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S Pecar

Publications and source records attributed to S Pecar.

30 records · Page 2Linked to original sources
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

Apyrogenic synthetic desmuramyldipeptide, LK-409, with immunomodulatory properties.

The synthesis and some immunological characteristics of a new desmuramyl dipeptide 7-oxooctanoyl-L-alanyl-D-isoglutamine (LK-409) are presented. The effects of this compound were compared with those of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). The influence of LK-409 on the number of B and T cells in spleen and the number of peritoneal macrophages was studied; Jerne's plaque forming cells assay was performed to monitor the effect of B cell differentiation. The blast transformation of T cells stimulated with concanavalin A was used to detect the influences on T lymphocytes. The activation of macrophages was studied as well. In contrast to MDP, LK-409 was apyrogenic in the doses applied but had similar immunomodulatory properties. Tested immunological properties and the absence of pyrogenicity and low toxicity make LK-409 a candidate for an immunomodulatory drug and a model molecule suitable for studying and understanding the dual activity of the MDP and its analogues.

Acetylmuramyl-Alanyl-Isoglutamine↗
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

Cellular metabolism of proxyl nitroxides and hydroxylamines.

Previous data from model systems indicated that the proxyl nitroxides should be especially resistant to bioreduction and therefore could be an effective solution to this often problematic characteristic of nitroxides. Therefore, we investigated the rate of reduction by cells and by the usual model system, ascorbate, of four proxyl nitroxides and three reference nitroxides. We found that, while the rate of reduction by ascorbate of the proxyl nitroxides was slower than the rate of a prototypic pyrrolidine nitroxide (PCA), the reverse was true for reduction by cells. We also studied the rate of oxidation of the corresponding hydroxylamines. The rate of oxidation by cells of the proxyl hydroxylamines was relatively fast, especially for the most lipophilic derivative. These results indicate that: (i) proxyl nitroxides may not be unusually resistant to bioreduction by functional biological systems; (ii) accurate knowledge of relative rates of metabolism of nitroxides and hydroxylamines in cells and tissues will require direct studies in these systems because the rates may not closely parallel those observed in model (chemical) systems; and (iii) proxyl nitroxides show potential value as agents to measure oxygen concentrations by the rates of oxidation of their corresponding hydroxylamines.

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

QSAR analysis of acetylcholinesterase inhibitors by use of structure similarity methods.

QSAR analysis of acetylcholinesterase inhibitors by a computer system for classification of chemical compounds by use of structure similarity methods has shown that the activity of these compounds is highly correlated with the geometry and the shape of attached radicals. Best values of the biochemical properties were obtained for compounds containing an optimal length of the attached radicals R1 and R2. The predicted values of the bimolecular rate constant for four compounds agree very well with the experimental results.

Acetylcholinesterase↗
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↗
PubMed1982

Spin-labeled phorbol esters and their interaction with cellular membranes.

A series of homologous spin-labeled fatty acid analogs of the type 12-O-FASL (n,m)-phorbol-13-acetate [(n,m)PA] of 12-O-tetradecanoylphorbol-13-acetate (TPA) with variable chain length N of the fatty acid moiety and various positions of the nitroxide group were synthesized. Their irritant doses 50 (ID50) on the ear and their initiation-promoting activities on the back skin of NMRI mice were determined. The irritancy 1/ID50 of the (n,m)PA follows the simple linear relation ln(1/ID50) = alpha N + beta. In an "equilibrium" approach, the electron paramagnetic resonance (EPR) spectra measured in absence and in presence of erythrocytes (E) were used to determine the partition coefficients KM,0 between the erythrocyte membrane (M) and the extracellular solution (o). The KM,0 depend on the chain length N of the fatty acid moieties following the equation ln KM,0 = aN + b and show that the (n,m)PA are accumulated preferentially in the erythrocyte membrane. In a "kinetic" approach, the decay rate J(t)/J(o) of the EPR signal intensity of (n,m)PA molecules in absence and in presence of equilibrated E was measured during oxidation with ferricyanide and reduction with ascorbate, respectively. From the data obtained by the "kinetic" approach, a "two-compartmental model" is shown to be the most simple approximation to describe the system, in which finite concentrations of the (n,m)PA are postulated for the extracellular and the membrane compartment only. EPR data indicate that generally the (n,m)PA are buried with their acyl chain in the E membrane with the phorbol moiety close to the outer membrane surface. Apparently only in case of (1,12)PA is the acyl chain long enough to be anchored in the lipid layer of the membrane in a way similar to the tetradecanoyl residue of TPA. A possible relationship with biological activities is discussed.

Animals↗
PubMed1979

Stereochemical properties of the binding site of liver microsomal cytochrome P-450 as studied by substrate analogous spin labels.

For the characterization of the substrate binding site optical and EPR measurements with spin labelled substrates on solubilized and pure cytochrome P-450 were performed. Analogously to the unlabelled derivatives spin labelled n-alkylamines and isocyanides with different chain lengths are type II substrates. The Ks-values evaluated from optical (P-450 = 1.98 . 10(-6) M) and ESR (P-450 = 1.98 . 10(-4) M) measurements are very similar indicating no concentration dependences. Contrary to the unlabelled n-alkylamines the spin labelled compounds show an affinity almost independent of the chain lengths. The SL-substrates with a short distance between the functional group and the NO-group bound to P-450 induce pronounced changes of the ligand field of the heme iron and a large broadening of the signal of the immobilized nitroxide indicating intensive interactions between the unpaired electron of the nitroxide group and the paramagnetic heme iron. Elongation of the alkyl chains results in spectra of the Fe3+ complexes with only slight modification and a remained unbroadened signal of the immobilized nitroxide. The binding of the substrate through their functional groups together with a 1:1 stoichiometry of the P-450 SL-IC-complex give evidence for the same binding site in the near vicinity of the heme iron.

Animals↗