Activation of aqueous hydrogen peroxide for non-catalyzed dihydroperoxidation of ketones by azeotropic removal of water.

Cyclic and acyclic ketones were selectively converted to gem-dihydroperoxides in 72-99% yield with 30% aq. hydrogen peroxide by azeotropic distillation of water from the reaction mixture without any catalyst. The reactions were more selective than with 100% H2O2 and due to neutral conditions also less stable products could be obtained.

Influence of desmuramylpeptides from LK-409 series on the cytokine production in the mouse spleen cells.

LK-409 (7-oxooctanoyl-L-Ala-D-iGln) was found to modulate immune response. To study the impact of minimal structural changes in the LK-409 molecule on the immunomodulatory activity a series of LK-409 analogues was prepared and their activities were evaluated. After the treatment of NmRI mice at a dose of 25 microg daily for three consecutive days the isolated spleen cells were stimulated with concanavaline A and phorbol 12-myristate 13-acetate (PMA) or ionomycin and PMA, and the production of IL-2, IL-4,-IL-6, IL-10 and IFN-gamma studied as a function of the different LK-409 analogues. All the compounds modulate the Th1/Th2 cytokine response, especially in the presence of ConA&PMA activation. The minimal structure modification of LK-409 strongly influences the regulation of the cytokine production.

Analysis of the causes and consequences of decreased antibiotic consumption over the last 5 years in Slovenia.

OBJECTIVES: Compared with European countries, the use of antibiotics in Slovenia is moderate. In the period 1999-2002 an 18.67% decrease in outpatient antibiotic consumption was noted. The aim of the present study was to analyse this decrease and its consequences. METHODS: The data on outpatient antibiotic consumption were obtained from the Institute of Public Health and Health Insurance Institute of Slovenia and expressed in defined daily doses (DDD)/1000 inhabitant-days. The number of media publications on 'antibiotic drugs' and 'bacterial resistance' during the study period was obtained. In 2000, the prescription of co-amoxiclav and fluoroquinolones was restricted because of a constant increase in the consumption of these drugs. The data on incidence of acute mastoiditis and penicillin resistance among invasive pneumococci were obtained. RESULTS: The total outpatient consumption of antibacterials increased from 15.21 DDD/1000 inhabitant-days in 1996 to 20.08 in 1999, and decreased to 16.97 in 2003. The consumption of restricted antibiotics decreased from 7.29 in 1999 to 5.25 DDD/1000 inhabitant-days in 2003. There was a positive correlation between antibiotic consumption and the number of newspaper articles (r=0.92), and a negative correlation between the number of diagnostic tests and antibiotic consumption (r=-0.73 for the C-reactive protein test and -0.68 for the streptococcal antigen detection test). Reduced antibiotic consumption was paralleled by a decrease in penicillin resistance among invasive pneumococci. No increase in mastoiditis cases was observed in spite of reduced antibiotic consumption. CONCLUSION: Restriction of antibiotic prescription proved to be effective in reducing outpatient antibiotic consumption. The effect was prolonged and affected restricted antibiotics as well as non-restricted drugs.

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.

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.

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.

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.

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.

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.

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.

N-[trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl]-L-alanyl-D-glutamic acid: a novel immunologically active carbocyclic muramyl dipeptide analogue.

A novel non-pyrogenic carbocyclic muramyl dipeptide (MDP) analogue, N-¿trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl¿-L-alanyl-D-glutamic acid, was obtained by replacement of the N-acetylmuramic acid part and the D-isoglutamine residue of the MDP molecule by a trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl moiety and D-glutamic acid, respectively. The title compound was selected as a promising candidate for further evaluation among several related analogues on the basis of an immunorestoration test in mice. This novel nor-MDP analogue protects mice against the immunosuppressive effect of cyclophosphamide and increases the nonspecific resistance of mice against fungal infection. It is an immunomodulator which enhances the maturation of lymphocytes B to plasma cells and increases the activity of lymphocytes B and lymphocytes T as well as that of macrophages but does not alter the number of these cells.

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.

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.

Allosteric effects of phenyltrimethylammonium and propidium on acetylcholinesterase active site.

Different spin labelled fluorophosphates and fluorophosphonates with different chain length were investigated with respect to their sensitivity to the allosteric changes of acetylcholinesterase active site produced by phenyltrimethylammonium (Pta) or d-tubocurarine (TC); only fluorophosphates were found to be sensitive to these changes. Therefore fluorophosphates were chosen also for the study of allosteric effects of propidium. The addition of Pta and propidium to spin labelled membrane acetylcholinesterase of the Torpedo marmorata electric organ decreased maximal hyperfine splitting of the EPR spectrum, indicating that the microgeography of the acetylcholinesterase active site is usually changed in a way which increases the freedom of motion of the spin label's piperidine ring. TC alone did not change the EPR spectrum, but it prevented the influence of Pta and not that of propidium.

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).

Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance.

Both propidium and monoclonal antibody (mAb) 25B1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.