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Biomedical subjects

M Schara

Publications and source records attributed to M Schara.

At least 37 records · Page 2Linked to original sources
PubMed1995

Transport and dynamics of molecules dissolved in maize root cortex membranes.

Translational diffusion of a fluorescent sterol probe was measured in the plasma membranes of protoplasts isolated from cortical cells of the primary root of maize seedlings. The apparent lateral diffusion coefficient was typically observed to be nearly insensitive to temperature, while the mobile fraction increased with increasing temperature. These fluorescence photobleaching recovery (FPR) measurements were compared with the electron paramagnetic resonance (EPR) spectra of the methyl ester of 13-doxyl palmitic acid in membranes of corn root tissue in situ. The complex spectra observed with this probe were analyzed as weighted sums of simpler spectra of various order parameters and rotational correlation times. The reconstituted spectra calculated from the model show that EPR also detects a mobile (less ordered, fluid) fraction, distinguished by the order parameter S = 0.1 to 0.2, which becomes more abundant as temperature increases and is qualitatively comparable to the mobile fraction determined by the FPR method. The observed results on the mobile fractions and the diffusion rates for translational (FPR) as well as rotational (EPR) motions are interpreted in terms of membrane organization, thus providing information on the population and structural patterns of the coexisting domains with a special emphasis on the response of the membrane to temperature changes.

Biological Transport↗
PubMed1995

Reduction of nitroxides by anthralin and some of its derivatives.

In DMSO solution, anthralin and its C-10 monosubstituted derivatives reduce nitroxides to the corresponding hydroxylamine derivatives, which are not further transformed. The reaction rate depends on the solvent used, the nitroxide, and the structure of the reducer. It is faster in DMSO than in DMF, piperidine type of nitroxides are reduced faster than the pyrrolidine type, and the substitution on C-10 of anthralin has a significant influence on the reaction rate. Anthralin derivatives without protons at C-10 are not able to reduce nitroxides.

Administration, Topical↗
PubMed1995

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).

Animals↗
PubMed1994

The ESR characterization of molecular mobility in the lipid surface layer of human serum lipoproteins.

Three different nitroxides were used to probe either the head group (Tempil stearate) or acyl chain region (Spin labeled cholestane (ChSl) and methyl ester of 5 doxyl palmitate (MeFASL(10,3))) of human plasma low density lipoproteins (LDL) and very low density lipoproteins (VLDL). The ESR data were compared with the simulated spectra which assume rapid anisotropic motion of nitroxide. The results indicate that in the head group region of both LDL and VLDL only the slowing down of the rotational motion occurred when temperature was lowered and the whole region showed up as a unique compartment. On the other hand, the acyl chain region, probed with MeFASL(10,3), behaved as one compartment at physiological temperatures, while at lower temperatures coexistence of fluid and immobilized components were observed. The ESR spectra of lipoproteins labeled with Cholestane showed even higher sensitivity to the mobility constraints. Here, the LDL spectra revealed a drastic immobilization of ChSl axial rotation already at physiological temperatures. The results of these experiments were discussed in terms of core phase transition and/or lipid-protein interactions.

Electron Spin Resonance Spectroscopy↗
PubMed1993

Irreversible structural changes in thylakoid membranes at high temperatures. Detection by luminescence and EPR.

The character of structural changes in thylakoid membranes caused by temperature variation was investigated. Experiments were performed on maize leaf segments in vivo and in a closed temperature cycle 24-50-24 degrees C. Two biophysical methods were used for detection: luminescence and EPR. Arrhenius plots of delayed fluorescence (DF) versus reciprocal temperature revealed two break points, T1 and T2. The MeFASL (10.3) spin probe monitoring properties close to membrane surface detected only T2 transition temperature. The results were interpreted in terms of a fluidity change which starts in a membrane centre at T1 and gradually displaces toward the surface at T2. The T1 and T2 transition temperatures are sensitive to pretreatment history of plants indicating that high temperature and drought-induced membrane alterations are irreversible. Activation energies E1, E2 and E3 were determined for temperature regions below T1 between T1 and T2, and above T2, respectively. The E1 and E3 activation energies showed greater sensitivity to stress than did E2. There are some indications that the DF method could be used to screen temperature sensitive and temperature resistant genotypes.

Biophysical Phenomena↗
PubMed1993

An ESR study of manganese binding in plant tissue.

Two different fractions of manganese were found in the maize plant root apoplasm (intercellular space containing cell walls) after soaking the roots in MnCl2 solutions (concentration range 0.01-10 mmol.l-1): (a) an Mn2+ fraction in the water free space (WFS) which gave a characteristic six-line spectrum, and (b) an immobilized fraction that gave no detectable ESR spectrum. Both fractions affect proton NMR relaxation (T1) of the tissue water through water exchange across cell membranes. ESR spectra of free and total manganese of the root tissue treated with MnCl2 also revealed different time courses for saturation of WFS and DFS with Mn2+. Binding of manganese in the extracellular space of the tissue seems to be the rate limiting step in permeation of Mn2+ across the root cell membranes.

Electron Spin Resonance Spectroscopy↗
PubMed1992

Spin-labeled phorbol esters and their interactions with cellular membranes--IV. Lipophilic binding and molecular orientation of spin-labeled phorbol-12,13-diesters in human erythrocyte membrane.

In human erythrocyte membranes, membrane binding of spin-labeled TPA-analogous phorbol (doxyl)esters [(n,m)PA] was investigated during measurement of the kinetics of the decay of their electron paramagnetic resonance signal by ascorbate reduction. In membrane-bound (n,m)PA the reduction rate was dependent of the position of doxyl in the aliphatic chain of their 12-O-acyl moiety. To describe quantitatively the reaction kinetics observed, two hypotheses (models) were developed and used. Model 1 is based on the assumption that ascorbate reduction takes place in the extracellular space. In this case the experimental data could be fitted by the partition and permeability coefficients of (n,m)PA determining model 1 only, if non-realistic values of these parameters were used. The more refined model 2, corresponding to a bilayer membrane structure, assumes the reduction to take place in the hydrophilic region of the membrane. Assuming a finite probability of finding the doxyl group within the hydrophilic membrane region, model 2 describes quantitatively the dependence of the reduction rate on the position of the doxyl in the aliphatic chain of the (n,m)PA used. From the validity of this model it may be postulated that the molecular orientation of TPA-analogous (n,m)PA in the bilayer membrane is determined by an anchoring of their lipophilic ester moiety in the lipophilic region of the membrane bilayer, thus locating the hydrophilic phorbol moiety within the hydrophilic region of the membrane. With regard to the well-known categories of non-specific versus specific binding of bioactive phorbol esters to protein kinase C/membrane complexes it is deduced that anchoring of (n,m)PA (and hence TPA) in the hydrophobic interior of the membrane structure may be the molecular equivalent of their non-specific binding.

Ascorbic Acid↗
PubMed1992

Spin-labeled phorbol esters and their interactions with cellular membranes--V. Electron paramagnetic resonance of spin-labeled phorbol-12,13-diesters bound to their receptors in mouse brain particulate fraction.

The relatively small concentrations required for in vivo bioactivity of diterpene ester skin irritants and promoters (approximately 10 nmol per animal; approximately 10 nM in cell cultures) has discouraged studies of EPR spectra of bioactive, TPA-analogous, spin-labeled phorbol-12,13-diesters [(n,m)PA] bound to their membrane receptors, protein kinases C (PKC). To meet the requirements of present EPR spectrometers, particulate fraction from mouse brain containing at least 25 x 10(-12) mol of receptors/mg protein (PKC species) were employed together with certain (n,m)PA selected to give an optimal ratio of specific to non-specific binding. For selection and optimization of experimental conditions, a theoretical model was developed that considers all characteristic parameters of the system. By fitting the model calculations to the experimental data of competitive agonist displacement from the particulate fraction of tritium-labeled TPA, the dissociation constants Kd for four selected (n,m)PA used as antagonists were determined. Optimal experimental conditions are met by (5,6)PA and by (5,8)PA, in that for both compounds the relative amount of displaced (n,m)PA is in accordance with the predictions derived from the model. Moreover, the model turned out also to be reliable for samples containing either small or large amounts of membranes. To obtain an EPR spectrum of an agonist bound to brain particulate fraction, the (5,6)PA was used. It shows a broad EPR spectrum typical for an immobilized molecule. The spectrum changes if an excess of TPA is added to the system; the slight differences in shape are due to displacement of (5,6)PA from specific receptor sites by non-labeled TPA and show up as a decreased central peak amplitude. This is the first time that the agonist/receptor interaction of a diterpene ester type irritant and tumor promoter has been demonstrated by direct spectroscopic measurement.

Animals↗
PubMed1991

Molecular motion of drugs in hydrocolloids measured by electron paramagnetic resonance.

The effects of a polymer, the Li-salt copolymer of methylmethacrylic acid, and its methyl ester on the motion of drug molecules in hydrocolloids were studied. The investigation was carried out by means of electron paramagnetic resonance (EPR) using the model nitroxide tempol, and the spin-labeled drugs lidocaine (sl-lid) and dexamethasone (sl-dex). Synthesis of sl-dex was performed. Spin-labeled molecules dissolved in hydrocolloids undergo a fast reorientation motion. The decreasing order of rotational correlation times (tau)--sl-dex greater than sl-lid greater than tempol--suggests that the size and the shape of the molecules strongly affect their motion. The inhibition of motion of larger molecules depends also on their flexibility. The tau values indicate proportionality of the microviscosity of hydrocolloids to the polymer concentration. Rotational motion is dependent on the local environment conditioned by the free spaces between polymer molecules.

Colloids↗
PubMed1989

Amphiphilic derivatives of betaine esters as modifiers of macrovesicular BLM.

A series of amphiphilic derivatives of betaine esters (V-n), with the chemical structure (CH3)3N+COOCnH2n + 1Cl- (n = 10, 12, 14 or 16) were studied with respect to their effects on the electrical properties of lecithin macrovesicular membranes. Normalized resistance and breakdown voltage were found to depend on the V-n concentration in the membrane and on the alkyl chain length (n). Resistance decreases up to about 10(4) ohm.cm2 and breakdown voltage decreases by 111 mV were detected in the V-n: lecithin molar ratio range measured (0.005-0.05). Maximal decrease in breakdown voltage was observed for V-14. These findings together with the featured anionic selectivity suggest that, due to the interaction of V-n with phospholipids, hydrophilic pores are formed in the lipid bilayers. This assumption is supported by the results obtained by electron paramagnetic resonance (EPR) measurements which showed no collective changes in bilayer dynamics or ordering. In particular, rotational correlation times and order parameters of the spin probe molecules dissolved in the membrane did not change in the concentration range tested. Since a large number of defects in the membrane can be expected to influence the collective ordering and dynamics, this observation also suggest that the number of pores formed is small.

Betaine↗
PubMed1988

Spin probe reduction in cells and tissues.

The reduction rates of different nitroxides in rat lung tissue were measured by electron paramagnetic resonance. We confirmed that the reduction rate of nitroxide spin probe molecules is coupled with their structure and transport characteristics. Oxygen was found to slow down the reduction rate of nitroxides in rat lung tissues. On the basis of these findings it can be concluded that most nitroxide properties described for homogeneous systems--cells and tissue homogenates--are also valid for heterogeneous systems, which is important for the application of nitroxides as metabolically active NMR contrast agents.

Aerobiosis↗
PubMed1988

Spin-labeled phorbol esters and their interactions with cellular membranes. III. Skin irritant and tumor-promoting activities of spin-labeled phorbol-12,13-diesters and relationships to their particular structures.

Sixteen 'doxyl' spin-labeled 12,13-(acetate, acylates) of phorbol were assayed in NMRI mice for irritant and for tumor-promoting activity. The spin-labeled positionally isomeric (n,m)PA- and AP(n,m)-type esters carry straight aliphatic acyl chains of different overall lengths N (number of C atoms). Within the chains the 'doxyl' label is located in different positions (n,m). The potency of some of the esters as irritants and as promoters is comparable to or even higher than that of the prototype diterpene ester promoter 12-O-tetradecanoylphorbol-13-acetate and its positional isomer 12-O-acetylphorbol-13-tetradecanoate. Their irritancies on the ear and their promoting activities on the back skin depend strongly on the structural features of the acyl chain carrying the spin label. Based upon the bioactivities of individual esters biologically meaningful probes were defined for investigations of the molecular interaction of phorbol-ester-type promoters with cellular targets for electron paramagnetic resonance.

Animals↗
PubMed1988

Influence of vinblastine containing chemotherapy on tumor tissue membrane fluidity; an EPR study. A case report.

The influence of vinblastine (VLB) in combined chemotherapy on tumor cell membrane fluidity of a patient with exulcerated soft tissue sarcoma was studied by the electron paramagnetic resonance method (EPR). Tissue samples were taken before and after applications of VLB, Cis-Platinum (CDP) and combined intraarterial chemotherapy (IAC) and incubated with the spin probe, the palmitic acid methylester spin label analog, MeFasl (10, 3) incorporating into the cell membranes. EPR spectra of the labelled tissue samples were measured. Within few days after application of the chemotherapeutic drugs some oscillations of the membrane fluidity in the tissue were observed, while after a longer period of few weeks the relative amount of cells with greater membrane fluidity was found to decrease. Furthermore, an unexpected gradual increase in the EPR signal intensity after incubation of the tissue with the spin probe, probably due to an oxidative process in this particular sarcoma tissue was observed. These data could be useful for understanding of drug induced changes in cell membranes and for future timing of drug treatments in combined schedules.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Effect of temperature on Ca-ATPase from sarcoplasmic reticulum membranes: ESR studies.

Using spin-labeled fatty acid derivatives and maleimide, the effect of temperature on the structural state of various parts of the lipid bilayer of sarcoplasmic reticulum (SR) membranes and the segmental motion of the Ca-ATPase molecule were investigated. The mobility of the spin probes localized in the hydrophobic zone and the outer part of the SR membrane was shown to increase with a rise in temperature from 4 to 44 degrees C, the temperature of 20 degrees C being critical for these changes. In the presence of ATP, critical changes in the spin probe mobility occur at lower temperatures, while in the presence of ATP and Ca2+ they are observed at 20 degrees C for a spin probe localized in the outer part of the SR membrane. The mobility of a spin probe localized in the hydrophobic part of the membrane increases linearly with a rise in temperature. In the absence of ligands, the segmental motion of Ca-ATPase changes linearly within a temperature range of 10-30 degrees C. However, when ATP alone or ATP and Ca2+ are simultaneously added to the incubation mixture, the protein mobility undergoes critical changes at 20 degrees C. The Arrhenius plots for ATPase activity and Ca2+ uptake rate in SR membrane preparations also have a break at 20 degrees C. It is assumed that changes in the structural state of membrane lipids produce conformational changes in the Ca-ATPase molecule; the enzyme seems to be unsensitive to the structural state of the membrane lipid matrix in the absence of the ligands.

Animals↗
PubMed1983

Proton magnetic resonance characterization of brain tumours.

A series of different human brain tumours have been characterized with respect to the proton spin-lattice relaxation time (T1) and the water content. In malignant tumour tissue the mean T1 values are significantly larger than in benign ones. The tumours of different histological types showed notable differences in the relationship between proton relaxation rate (1/T1) and the corresponding water content (r). Malignant tumours exhibited a large variation in the water content in the presence of relatively small changes in relaxation rates, whilst just the opposite was true of the benign tumours and normal tissue. Taking into consideration the fast exchange between the intracellular and extracellular fluid, the (1/T1)/r relationship observed in malignant tissue is explainable by the variation in water content in the extracellular fluid due to oedema, which is most pronounced in malignant tumours. The individual response of patients to the malignant growth seems to determine the tissue water content and hence the measured relaxation time, whilst it appears that the relaxation rate and the water content of the malignant cells of the same tumour type do not vary appreciably from one patient to another. On the other hand, benign and non-tumourous tissue cells from different patients show larger variations in T1, whilst the variations in the extracellular water content are obviously smaller than in malignant tissues.

Brain Neoplasms↗
PubMed1983

Free radicals in tetanic activity of isolated skeletal muscle.

1. The concentration of the free radicals in muscle tissue mitochondria changes with the phase of muscle contraction. 2. Potassium cyanide (KCN) influences the shape of the isotonic tetani mechanograms; the contraction is still strong and qualitatively comparable in amplitude to untreated controls. 3. On the other hand, the correlation between the free radical concentration and muscle contraction is lost. 4. The free radical concentration in isolated mitochondria is influenced by KCN and potassium ferricyanide. 5. The free radical concentration changes due to the electron flow in the respiratory chain. 6. Inhibition of the respiratory phosphorylation by KCN could affect the free radical level.

Animals↗