Dithranol reaction with nitroxide radicals in DMSO, a HPLC study.

Dithranol (1,8-dihydroxy-9-anthrone), an efficient drug for the topical treatment of psoriasis undergoes a complex chemical transformation after topical application. An absorption phase HPLC method has been developed and validated to follow the appearance of its oxidative products in a DMSO solution. In DMSO solution dithranol, chrysazin, and biantrone were monitored simultaneously by HPLC during autooxidation process, as well as in the presence of nitroxide radicals which increase the reaction rate. The kinetics of the very early stage of dithranol transformation is presented for the first time and discussed. Two unknown dithranol-derived intermediates were found and partially characterised.

New EPR method for cellular surface characterization.

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.

An EPR study of the secretion of G-CSF heterologous protein from Pichia pastoris.

The biologically active protein known as human granulocyte colony stimulating factor (G-CSF) can be efficiently secreted from the transformed GS115 Pichia pastoris (GS115/pPIC9/G-CSF), which contains an alpha-mating-factor prepro signal sequence and an alcohol oxidase I promoter, both introduced using the pPIC9 plasmid. To study the P. pastoris G-CSF protein-secretion process, changes to the plasma membrane's lateral domain structure were monitored using electron paramagnetic resonance (EPR). The GS115 and its transformed analogue show that the plasma membrane can be described by fluid-disordered and fluid-ordered lateral domains. The relative proportion of these domains is a sensitive parameter that is able to describe the membrane's involvement in the protein-excretion process. Here, P. pastoris GS115 served as a control for us to compare with the GS115/pPIC9/G-CSF heterologous protein-secreting cells. Electron paramagnetic resonance studies using the spin-probe 5-doxyl methyl ester of palmitic acid [MeFASL (10,3)] showed that during cultivation in a glycerol medium all types of cells had a relatively high proportion of cell-membrane fluid-disordered domains, while during the production phase the G-CSF protein-secreting cells showed a decrease in the proportion of fluid-disordered domains. We ascribe this effect to the vesicular lipid exchange caused by the fusion of secretary vesicles with the plasma membrane during exocytosis. Using electron microscopy and immunocytochemistry intracellular vesicles containing the G-CSF protein were detected. Our studies support the exocytotic mechanism of the heterologous protein secretion.

Antioxidative properties of pig vesical mucosa: a comparison with gastric and intestinal mucosa.

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.

The action of mercury on cell membranes.

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.

Fluidity of the myelin sheath in the peripheral nerves of diabetic rats.

In the present study we examined the structural integrity of the myelin sheath in the peripheral nerves from short-term streptozotocin (STZ)-treated diabetic rats, using ESR spectroscopy as a tool in determining the dynamic state and the structure of the myelin lipid phase. Experiments were performed on spin-labeled sciatic and sural nerves from STZ-treated Hannover-Wistar rats and age-matched controls. The spectrum analysis employed a numerical simulation model with the set of fitting parameters that in the same time relate the ESR line shape and structure and dynamics of the probed environment. The simulation considered three spectral components weighted and summed in the composite spectrum. The comparative analysis of results showed the fraction of the spectral component II to be significantly increased in the spectra of diabetic rats, indicating the significant increase in overall fluidity of the myelin structure. The origin of fluidity changes was further investigated using an experimental model for demyelination (local injection of ethidium bromide in vivo), proteolytic action of trypsin in vitro, and osmotic myelin swelling in vitro. Analysis and comparison of the results suggested a conclusion in terms of changed biophysical properties of the myelin lipid phase in peripheral nerves in the pathology of diabetes.

Structural aspects of thiol-specific spin labeling of human plasma low density lipoprotein.

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.

The EPR study of LDL perturbed by alcohols with different molecular architecture.

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.

Fast and accurate characterization of biological membranes by EPR spectral simulations of nitroxides.

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 vitro permeability and scanning electron microscopy study of acid-etched and ground enamel surfaces protected with dental adhesive coating.

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.

Lipid domains in the exoplasmic and cytoplasmic leaflet of the human erythrocyte membrane: a spin label approach.

The existence of different lipid domains in the monolayers of the human erythrocyte membrane was investigated at 4 degrees C by employing spin-labelled phospholipid analogues. Spectra of analogues located exclusively either in the exoplasmic or in the cytoplasmic leaflet of erythrocyte membranes were recorded. Spectra were simulated by variation of order parameter describing the average amplitude of motion of the long molecular axis of the nitrogen 2 p pi orbital of the spin label and of the respective correlation times. For both leaflets at least three components were required to fit the experimental spectra, differing mainly in the order parameter. While the parameters of each component are not very different between both membrane halves, the relative contribution of each component to the spectrum is different between the exoplasmic and cytoplasmic leaflet. The order parameter of the most fluid component, presumably resembling the lipid bulk phase, is smaller in the cytoplasmic leaflet in comparison to the exoplasmic one. The lateral coexistence of different lipid domains in the human red blood cell membrane is concluded. The molecular nature of those domains is discussed.

Membrane fluidity characteristics of human lung cancer.

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.

Melatonin and oxidative damage in mice liver induced by the prooxidant antitumor drug, adriamycin.

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.

The permeability of human cementum in vitro measured by electron paramagnetic resonance.

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.

Anthranoid free radicals found in pseudomelanosis coli.

Pseudomelanosis coli occurs after prolonged intake a anthranoids. After discontinuation of intake the pigmentation disappears apparently without noxious effects, including carcinogenicity and genotoxicity. We are presenting ESR spectra of pseudomelanosis coli specimen, compared to ESR spectra of pigmented skin scales taken from psoriatic patients treated topically with anthralin, and with ESR spectra of anthralin brown material formed in vitro. The ESR spectra show comparable g values within the accuracy of measurements. The examined specimens reveal remarkable stability: the intensity of the ESR signal remained practically constant over the period of four years. The chemical and physicochemical properties of the brown pigments formed from anthranoids explain the observed bio-inertness of these materials including that of melanosis coli pigment derived from anthranoids.

Crystallite arrangement of hydroxyapatite microcrystals in human tooth cementum as revealed by electron paramagnetic resonance (EPR).

Human dental cementum was analyzed by electron paramagnetic resonance (EPR). The measured EPR powder spectra of gamma-irradiated cementum resembled those of gamma-irradiated enamel. Both spectra were characterized by the same line shapes and g values. The position of the extreme first derivate peaks can be described by g1 = 2.0023 and g2 = 1.9971 +/- 0.0002, and are assignable to the CO3(3-) center. The angular dependence of the cementum EPR spectra indicates a different arrangement of the hydroxyapatite microcrystals compared to that of enamel. A corresponding model of cementum microcrystal alignment has been proposed. The methodology presented can be utilized for studying the mineralization process of root cementum and other mineralized tissues.

The interaction of lower alcohols with apoB in spin labeled human plasma low density lipoproteins (LDL).

In this study the interaction of alcohol with the macromolecular lipid-protein assembly represented by human plasma low density lipoproteins (LDL) was investigated. The spin label which covalently binds to the side chain amino group of lysines as well as terminal amino groups was attached to the spin labeled apoprotein (apoB) of native LDL in order to observe the protein component in the electron spin resonance (ESR) spectrum. The interaction of different lower alcohols (methanol, ethanol, propanol and butanol) with the spin labeled LDL was studied for two alcohol concentrations (0.3 and 3.0 M). The ESR spectra indicate a decrease of the hyperfine splitting and narrowing of the linewidth upon the action of alcohol that leads to the conclusion that alcohol provokes a change in the apoB conformation. These findings are explained by following the arguments of the phospholipid mediated mechanism of alcohol action, through the modulation of the lipid packing free volume which results in the protein conformational change.

Enhanced permeability of acid-etched or ground dental enamel.

STATEMENT OF PROBLEM: Acid etching creates retentive microcraters on enamel surfaces. Designing of a partial denture often involves reshaping the supporting and retentive teeth by grinding the enamel. Unfortunately, both these procedures damage the enamel surface. In vivo such surface damage takes several months to recover. PURPOSE: This study evaluated the effect of 1-minute etching, prolonged etching, and grinding on the permeability of dental enamel for water-soluble molecules. MATERIAL AND METHODS: With the electron paramagnetic resonance and a two-chamber diffusion cell, the influence of etching and grinding on the diffusion of spin label molecules through the enamel was studied quantitatively. The enamel permeability was measured in 30 sound enamel samples, of which 10 samples were exposed to 1-minute etching with 37% phosphoric acid, 10 samples were etched for 5 minutes, and 10 samples were ground with a diamond bur. RESULTS AND CONCLUSIONS: All procedures significantly increased the permeability of dental enamel. These results demonstrate that in vivo the acid-etched and ground dental enamel surfaces are less protected and consequently, unless the tooth is properly protected, are more susceptible to carious lesions. Therefore ground or accidentally etched enamel should be protected.

Nitroxide reduction with ascorbic acid in spin labeled human plasma LDL and VLDL.

The LDL and VLDL were spin labeled with Tempo which partitions both in the aqueous and lipid phase. The ESR spectra were measured in the equilibrium state as well as during the reduction of the spin label with ascorbic acid. The kinetics of the concentration decay curves was parametrized with two exponentials. The theoretical simulation of the experimental spectra revealed a drastic linewidth narrowing in the VLDL samples exposed to the ascorbic acid. Since the transport properties of the specific monolayer are reflected in the observed reaction rates, the analysis of the fatty acid composition of phospholipids, triglycerides and cholesterol esters in LDL and VLDL was performed. It is concluded that different lipid packing at the surface of LDL and VLDL might be the consequence of different intermolecular forces between phospholipids and cholesterol. This finding was connected to the experimentally detected different reaction kinetics in LDL and VLDL as well as their different susceptibility to the ESR linebroadening effects during the nonequilibrium conditions of the spin label reduction with ascorbic acid.

Spectroscopic characterization of specific phorbol ester binding to PKC-receptor sites in membranes in situ.

Electron paramagnetic resonance (EPR) spectra were measured for the spin labelled phorbol-12,13-diester [5,6]PA bound to membranes of the particulate fraction of mouse brain tissue rich in PKC receptors. [5,6]PA is a bioactive derivative of the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), carrying the spin label in a doxyl group in position 7' of the 12-O-tetradecanoyl residue. A mathematical model based on special algorithms (Griffith, O.H. and Jost, P.C. Spin labeling: theory and applications, 1976) allows a satisfactory reconstruction of the experimental spectrum. It reveals that in the experimental spectrum the signal from the [5,6]PA molecules bound non-specifically to the lipid bilayer of the membranes is superimposed by the signal of [5,6]PA molecules bound specifically, i.e. to the active site of PKC (approximately 10% of total EPR signal intensity). Moreover, interpretation of spectral parameters indicates that in [5,6]PA molecules bound specifically the tetradecanoyl chain exhibits a larger motional freedom compared to that in [5,6]PA bound non-specifically. These new findings are in accordance with views featured independently by two recent molecular models of interaction of PKC with cellular membranes (8,9).