Direct measurement of interaction forces in free thin liquid films stabilized with phosphatidylcholine

Microscopic foam films from suspensions of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles have been obtained. The film formation is facilitated at temperatures above the gel-liquid crystalline transition of DMPC. A detailed study of the dependence of equilibrium thickness of DMPC foam films on electrolyte concentration at constant capillary pressure and a direct measurement of the disjoining pressure isotherm has been carried out. Formation of thick equilibrium horizontal microscopic films stabilized with DMPC at low external pressures and low electrolyte concentrations was found and interpreted as being due to the existence of long-range electrostatic interactions in these films. A diffuse electric layer potential of 36 mV has been calculated. The DMPC films have been compared to films obtained from non-ionic surfactant solutions where the long range electrostatic repulsion is explained as being due to specific adsorption of OH^– at the film interfaces. However, unlike the results obtained for surfactant films, in this study formation of common black films and thinning of the DMPC Newton film with pressure have been observed. Offprint requests to: D. Exerowa     

Direct measurement of free radicals in the brain cortex and the blood serum after nociceptive stimulation in rats

The concentrations of ROS were measured in samples of the sensorimotor brain cortex and in the rat blood. We measured the following parameters: The six lines spectra, nitroxide radical, free hydroxyl radical and singleton oxygen. Their concentration was measured under physiological conditions, after the nociceptive stimulation and after the application of melatonin, both in normal and stimulated animals. In the brain cortex only the singleton oxygen decreased after the nociceptive stimulation, whereas the nitroxide radicals and six lines spectra increased. The free hydroxyl radicals did not change significantly. In the blood serum the six lines spectra and nitroxide radical increased, the concentration of the free hydroxyl radicals did not change. Melatonin increased both the hydroxyl and nitroxide radicals. There was a non-significant decrease in the six lines spectra. The estimation of ROS can be used as a tool for detecting metabolic changes and the consequences of different environmental influences, in our case the influence of nociception and melatonin.     

Induction of free radicals in hepatocytes,mitochondria and microsomes of rats by ochratoxin A and its analogs

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and Oα; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe³⁺ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe³⁺. The spin adduct hyperfine splitting constants indicated the presence of α-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.     

Formation of free radicals during interaction of adriamycin and carminomycin with xanthine oxidase

Xanthine oxidase reduces carminomycin and adriamycin to the semiquinones which have been detected by ESR technique. The steady state carminomycin semiquinone concentration is some tens times higher than the corresponding value for adriamycin. This effect appears to be a result of carminomycin semiquinone stabilization due to internal hydrogen bonding.     

Interactions of carnosine and superoxide radicals in aqueous solutions

Carnosine was discovered to be able to interact with superoxide-anion and active hydroxyl radicals, using carnosine aqueous solutions. This interaction is specific and can be detected at carnosine concentrations more than 0.02 mM. Interaction of carnosine with O2 results in occurring of charge translocation complex with absorbtion maximum at 265 nm. Stability of this complex is dependent on the medium pH, decreasing with its acidification.     

In vivo ESR measurement of free radicals in whole mice

The in vivo measurement of nitroxide radicals in whole mouse was carried out by L-band ESR spectroscopy. Spectra were successively observed in hepatic and bladder domains of female mice after intravenous administration of spin-labeled compounds (CPROXYL or TEMPOL). The signal intensities from both domains decreased gradually. The kinetic constants of clearance in the hepatic domain were 0.09/min for CPROXYL and 0.71/min for TEMPOL. The clearance constants in the bladder domain coincided with those in the hepatic domain within experimental error, whereas the constants in collected blood were 1/7-1/10 of those in the hepatic or bladder domains. The mechanism of clearance of nitroxide radicals in whole mice is discussed.     

The interaction of hypochlorite with fatty acid hydroperoxides results in the generation of free radicals

The interaction of hypochlorite with linoleic acid hydroperoxides was studied by the coumarin C-525-enhanced chemiluminescence and ESR spin trapping techniques. Linoleic acid hydroperoxide was obtained in the reaction of lipoxygenase and linoleic acid. Alpha-(4-pyridyl-1-oxyl)-N-tert Butylnitron was used as a spin trap. It was shown that the addition of hypochlorite to the incubation media containing linoleic acid and lipoxygenase resulted in an intensive chemiluminescence flash. The intensity of this flash correlated with the hydroperoxide concentration. The analysis of ESR spectra of spin adducts produced in the reaction of hypochlorite with linoleic acid hydroperoxide showed the presence of O-centered, most likely peroxyl, radical with the splitting constants alphabetaH = 0.260 mT aN = 1.662 mT and C-centered penthyl radical with the splitting constants alphabetaH = 0.260 mT; aN = 1.662 mT. These data suggest that hypochlorite produced by phagocytes in vivo can induce the generation of free O- and C-centered radicals, promoters of free radical processes.     

Oxygen-derived free radicals in acute experimental pancreatitis]

A marked decrease in the activity of superoxide dismutase together with an increase in pancreatic tissue lipid peroxidation have been found in rats with cerulein-induced acute pancreatitis (CRAP). It has been suggested, that the observed imbalance between oxygen-derived free radicals generation and inactivation may cause an injury to the pancreas. Preventive effect of hydroxyl radical scavenger in CRAP has been seen.     

The interaction of 5,5-dimethyl-1-pyrroline-N-oxide with human myeloperoxidase and its potential impact on spin trapping of neutrophil-derived free radicals

Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl-. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl- to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl- required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO/H2O2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (greater than 10 mM) significantly decreased MPO/H2O2/Cl- -mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.     

The interaction of benzhydroxamic acid with horseradish peroxidase and its fluorescent analogs

Horseradish peroxidase (HRP) reconstituted with protoporphyrin IX or zinc protoporphyrin IX binds benzhydroxamic acid with affinities of 1.54 × 10⁴ and 8 × 10²m^?1, respectively. This interaction is competitive with respect to hydrogen donor substrates of peroxidase. The steady-state oxidation of benzhydroxamic acid by HRP was studied by monitoring the disappearance of the hydroxamate function and the formation of nitrite. The inhibition by benzhydroxamic acid of oxidation of HRP substrates may be classified as an inhibition by a competing substrate. In the case of HRP-catalyzed oxidation of ferrocyanide a marked activating effect of benzhydroxamic acid was observed. Mechanisms responsible for this effect are discussed.     

Synthesis of the tripeptide glycyl-L-leucyl-L-phenylalanine and its analogs]

Peptide Gly-L-Leu-L-Phe and its derivatives were synthesized by the C-end elongation utilizing DCC/HOBT technique and by enzymatic route with the help of papain using esters of N-benzyloxycarbonyl-glycine and -L-leucine as acyl donors have been suggested. The chemical, similarly to the enzymatic, synthesis was not accompanied by racemization. Conditions for HPLC separation and preparative isolation of the enzymatic reaction products were developed.     

Kinetic studies of the interaction of synthetic RNAs with quinacrine and its analogs.

Temperature-jump relaxation method has been used to study the interaction of synthetic RNAs with quinacrine (QAC) and its analog. Two relaxation times were observed. The dependence of relaxation times on the RNA concentration and optical properties of the RNA-dye complexes suggests that (1) QAC binds to poly-(rA).poly(rU) through two bimolecular reactions including isomerization from one complex form to another and (2) the 2-methoxy group of the acridine ring plays a significant role in the isomerization.     

Spectroscopic studies of the interaction of synthetic RNAs with quinacrine and its analogs.

The interaction of synthetic RNAs with quinacrine (QAC) and its analogs has been studied by absorption, fluorescence and circular dichroism (CD) measurements. The results indicate that the 2-methoxy group of the acridine ring plays an important role in the appearance of the new absorption band upon binding of QAC to poly-(rA).poly(rU).     

A comparative study of synthetic carnosine analogs as antioxidants

1. The antioxidative activity of carnosine and 16 related compounds, both synthetic and natural, was determined. 2. The antioxidative effect was estimated by the ability of the dipeptides to prevent MDA accumulation in the course of LPO induced in rabbit sarcoplasmic reticulum membranes by the Fe2+ ascorbate system. 3. It was found that the antioxidative effect comparable to that of carnosine was exerted by water-soluble (cyclo-L-histidyl-L-proline) and alcohol-soluble (cyclo-L-histidyl-L-phenilalanine) dipeptides as well as by the histidine-free cyclodipeptides (cyclo-L-tyrosyl-L-proline). 4. However, in contrast to its synthetic analogs, carnosine not only inhibited the LPO, but also diminished the level of products accumulated during membrane lipid peroxidation.     

Thiyl and phenoxyl free radicals and NADH. Direct observation of one-electron oxidation.

Absolute rate constants for the reaction of NADH with thiyl free radicals derived from various sulphur-containing compounds of biological significance were measured by using the technique of pulse radiolysis. These and related reactions with phenoxyl free radicals are believed to occur through one-electron-transfer processes. Further evidence comes from studies with deuterated NADH. The results support the possibility that, in biochemical systems, thiols may act as catalysts linking hydrogen-atom and electron-transfer reactions.     

Probing the TRAP-RNA interaction with nucleoside analogs

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2-3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP-RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5'-(UAGCC)-3' repeated 11 times and measured their effect on the TRAP-RNA interaction. Deoxyribonucleoside substitutions revealed that a 2'-hydroxyl group (2'-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP-RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2'-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.     

Reactivity of ubiquinones and ubiquinols with free radicals

The reactivity of quinones 1–4 and of the corresponding quinols 5–8 towards carbon- and oxygen-centred radicals were studied. All quinones bearing at least one nuclear position free, readily react with alkyl and phenyl radicals to afford the alkylated quinones 12–24; however, quinones 1 and 3 reacted with 2-cyano-2-propyl radical to yield products (the mono- and di-ethers 9–11) derived from the attack on the carbonylic oxygen. The reactions carried out on quinones with the benzoyloxy radical led to no reaction products and in the case of Q₁₀, the isoprenic chain also remained unchanged. Quinols 5–8 reacted only with oxygencentred radicals (benzoyloxy and 2-cyano-2-propylperoxy radicals) to give the corresponding quinones. The isoprenic chain of Q₁₀ did not undergo attack even with peroxy radicals. Carbon-centred radicals resulted unable to abstract hydrogen from the studied quinols.