Microdialysis study of ischemia-induced hydroxyl radicals in the canine heart

A new experimental approach for spin-trapping of oxygen radicals in a selected region of the heart in situ is described. This approach is based on microdialysis, and it permits the detection of oxygen radicals in conditions of local ischemia and restoration of normal blood flow. Increased hydroxyl radical generation in an ischemic area of canine myocardium, as a result of 40 min local occlusion, has been studied.     

Effects of catalase,peroxidase, superoxide dismutase and 10 scavengers of oxygen radicals in carrageenin edema and in adjuvant arthritis of rats

Summary Catalase, peroxidase and superoxide dismutase were found to inhibit significantly carrageenin edema and the primary phase of adjuvant arthritis in rats after i.v. injection. Heat-inactivated enzymes were as effective as the native enzymes. None of 10 scavengers of oxygen radicals inhibited the adjuvant arthritis at any time. Accordingly, no evidence for a participation of oxygen radicals in the secondary arthritis phase could be found, whereas a role of oxygen radicals in the primary arthritis phase and in carrageenin edema cannot be ruled out.     

Thiyl radicals in biosystems: effects on lipid structures and metabolisms

Thiyl radicals are intermediates of enzyme- and radical-driven biochemical processes, and their potential as reactive species in the biological environment has been somehow underestimated. From organic chemistry, however, it is known that thiyl radicals isomerize the double bonds of unsaturated fatty acids to a mixture with very dominating trans isomers. Recently, this reaction has been particularly studied for biosystems, focusing on the effect of thiyl radicals on the natural all-cis double bonds of unsaturated phospholipids, which undergo a conversion to the unnatural trans form. In this paper we report briefly the role of thiyl radicals in biosystems, describe the main features of the radical-induced cis-trans isomerization process under both in vitro and in vivo conditions, and reflect on some consequences for membrane structures, lipid metabolism and enzymatic reactions.Received 29 October 2004; received after revision 3 December 2004; accepted 4 January 2005     

A new cellular function for peroxisomes related to oxygen free radicals?

Although in cell biology peroxisomes are still 'young' organelles, it is becoming increasingly clear that they are involved in important cellular functions. Recent results have indicated the presence of the metalloenzyme superoxide dismutase in peroxisomes and the production of superoxide free radicals (O2-) in these oxidative organelles. These findings, together with other experimental evidence, point towards the existence of new roles for peroxisomes in cellular active oxygen metabolism, something that has a potential impact in multiple areas of cell biology, particularly in biochemistry and biomedicine.     

Nitric oxide: a radical molecule in quest of free radicals in proteins

A number of enzymes use an amino acid free radical cofactor. Tyrosyl and tryptophanyl radicals react with nitric oxide (NO) with an almost diffusion-limited rate. The catalytically competent tyrosyl radical in ribonucleotide reductase (RR) and prostaglandin H synthase (PGHS) recombines with NO in a radical-radical reaction. The unstable adduct formed can dissociate to regenerate the tyrosyl radical. However, upon prolonged incubation with NO, the diiron center of mouse RR leaks out, while the adduct is sucessively oxidized into an iminoxyl radical and a nitrotyrosine in PGHS. These data provide a plausible mechanism for the physiological inactivation of RR observed in various models, and may help in understanding the inhibition of PGHS reported in some cases. Reversible combination with NO is an intrinsic property of tyrosyl radicals, which also occurs with Y_D ^• and Y_Z ^• in photosystem II, where NO has been useful in the analysis of the oxygen-evolving complex.     

Stabile freie Radikale in Huminsäuren

Summary The electron spin resonance was measured with several humic acids. The preparations used here contain stable free radicals. Grey humic acids contain more radicals than brown humic acids.

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Herrn Dr.Seifert und Fräulein Dr.Ebert (Deutsche Akademie der Wissenschaften Berlin-Adlershof, Arbeitsgruppe: Physikalische Methoden der Analytischen Chemie, Labor für Hochfrequenz-Spektroskopie; Dir.: Prof. Dr.Kriegsmann) danken wir für die Unterstützung bei der Durchführung der Messungen.     

Inhibition of nitrosamine formation by ascorbic acid: participation of free radicals in its anaerobic reaction with nitrite

Summary The participation of semiquinone free radicals during the reaction of ascorbic acid with acidified sodium nitrite has been demonstrated by ESR spectroscopy unambiguously for the first time. Scavenging of the nitrosating agent, reflected by the observed free radical concentration, unexpectedly occurs with scarcely varying efficiency over the pH range 0.1–4.5.     

Hydroxyl radicals are not involved in NADPH dependent microsomal lipid peroxidation

Summary NADPH dependent H₂O₂ formation in microsomes in the presence of chelated iron leads to formation of hydroxyl radicals. Enhancement of hydroxyl radical generation (via ferric-EDTA or sodium azide) did not result in a concomitant increase in lipid peroxidation; rather, a decrease was observed. Moreover, the hydroxyl radical scavenger DMSO did not inhibit lipid peroxidation. This comparison of hydroxyl radical formation with lipid peroxidation suggests that hydroxyl radicals do not play a part in NADPH-dependent lipid peroxidation.     

The origin,diversity, and structure function relationships of insect luciferases

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes. Received 4 March 2002; received after revision 13 May 2002; accepted 21 May 2002     

Hydroxyl radicals are not involved in NADPH dependent microsomal lipid peroxidation

NADPH dependent H2O2 formation in microsomes in the presence of chelated iron leads to formation of hydroxyl radicals. Enhancement of hydroxyl radical generation (via ferric-EDTA or sodium azide) did not result in a concomitant increase in lipid peroxidation; rather, a decrease was observed. Moreover, the hydroxyl radical scavenger DMSO did not inhibit lipid peroxidation. This comparison of hydroxyl radical formation with lipid peroxidation suggests that hydroxyl radicals do not play a part in NADPH-dependent lipid peroxidation.     

Molecular enigma of multicolor bioluminescence of firefly luciferase

Firefly luciferase-catalyzed reaction proceeds via the initial formation of an enzyme-bound luciferyl adenylate intermediate. The chemical origin of the color modulation in firefly bioluminescence has not been understood until recently. The presence of the same luciferin molecule, in combination with various mutated forms of luciferase, can emit light at slightly different wavelengths, ranging from red to yellow to green. A historical perspective of development in understanding of color emission mechanism is presented. To explain the variation in the color of the bioluminescence, different factors have been discussed and five hypotheses proposed for firefly bioluminescence color. On the basis of recent results, light-color modulation mechanism of firefly luciferase propose that the light emitter is the excited singlet state of OL⁻ [¹(OL⁻)*], and light emission from ¹(OL⁻)* is modulated by the polarity of the active-site environment at the phenol/phenolate terminal of the benzothiazole fragment in oxyluciferin.     

Free radicals produced in a nitrosofluorene-unsaturated lipid reaction

Summary We report here the first demonstration that the carcinogen 2-nitrosofluorene reacts directly with lipid molecules containing carbon-carbon double bonds to yield free radicals which appear to be the nitroxyl free radical of the carcinogen covalently bound to the lipid.This investigation was supported in part by the U.S. Public Health Service Grant 5 RO1 CA18591-01 from the National Institutes of Health.     

Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin

Depending on the availability of suitable reducing agents, alloxan can be either a prooxidant or an antioxidant. Alloxan and its reduced derivative, dialuric acid, act as a redox couple, driven by reduced glutathione (GSH) or L-cysteine, generating in vitro in the presence of oxygen, both superoxide radical and hydrogen peroxide. The production of superoxide radicals was shown by the appearance of lucigenin chemiluminescence (CL) as well as by the generation of formazan from nitroblue tetrazolium (NBT). The lucigenin CL as well as the NBT reduction was inhibited by superoxide dismutase and partially by catalase. Melatonin inhibited alloxan-mediated CL. In contrast, in the absence of reducing agents, alloxan is a scavenger of superoxide radicals formed by other reactions. Because of the high content of reducing compounds in the cell (e.g. glutathione), it is suggested that alloxan acts in vivo mainly as a generator of reactive oxygen species. Received 9 November 1998; received after revision 15 January 1999; accepted 15 January 1999     

Gonyauline: A novel endogenous substance shortening the period of the circadian clock of a unicellular alga

Summary The circadian clock in the unicellular algaGonyaulax polyedra is accelerated by a substance in extracts from the cells themselves. The extracts have been fractionated using the circadian rhythm of bioluminescence as bioassay. The active substance, termed gonyauline, has been isolated and characterized as a novel low molecular weight cyclopropanecarboxylic acid (S-methyl-cis-2-(methylthio) cyclopropanecarboxylic acid). Synthetic gonyauline has a similar shortening effect on the period of the circadian clock.     

Effect of antioxidant substances on the level of free organic radicals naturally present in the rat diaphragm]

Using the electron spin resonance method, the effect of antioxydants on the number of natural organic free radicals is studied in the case of the diaphragm of the Rat. The effect observed shows an important decrease in the free radical concentration, which may, in the case of ascorbic acid and BHT, lead to their complete disappearance. These results are discussed in the light of the hypothesis according to which antioxydants play a role in the lengthening of the life span because of their fundamental character of inhibitors of free radicals.     

N-Monomethyl-arginine and nicotinamide prevent streptozotocin-induced double strand DNA break formation in pancreatic rat islets

The impact of short term in vitro exposure to the diabetogenic drug streptozotocin on pancreatic islet glucose metabolism, insulin secretion, DNA fragmentation and cell viability, was studied. Streptozotocin impaired cell viability as well as insulin secretion and the oxidation of glucose. These effects were partially counteracted by inhibition of the inducible form of nitric oxide synthase with N-monomethyl-arginine and by scavenging oxygen free radicals with nicotinamide. Isolated islets underwent double strand DNA fragmentation after 24 h in culture. The degree of DNA breakdown was strongly enhanced by exposure of the islet DNA was obtained with N-monomethyl-arginine and nicotinamide. These data suggest that the generation of reactive oxygen and nitrogen species is involved in the deleterious action of streptozotocin on pancreatic islet tissue. A role for oxygen radicals generated during streptozotocin-induced islet cell damage as possible mediators of the expression of the inducible form of nitric oxide synthase and the scavenging action of nicotinamide on these radicals, is then proposed.     

Biochemical events associated with rapid cellular damage during the oxygen-and calcium-paradoxes of the mammalian heart

Summary The O_2– and Ca²⁺-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H⁺ and e^– and also oxygen radicals or recox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca²⁺ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised condition, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca²⁺ or by raising [Ca²⁺]_i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca²⁺-paradox by the membrane perturbation associated with removal of extracellular Ca²⁺; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca²⁺]_i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca²⁺-paradox which continues under anoxia. Massive entry of Ca²⁺ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

A new simple temperature-controlled membrane oxygenator for the perfusion of isolated rat livers

Summary A new temperature-controlled membrane oxygenator for perfusing isolated rat livers was assembled using a combination of heat-exchangeable rubber tubing and silicon rubber tubing. The apparatus supplied enough oxygen to satisfy the requirements of hemoglobin-free perfused livers.Acknowledgment. One of the authors (MO) would like to thank to Prof. Dr R. Scholz, Institut für Physiologische Chemie der Universität München, for his kind permission to observe his perfusion experiments and also for his valuable discussion.     

Biochemical events associated with rapid cellular damage during the oxygen- and calcium-paradoxes of the mammalian heart

The O2- and Ca2(+)-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H+ and e- and also oxygen radicals or redox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca2+ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised conditions, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca2+ or by raising [Ca2+]i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca2(+)-paradox by the membrane perturbation associated with removal of extracellular Ca2+; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca2+]i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca2(+)-paradox which continues under anoxia. Massive entry of Ca2+ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.