Slavko Pečar

Abuse of Clenbuterol and its Detection

Clenbuterol and other beta-agonists are commonly misused as repartitioning agents in meat production and as doping substances to improve athletic performance. Numerous reports on food poisoning throughout Europe prompted the EU regulatory offices and FDA to implement a ban on the use of beta-agonists as growth promoters. Several analytical methods have been developed for detecting illegal administration of these compounds, based mainly on chromatography and immunoassay screening. This article deals with the pharmacological aspect of beta-agonists in growth promotion, control of their abuse and methods of analysis.

Skin protection against ultraviolet induced free radicals with ascorbyl palmitate in microemulsions

UV irradiation induces free radical formation in the skin. UV filters and antioxidants can be used for protection. In the present work, the amphiphilic antioxidant ascorbyl palmitate has been investigated and its effectiveness against free radical formation in porcine skin determined with electron paramagnetic resonance (EPR) spectroscopy with a spin trapping technique. 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was used as spin trap. In this study, three different radicals were identified in UV irradiated porcine ear skin: two originated from sulphur centred radicals (SO(3)*), while the third was the carbon-centred acyl (C=O*) radical. Ascorbyl palmitate applied on the skin decreased the level of formation of free radicals. Its effectiveness depended significantly on the carrier system - the type of microemulsion and its concentration, while the time of application had no influence on its effectiveness. Oil in water microemulsions delivered ascorbyl palmitate to the skin significantly better than water in oil microemulsions. In both types of microemulsions, the effectiveness increases at higher concentrations of ascorbyl palmitate.

Influence of spin probe structure on its distribution in SLN dispersions.

Solid lipid nanoparticles (SLN) are drug carrier system composed of biodegradable substances, which are solid at room temperature. The physico-chemical properties and structure of the incorporated compounds can affect their partitioning in SLN dispersions. In this work the influence of lipophilicity and structure of different SP on its location in SLN were studied. By electron paramagnetic resonance (EPR) measurements it was found that lipophilic SP distribute between a solid glyceride core and a soft phospholipid layer, with the more polar part (piperidine ring or methylcarboxylic groups) oriented toward the water-lipid interface. The majority of SP is located in the phospholipid layer, but the portion in the solid lipid core increases with SP lipophilicity. The hydrophilic Tempol does not incorporate into SLN.

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.

Design and Synthesis of Novel N-Benzylidenesulfonohydrazide Inhibitors of MurC and MurD as Potential Antibacterial Agents

A series of novel N-benzylidenesulfonohydrazide compounds were designed and synthesized as inhibitors of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) and UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) from E. coli, involved in the biosynthesis of bacterial cell-walls. Some compounds possessed inhibitory activity against both enzymes with IC50 values as low as 30 μM. In addition, a new, one-pot synthesis of amidobenzaldehydes is reported.

An ESR characterization of micelles and vesicles formed in aqueous decanoic acid/sodium decanoate systems using different spin labels.

Aqueous decanoic acid/sodium decanaote systems were studied as a function of pH and concentration, up to 0.3 M decanoic acid/sodium decanoate, by electron spin resonance (ESR) spectroscopy using three different amphiphilic spin labels. The distribution of the spin labels between vesicles and micelles as well as their dynamic properties were determined by quantitative analysis of the ESR spectra using two novel simulation software packages. Rotational correlation time of the labels in micelles was found to increase with decreasing pH, with substantial increase in the region where vesicles were formed (7.8<pH<8). In the interval 6.5<pH<7.8, the coexistence of vesicles and micelles was observed. Presence of vesicles was confirmed by the captured aqueous volume, determined independently with a hydrophilic spin label. The ESR measurements indicate that decanoic acid vesicle formation observed as the concentration is increased between 0.01 M and 0.03 M at pH 7.0 most likely occurs via the formation of micelles which remain in coexistence with the vesicles, even if the concentration is well above these values.

Nitroxide–fluorophore double probes: a potential tool for studying membrane heterogeneity by ESR and fluorescence

A serious drawback of ESR, particularly in its application to cells, is the lack of information on the location of spin probes in the system. In order to realize real time tracking, a spin probe was combined with a fluorophore in a new kind of nitroxide-fluorophore double probe which, in addition to information about lipid dynamics, enables visualization by fluorescence microscopy. The two sets of probes synthesized are based on an amino-alkyne-functionalized sugar that serves as a central polar group and as a linker between the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore and the derivative of the spin labelled fatty acid. In this setting, the location of the fluorophore is restricted to the water-lipid interface, while the nitroxide is located deep in the lipid bilayer. Preliminary tests on cells show preferential localization of both probes in the plasma membrane, with a relatively slow redistribution to other membranes of the cell. We believe that such double probes would be particularly useful for studies of plasma membrane heterogeneity and associated cellular processes.

Are calculated log P values for some guanine derivatives by different computer programs reliable

The log P values of n-octanol/water for some guanine derivatives, acyclovir, deoxyaciclovir and their acetyl congeners, were calculated by some commercially available computer programs for log P calculation. These values were compared with those obtained by the conventional shake-flask method. It was established that the calculations of log P values for examined guanine derivatives by these computation programs do not give reliable results.

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 paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site

Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim.