course of periodontal disease. Knowledge of molecular diffusion across the tooth cementum/dentine structure is likely to be important for planning new treatments for periodontal disease.

Interaction of methanol, ethanol, propanol and butanol with human plasma low density lipoproteins (LDL) was studied in this work. The surface lipid monolayer of LDL was spin labeled and the electron spin resonance (ESR) spectra were measured in the presence and absence of alcohols. The decomposition of the complex ESR spectra was performed via theoretical simulations of experimental data. The results gained from this study indicate that the influence of alcohol could be observed through the changes of Lipid ordering in the surface of LDL monolayer. This observation supports the hypothesis on the mechanism of alcohol action through its interference with lipid-protein interactions at the level of macromolecular surface.