Inhibition of Photosystem II by the singlet oxygen sensor compounds TEMP and TEMPD

2,2,6,6-tetramethylpiperidine (TEMP) and 2,2,6,6-tetramethyl-4-piperidinone (TEMPD) have earlier been used to quantify singlet oxygen produced by plant material. Both compounds were found to cause severe side effects on Photosystem II. Addition of TEMP or TEMPD to thylakoids immediately stabilized the reduced state of the Q(A) electron acceptor and destabilized the reduced state of the Q(B) acceptor, causing decrease in the driving force of forward electron transfer. Oxygen evolution, thermoluminescence and fluorescence measurements indicated that the number of functional PSII units decreased during incubation of thylakoids with TEMP or TEMPD. Singlet oxygen determinations in photosynthetic systems with piperidine derivatives should be interpreted with care.     

Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II

Electron spin resonance spectroscopy and liquid chromatography have been used to detect radical formation and fragmentation of polypeptides during photoinhibition of purified major antenna proteins, free of protease contaminants. In the absence of oxygen and light, no radicals were observed and there was no damage to the proteins. Similarly illumination of the apoproteins did not induce any polypeptide fragmentation, suggesting that chlorophyll, light and atmospheric oxygen are all participating in antenna degradation. The use of TEMP and DMPO as spin traps showed that protein damage initiates with generation of (1)O(2), presumably from a triplet chlorophyll, acting as a Type II photosensitizer which attacks directly the amino acids causing a complete degradation of protein into small fragments, without the contribution of proteases. Through the use of scavengers, it was shown that superoxide and H(2)O(2) were not involved initially in the reaction mechanism. A higher production of radicals was observed in trimers than in monomeric antenna, while radical production is strongly reduced when antennae were organized in the photosystem II (PSII) complex. Thus, monomerization of antennae as well as their incorporation into the PSII complex seem to represent physiologically protected forms. A comparison is made of the photoinhibition mechanisms of different photosynthetic systems.     

Small CAB-like proteins prevent formation of singlet oxygen in the damaged photosystem II complex of the cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterial small CAB-like proteins (SCPs) are single-helix membrane proteins mostly associated with the photosystem II (PSII) complex that accumulate under stress conditions. Their function is still ambiguous although they are assumed to regulate chlorophyll (Chl) biosynthesis and/or to protect PSII against oxidative damage. In this study, the effect of SCPs on the PSII-specific light-induced damage and generation of singlet oxygen ((1)O(2)) was assessed in the strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking PSI (PSI-less strain) or lacking PSI together with all SCPs (PSI-less/scpABCDE(-) strain). The light-induced oxidative modifications of the PSII D1 protein reflected by a mobility shift of the D1 protein and by generation of a D1-cytochrome b-559 adduct were more pronounced in the PSI-less/scpABCDE(-) strain. This increased protein oxidation correlated with a faster formation of (1)O(2) as detected by the green fluorescence of Singlet Oxygen Sensor Green assessed by a laser confocal scanning microscopy and by electron paramagnetic resonance spin-trapping technique using 2, 2, 6, 6-tetramethyl-4-piperidone (TEMPD) as a spin trap. In contrast, the formation of hydroxyl radicals was similar in both strains. Our results show that SCPs prevent (1)O(2) formation during PSII damage, most probably by the binding of free Chl released from the damaged PSII complexes.     

Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II

Electron spin resonance spectroscopy and liquid chromatography have been used to detect radical formation and fragmentation of polypeptides during photoinhibition of purified major antenna proteins, free of protease contaminants. In the absence of oxygen and light, no radicals were observed and there was no damage to the proteins. Similarly illumination of the apoproteins did not induce any polypeptide fragmentation, suggesting that chlorophyll, light and atmospheric oxygen are all participating in antenna degradation. The use of TEMP and DMPO as spin traps showed that protein damage initiates with generation of ¹O₂, presumably from a triplet chlorophyll, acting as a Type II photosensitizer which attacks directly the amino acids causing a complete degradation of protein into small fragments, without the contribution of proteases. Through the use of scavengers, it was shown that superoxide and H₂O₂ were not involved initially in the reaction mechanism. A higher production of radicals was observed in trimers than in monomeric antenna, while radical production is strongly reduced when antennae were organized in the photosystem II (PSII) complex. Thus, monomerization of antennae as well as their incorporation into the PSII complex seem to represent physiologically protected forms. A comparison is made of the photoinhibition mechanisms of different photosynthetic systems.     

Antioxidant properties of tea investigated by EPR spectroscopy

The antioxidant properties of green, black and mixed (fruit) tea samples of different origin were investigated by means of EPR spectroscopy. A six line EPR spectrum of solid tea samples indicates the presence of Mn(II) ions and it is superimposed with a sharp singlet line attributed to semiquinone radical species (Delta H(pp)=1 mT; g=2.0022). Antioxidant properties of aqueous tea extracts in H(2)O(2)/NaOH/dimethylsulfoxide system generating reactive radicals (*OH, O(2)*-), *CH(3)) were followed by spin trapping technique. In addition, antioxidant capacity of these samples was assessed using stable radicals 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPOL). Typically, the highest antioxidant potential to terminate superoxide radicals was found in green teas, followed by black and fruity teas. The pro-oxidant activity of green teas evidenced by spin traps was promoted in samples with higher Mn(II) and ascorbic acid concentrations. Various sources of free radicals used in the antioxidant tests due to their specific action show different termination rates in the presence of the individual tea samples.     

EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines

Superoxide (O???) has been implicated in the pathogenesis of many human diseases, but detection of the O(2)(?-) radicals in biological systems is limited due to inefficiency of O??? spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O??? in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O???, producing stable nitroxides and allowing site-specific cO??? detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O???. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O??? both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O??? stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O???. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O??? radical in extracellular or intracellular compartments, cytoplasm or mitochondria.     

Scavenging of reactive oxygen species by some nonsteroidal anti-inflammatory drugs and fenofibrate

Ketoprofen and tolmetin are widely used nonsteroidal anti-inflammatory drugs, whereas fenofibrate belongs to a family of hypolipidemic drugs used in the prevention of cardiovascular diseases. The aim of this study was to assess effect of these drugs on reactions generating reactive oxygen species (ROS). The following generators of ROS were used: 18-crown-6/KO(2) dissolved in DMSO as a source of superoxide radical (O(.-)(2), the Fenton-like reaction (Cu/H(2)O(2)) for hydroxyl radical (HO(.)), 2,2'-azobis (2-amidino-propane) dichloride (AAPH) as peroxyl radical (ROO(.)) generator, and a mixture of alkaline aqueous H(2)O(2) and acetonitrile for singlet oxygen ((1)O(2)). Measurements were done using chemiluminescence, fluorescence, and spin-trapping with 2,2,6,6-tetramethylpiperidine combined with electron spin resonance spectroscopy (ESR), and a deoxyribose assay based on the spectrophotometry. The results obtained demonstrated that all tested drugs were active against O(.-)(2). There was a clear ranking of drug inhibition effects on chemiluminescence from the O(.-)(2) system: ketoprofen > tolmetin > fenofibrate. The examined compounds inhibited the HO(.)-dependent deoxyribose degradation and scavenged the ROO(.) concentration dependently with an order of potencies similar to that of the superoxide radical system. Hence, these results indicate that the studied drugs show broad ROS scavenging property and, as a consequence, might decrease tissue damage due to the ROS and thus to contribute to anti-inflammatory therapy.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.     

Vasoactive intestinal peptide, a singlet oxygen quencher

The neuropeptide vasoactive intestinal peptide (VIP), a highly basic 28-amino acid peptide, has a widespread distribution in the body. The functional specificity of this peptide not only includes its potent vasodilatory activity, but also its role in protecting lungs against acute injury, in preventing T-lymphocyte proliferation and in modulating immune function. We have investigated the possible antioxidant properties of VIP and found that VIP does not have significant O2-, OH., or H2O2 scavenging ability. However, VIP was found to inhibit, in a dose-dependent manner, the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation. 1O2 was produced in photosensitizing systems using rose bengal or methylene blue as sensitizers and was detected as TEMP-1O2 product (TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. The formation of TEMPO signal was strongly inhibited by known singlet quenchers, e.g. beta-carotene, histidine as well as azide, but not by catalase (20 micrograms/ml) which removes H2O2 and mannitol (6 mM) or ethanol (5.9 mM) which remove OH.. Superoxide dismutase (2.5 micrograms/ml) inhibited the photoreaction up to 20% by removing O2- and most probably by blocking the secondary charge transfer pathway of 1O2 formation. These results suggest that the formation of nitroxide radical by 1O2 attack on TEMP may be used as a simple and specific assay for 1O2, and VIP can serve as an effective 1O2 scavenger/quencher, thus it may modulate the oxidative tissue injury caused by this reactive species of oxygen.     

Effect of proline on the production of singlet oxygen

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.     

The effects of derivatives of the nitroxide tempol on UVA-mediated in vitro lipid and protein oxidation

Derivatives of tetramethylpiperidines are extensively employed in polymers to prevent photooxidation, and their stabilizing effect is attributed to the activity of the nitroxide radical derived from the parent amine. In this study, we examined the photoprotective effect of a commercial polymer photostabilizer, HALS-1, its corresponding nitroxide, bis(2,2,6,6-tetramethyl-piperidine-1-oxyl-4-yl)sebacate (TINO), and two derivatives of the piperidine nitroxide TEMPOL, 2,2,6,6-tetramethyl-piperidin-4-acetyloxy-1-oxyl (TEMP2) and 2,2,6,6-tetramethyl-piperidin-4-octanoyloxy-1-oxyl (TEMP8) synthesized by us, in liposomes exposed to ultraviolet A (UVA) radiation. For comparison, the UVA-absorber, 4-tert-butyl-4'-methoxydibenzoylmethane (Parsol 1789) used in many suncream formulations, was also included. The nitroxide TINO resulted extremely efficient at inhibiting aldehydic breakdown products deriving from 30 min exposure of liposomes to UVA and the protection was dose-dependent (10-100 microM). The corresponding amine HALS-1 was the least efficient while protection increased in the order: TEMP2 < Parsol 1789 < TEMP 8. HALS-1, TINO, and the two TEMPOL derivatives were also tested in a simple protein system consisting of bovine serum albumin (BSA) exposed to UVA. In this case, these compounds did not inhibit nor enhance UVA-mediated protein carbonyl formation in BSA. The differences in protection between the compounds are discussed in relation to their chemical reactivity, UVA-absorbing capacities, and their molecular structure. Overall, the results obtained envisage the potential use of nitroxide compounds as topical antioxidants.     

Vascular effects of singlet oxygen (1O2) generated by photo-excitation on adrenergic neurotransmission in isolated rabbit mesenteric vein

It has been suggested that reactive oxygen species (ROS) may be involved in the regulation of vascular tone. However, the nature of ROS effects on vascular sensitivity remains to be elucidated. The present study was designed to investigate the effects of ROS, especially 1O2, on neurotransmission at the sympathetic neurovascular junction. Basal noradrenaline (NA) release, release of NA induced by electrical stimulation (ES), and resting NA release at the sympathetic nerve terminals were determined using a superfusion technique. The amount of NA was determined by HPLC; isometric tension changes evoked by ES were also recorded simultaneously. 1O2 was generated from Rose Bengal by photo-activation. The generation of 1O2 in the superfusate was monitored by electron spin resonance (ESR) using the spin trap 2,2,6,6-tetramethyl-4-piperidinol throughout the experimental time course. The ESR results confirmed that 1O2 was generated by photo-activation of Rose Bengal via the formation of 2,2,6,6-tetramethyl-4-hydroxyl-piperidinyloxy. Exposure of helical strips of rabbit mesenteric vein to 1O2 induced a significant increase in tension and NA release during the basal period, but had no effect on ES-induced release. L-histidine, an 1O2 scavenger, significantly inhibited the observed effects on vascular tension and NA release in response to 1O2. These results suggest that 1O2 may induce NA-mediated vasoconstriction at the postjunctional site, and may be associated with Ca(2+)-independent NA release from the prejunctional site of adrenergic neurotransmission.     

Insulin-like growth factor I receptor is expressed at normal levels in Nijmegen breakage syndrome cells

Curcumin (diferuloylmethane) is a major component of food flavoring turmeric (Curcuma longa), and has been reported to be anticarcinogenic and anti-inflammatory. Although curcumin was shown to have antioxidant properties, its exact antioxidant nature has not been fully investigated. In this report we have investigated the possible antioxidant properties of curcumin using EPR spectroscopic techniques. Curcumin was found to inhibit the (1)O(2)-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose-dependent manner. (1)O(2) was produced in a photosensitizing system using rose bengal as sensitizer, and was detected as TEMP-(1)O(2) adducts by electron paramagnetic resonance (EPR) spectroscopic techniques using TEMP as a spin-trap. Curcumin at 2.75 microM caused 50% inhibition of TEMP-(1)O(2) adduct formation. However, curcumin only marginally inhibited (24% maximum at 80 microM) reduction of ferricytochrome c in a xanthine-xanthine oxidase system demonstrating that it is not an effective superoxide radical scavenger. Additionally, there was minor inhibition of DMPO-OH adduct formation by curcumin (solubilized in ethanol) when an ethanol control was included in the EPR spin-trapping study, suggesting that curcumin may not be an effective hydroxyl radical scavenger. Together these data demonstrate that curcumin is able only to effectively quench singlet oxygen at very low concentration in aqueous systems.     

Enhanced sensitivity and characterization of photosystem II in transgenic tobacco plants with decreased chloroplast glutathione reductase under chilling stress

Chloroplast glutathione reductase (GR) plays an important role in protecting photosynthesis against oxidative stress. We used transgenic tobacco (Nicotiana tabacum) plants with severely decreased GR activities by using a gene encoding tobacco chloroplast GR for the RNAi construct to investigate the possible mechanisms of chloroplast GR in protecting photosynthesis against chilling stress. Transgenic plants were highly sensitive to chilling stress and accumulated high levels of H?O? in chloroplasts. Spectroscopic analysis and electron transport measurements show that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrate that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses illustrate that PSII protein accumulation was decreased greatly in transgenic plants. Our results suggest that chloroplast GR plays an important role in protecting PSII function by maintaining the electron transport in PSII acceptor side and stabilizing PSII complexes under chilling stress. Our results also suggest that the recycling of ascorbate from dehydroascorbate in the ascorbate-glutathione cycle in the chloroplast plays an essential role in protecting PSII against chilling stress.     

Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Oxygen evolution and chlorophyll fluorescence from multiple turnover light pulses: charge recombination in photosystem II in sunflower leaves

Oxygen evolution and Chl fluorescence induction were measured during multiple turnover light pulses (MTP) of 630-nm wavelength, intensities from 250 to 8,000?μmol quanta m(-2)?s(-1) and duration from 0.3 to 200?ms in sunflower leaves at 22?°C. The ambient O(2) concentration was 10-30?ppm and MTP were applied after pre-illumination under far-red light (FRL), which oxidized plastoquinone (PQ) and randomized S-states because of the partial excitation of PSII. Electron (e ( - )) flow was calculated as 4·O(2) evolution. Illumination with MTP of increasing length resulted in increasing O(2) evolution per pulse, which was differentiated against pulse length to find the time course of O(2) evolution rate with sub-millisecond resolution. Comparison of the quantum yields, Y (IIO)?=?e ( - )/hν from O(2) evolution and Y (IIF)?=?(F (m)?-?F)/F (m) from Chl fluorescence, detected significant losses not accompanied by fluorescence emission. These quantum losses are discussed to be caused by charge recombination between Q (A) (-) and oxidized TyrZ at a rate of about 1,000?s(-1), either directly or via the donor side equilibrium complex Q(A)?→?P (D1) (+) ??TyrZ(ox), or because of cycling facilitated by Cyt b (559). Predicted from the suggested mechanism, charge recombination is enhanced by damage to the water-oxidizing complex and by restricted PSII acceptor side oxidation. The rate of PSII charge recombination/cycling is fast enough for being important in photoprotection.     

Mechanism of photoinhibition of photosynthetic water oxidation by Cl- depletion and F- substitution: oxidation of a protein residue

New evidence on the chloride requirement for photosynthetic O2 evolution has indicated that Cl- facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z+ radical. Illumination above 250 K of spinach PSII centers which are inhibited in O2 evolution by either Cl- depletion or F- substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca2+ only [Boussac et al. (1989) Biochemistry 28, 8984; Sivaraja et al. (1989) Biochemistry 28, 9459]. The physiological roles of Cl- and Ca2+ in water oxidation are thus linked. The characteristics include a nearly isotropic g = 2.00 +/- 0.005, a symmetric line shape with line width = 16 +/- 2 mT, almost stoichiometric spin concentration relative to Try-D+ = 0.6 +/- 0.3 spin/PSII, very rapid spin relaxation at all temperatures measured down to 6 K, and an undetectable change in magnetic susceptibility upon formation (less than 1 mu B2). The signal appears to originate from a spin doublet (radical) in magnetic dipolar contact with a transition-metal ion, most probably a photooxidized protein residue within 10 A of the Mn cluster (Mn-proximal radical). It is distinct from the three other protein-bound radical-type electron donors found in the PSII reaction center: Tyr-D+, Tyr-Z+, and C+. This signal photoaccumulates to a stable level under continuous illumination at 270 K and decays only after illumination stops.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins

Alterations in red blood cell (RBC) plasma membranes, i.e. in lipids and proteins, and osmotic fragility of these cells after treatment with Proteus mirabilis O29 endotoxin (lipolysaccharide (LPS)) were examined using a spin labelling method. At the highest concentration of LPS, insignificantly decreased fluidity of membrane lipids was observed. Changes in conformation of membrane proteins were determined by two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (MSL) and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The analysis of spectra of MSL and ISL showed modifications in membrane proteins in red blood cells treated with the highest concentration of lipopolysaccharide. On the other hand, in the case of isolated membranes, disturbances in membrane were observed for all concentrations of LPS. The alterations in membrane lipids and proteins are paralleled in a significant rise in osmotic fragility of RBCs upon endotoxin treatment. These results provide experimental evidence that P. mirabilis O29 LPS causes deleterious changes in membranes of human red blood cells. They show that action of lipopolysaccharide mainly concerns the membrane cytoskeleton.     

Spin trapping study of reactive oxygen species formation during bopindolol peroxidation

The generation of singlet molecular oxygen ((1)O(2)) and hydroxyl radicals (HO*) during peroxidation of bopindolol in the presence of Co(II) ions was studied using electron spin resonance (ESR) and spectrophotometry methods. 2,2,6,6-Tetramethyl-4-piperidone and 5,5-dimethyl-1-pyrroline-1-oxide were used as traps. The spectrophotometry determination of (1)O(2) was based on bleaching of p-nitrosodimethylaniline (RNO), which was caused by the product of the reaction of (1)O(2) with imidazole and was followed by monitoring the decrease in optical density at 440 nm. The effect of (1)O(2) quenchers and oxygen free radical scavengers on the ESR signal and the bleaching of RNO was studied. The data presented here give new evidence for generation of the reactive oxygen species during peroxidation of bopindolol.     

Irradiation of titanium dioxide generates both singlet oxygen and superoxide anion.

Although photoexcited TiO2 has been known to initiate various chemical reactions, such as the generation of reactive oxygen species, precise mechanism and chemical nature of the generated species remain to be elucidated. The present work demonstrates the generation of singlet oxygen by irradiated TiO2 in ethanol as measured by ESR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TMP) as a 1O2-sensitive trapping agent. Under identical conditions, the superoxide ion was also detected by spin trapping agent 5,5-dimethyl-pyrroline-N-oxide (DMPO). Kinetic analysis in the presence of both 4-oxo-TMP and DMPO revealed that singlet oxygen is produced directly at the irradiated TiO2 surface but not by a successive reaction involving superoxide anion. The basis for this view is the fact that DMPO added in the mixture increased the signals responsible for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), a reaction product of 4-oxo-TMP and 1O2. The detailed mechanism for the generation of 1O2 and superoxide ion by irradiated TiO2 and reactions between these species and DMPO are discussed.