The interaction of superoxide radicals with active dicarbonyl compounds

The interaction of superoxide radical anion (O₂ ^??) with active dicarbonyls (methylglyoxal, glyoxal, and malonic dialdehyde) was studied. It was demonstrated that glyoxal and methylglyoxal inhibited superoxide-dependent accumulation of formazan; however, malonic dialdehyde stimulated this process. The formation of O₂ ^?? in these experiments occurred during the decomposition of the SOTS-1 azo initiator. On the other hand, all of the studied dicarbonyls in this system of O₂ ^?? generation competed for superoxide with the TIR ON spin trap. These compounds also inhibited luminal-dependent chemiluminescence during the AIBN azo initiator-induced peroxidation of liposomes from the egg phosphatidylcholine. A mechanism for the antiradical and antioxidant effects of the studied dicarbonyls, assuming the production of free radical intermediates in their reactions with O₂ ^?? or its protonated form, is proposed.     

Formation of the N,N′-Dialkylpyrazine Cation Radical from Glyoxal Dialkylimine Produced on Reaction of a Sugar with an Amine or Amino Acid

The formation of an intermediate product, which could easily give a radical product, in an early stage of the Maillard reaction was confirmed commonly occur in various sugar-amino compound systems, by detection of the N,N′-dialkylpyrazine cation radical generated on the addition of ascorbic acid (AsA) to the reaction mixtures. This intermediate was produced immediately after to glycosylamine formation, prior to Amadori rearrangement, and completely parallel to the formation of glyoxal dialkylimine, which was identified by TLC as a main component of the extract. Authentic glyoxal dialkylimine was shown to produce an identical radical on treatment with both reducing agents and acids instead of AsA. It was thus demonstrated that the intermediate is glyoxaldialkylimine and that acid hydrolysis followed by reduction is required for production of the free radical.     

Radical acetoacetate oxidation by myeloperoxidase, lactoperoxidase, prostaglandin synthetase, and prostacyclin synthetase: implications for atherosclerosis

When the myeloperoxidase-catalyzed peroxidation of acetoacetate proceeds in the presence of piperidinooxy free radical, methyl glyoxal is formed, and the nitroxide group is reduced to the secondary amine. A mechanism is advanced wherein an alpha-carbon-centered acetoacetate radical, generated by the peroxidase, forms an unstable adduct with the nitroxide group, subsequently decomposing to the observed products. Formation of methyl glyoxal, detected as its bis-2,4-dinitrophenylhydrazone by radial thin-layer chromatography, represents a method of determining free radical acetoacetate peroxidation by other peroxidases. It is shown that lactoperoxidase, prostaglandin synthetase, and prostacyclin synthetase generate methyl glyoxal with requirements identical to those of myeloperoxidase. With prostaglandin synthetase, arachidonic acid could replace the supporting peroxide. Substantiation that the catalyst for the reaction in aortic microsomes was prostacyclin synthetase was obtained by showing that 15-hydroperoxyarachidonic acid strongly inhibited the activity (5). The finding that these peroxidases catalyze radical acetoacetate oxidation could have broad implications for cellular damage via lipid peroxidation (7). Specifically, radical oxidation of acetoacetate by prostacyclin synthetase is proposed to be a link between cardiovascular risk factors and the initiation of atherosclerosis.     

Two-step oxidation of glycerol to glyceric acid catalyzed by the Phanerochaete chrysosporium glyoxal oxidase

Glyoxal oxidase of P. chrysosporium is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H(2)O(2). In addition to known substrates, glycerol is also found to be a substrate for glyoxal oxidase. During enzyme turnover, glyoxal oxidase undergoes a reversible inactivation, probably caused by loss of the active site free radical, resulting in short-lasting enzyme activities and undetectable substrate conversions. Enzyme activity could be extended by including two additional enzymes, horseradish peroxidase and catalase, in addition to a redox chemical activator, such as Mn(III) (or Mn(II)+H(2)O(2)) or hexachloroiridate. Using this three-enzyme system glycerol was converted in glyceric acid in a two-step reaction, with glyceraldehyde as intermediate. A possible operation mechanism is proposed in which the three enzymes would work coordinately allowing to maintain a sustained glyoxal oxidase activity. In the course of its catalytic cycle, glyoxal oxidase alternates between two functional and interconvertible reduced and oxidized forms resulting from a two-electron transfer process. However, glyoxal oxidase can also undergo an one-electron reduction to a catalytically inactive form lacking the active site free radical. Horseradish peroxidase could use glyoxal oxidase-generated H(2)O(2) to oxidize Mn(II) to Mn(III) which, in turn, would reoxidize and reactivate the inactive form of glyoxal oxidase. Catalase would remove the excess of H(2)O(2) generated during the reaction. In spite of the improvement achieved using the three-enzyme system, glyoxal oxidase inactivation still occurred, which resulted in low substrate conversions. Possible causes of inactivation, including end-product inhibition, are discussed.     

Deprotecton Studies of Ethanoic Acid 5,6,7,9-Tetrahydro-3-/2-(Acetoxyemxy Iethyl-5-0-Acetyl-9-OXO-3H-IMIDAZO/1,2a/ Purine-6,7-Dioyol Ester (1) and Related Glyoxal-Guaniee Adduts

Abstract

Selective deprotection of fully protected 1 was achieved via sodium methoxide or cyanide catalyzed removal of acetate groups. The glyoxal protecting group diminished nucleophylisity of N7 regardless of the blocking of N1 position in guanine.     

Stimulation and inhibition of photosystem II electron transport in cyanobacteria by ions interacting with the cytoplasmic face of thylakoids

The mechanism by which suspension medium ions regulate the rate of photoinduced electron transport across photosystem II was investigated with ion permeabilized cells of the cyanobacterium Anacystis nidulans. Electron transport was measured as the reduction of the electroneutral acceptor dichlorophenol indophenol, whose surface concentration is independent of electrostatic membrane potential. Potassium salts stimulate photoinduced electron transport at low concentrations and inhibit it at higher concentrations. No inhibition is observed when an antichaotropic anion is associated with potassium, while the inhibition is more severe the stronger the chaotropic character of the anion. Neutralization of the surface charge by potassium ions ligated to negatively charged membrane sites at the cytoplasmic side is a prerequisite for the expression of the chaotropic inhibition of photosystem II electron transport.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DPC 1,5-diphenyl carbazide - FeCN ferricyanide anion - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - PS photosystem - TEC³⁺ tris ethylene diamine cobalt cation     

Deactivation ofCandida rugosa lipase

Summary Deactivation ofCandida rugosa lipase was found to be complex. Hydrophobic interaction induced by iso-octane influenced the initial phase of deactivation, and increased the turn-over rate of the intermediate in the transition phase. After urea-treatment the structure of the last phase was not further influenced by thermal treatment, whereas that of initial phase was more sensitive to temperature change. Charge interaction was important in maintaining the structure during the deactivation, and especially anion charge might be a major factor.     

Sulfate anion free radical formation by the peroxidation of (Bi)sulfite and its reaction with hydroxyl radical scavengers

The one-electron oxidation of (bi)sulfite is catalyzed by peroxidases to yield the sulfur trioxide radical anion (SO3-), a predominantly sulfur-centered radical as shown by studies with 33S-labeled (bi)sulfite. This radical reacts with molecular oxygen to form a peroxyl radical. The subsequent reaction of this peroxyl radical with (bi)sulfite has been proposed to form the sulfate anion radical, which is nearly as strong an oxidant as the hydroxyl radical. We used the spin trapping electron spin resonance technique to provide for the first time direct evidence for sulfate anion radical formation during (bi)sulfite peroxidation. The sulfate anion radical is known to react with many compounds more commonly thought of as hydroxyl radical scavengers such as formate and ethanol. Free radicals derived from these scavengers are trapped in systems where (bi)sulfite peroxidation has been inhibited by these scavengers.     

A H<Subscript>2</Subscript>O<Subscript>2</Subscript>-producing glyoxal oxidase is required for filamentous growth and pathogenicity in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H₂O₂. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P. J. PuntThe first two authors contributed equally to this workWe dedicate this work to the memory of Jeff Schell, a charismatic and outstanding person who loved science and respected people     

Mechanism(s) involved in meat mutagen formation and inhibition.

The Maillard reaction, which involves Amadori rearrangement as a key step, also results in sugar fragmentation and free radical formation. The imidazoquinoline meat mutagens (2-amino-3-methylimidazo[4,5-f]-quinoline, or IQ, and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, or MeIQ) are formed from a reaction mixture containing alkylpyridine free radicals and creatinine. The imidazoquinoxaline meat mutagens (2-amino-3,4-dimethylimidazo[4,5-f]-quinoxaline, or MeIQx, and 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline, or 4,8-DiMeIQx) may be produced by reacting a mixture containing dialkylpyrazine free radicals and creatinine. Two different pathways for free radical formation are proposed. One involves bimolecular ring formation from the enaminol form of the glycoaldehyde alkylimine and is followed by oxidative formation of the free radical. The other pathway involves formation of N,N1-dialkylpyrazinium ions from glyoxal monoalkylimine followed by reduction to produce the free radicals. The respective intermediates (glycoaldehyde alkylimine and glyoxal monoalkylamine) are formed by reacting glycoaldehyde and glyoxal with amino compounds. The glycoaldehyde system reacts faster and produces more free radicals than the glyoxal system. The reactions help to explain the formation of imidazoquinoxaline meat mutagens and their predominance in fried fish and why these mutagens are present in larger quantities in fried ground beef than the imidazoquinoline-type meat mutagens. These two pathways may not be the only mechanisms involved in formation of meat mutagens, but other free radical reactions may also contribute to meat mutagenicity and are mentioned briefly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenicity of and Formation of Oxygen Radicals by Trioses and Glyoxal Derivatives

Dihydroxyacetone, glyceraldehyde, glyoxal, methyl glyoxal, and glyoxylic acid were found to show mutagenicity on Salmonella typhimurium TA 100. The mutagenicities of these substances were inhibited by the addition of S-9 or some free radical scavengers. The alkaline buffered solutions of these mutagenic substances were found to reduce Nitro Blue tetrazolium chloride. DNA was degraded by the addition of these mutagenic substances. It has also been confirmed that free radicals derived from autoxidation of these substances are responsible for their mutagenicity.     

Receptors for the Malarial Parasite

Abstract

During the erythrocytic cycle of Plasmodium, the parasite develops within an enclosed space, the parasitophorous vacuole, formed by endocytosis of an invasive stage, the merozoite. Among the erythrocyte membrane proteins possibly acting as a receptor for the attachment of P. falciparum merozoites to human erythrocytes is glycophorin A Isolated glycophorin inhibits merozoite entry in a competitive manner, perhaps via association with a 155 kDa surface protein. Another protein that competitively inhibits merozoite invasion, is band 3, the erythrocyte anion transport protein. The protein bearing Duffy blood group antigens may act to modulate invasion, but does not behave as a receptor.     

Production and chemiluminescent free radical reactions of glyoxal in lipid peroxidation of linoleic acid by the ligninolytic enzyme, manganese peroxidase.

Glyoxal is a key compound involved in glyoxal oxidase (GLOX)-dependent production of glyoxylate, oxalate and H2O2 by lignin-degrading basidiomycetes. In this paper, we report that glyoxal was produced from a metabolite of ligninolytic fungi, linoleic acid, by manganese peroxidase (MnP)-dependent lipid peroxidation. In the absence of the parent substrate of linoleic acid, the dialdehyde was oxidized by MnP and Mn(III) chelate to start free radical reactions with emission of chemiluminescence at 700-710 nm. The spectroscopic profile of the light emission is distinguishable from (a) singlet oxygen, (b) triplet carbonyls from dioxetane and alpha-hydroxyperoxyl radicals, and (c) biacyl triplet formed by the coupling of two acyl radicals. The photon emission of glyoxal by MnP was activated by co-oxidation of tartrate. The MnP-dependent oxidation of glyoxal in tartrate buffers continued for 10 days without addition of exogenous H2O2. The importance of these results is discussed in relation to the free radical chemistry of lignin biodegradation by wood rot fungi.     

Radical scavenging activities of alpha-alanine C60 adduct

Water-soluble alpha-alanine C60 adduct was synthesized, and its scavenging abilities for superoxide anion O2- and hydroxyl radical *OH were studied by the spectrophotometry and chemiluminescence. It was found that alpha-alanine C60 adduct showed an excellent efficiency in eliminating superoxide anion and hydroxyl radical. The 50% inhibiting concentration (IC50) for superoxide was 184 microg/mL by spectrophotometry and 292 microg/mL by chemiluminescence. The IC50 for hydroxyl radical was 42 microg/mL. In different test systems, the results showed that alpha-alanine C60 adduct had comparable radical scavenging abilities as thiourea and ascorbic acid, and was proved to be an effective scavenger for superoxide anion and hydroxyl radical. It can be prospected that water-soluble alpha-alanine C60 adduct will be useful in radical related biomedical fields.     

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.     

Generation of free radicals induced by nifurtimox in mammalian tissues

Nifurtimox is reduced by rat liver microsomes to a nitro anion-free radical as indicated by ESR spectroscopy. This subcellular fraction gives a steady state radical concentration which is proportional to the square root of the protein concentration, suggesting that the nifurtimox anion radical is a necessary intermediate in the reduction and that the radical decays through a nonenzymatic second order process. The steady state concentration of the anion radical in the microsomal system is not decreased by superoxide dismutase or catalase, thus indicating that neither the superoxide anion nor hydrogen peroxide is an intermediary in the generation of the anion radical. The steady state concentration of the anion radical in the microsomal system is also not altered in the presence of metyrapone or CO and is decreased in the presence of NADP+ and p-chloromercuribenzoate. This observation suggests that the formation of nifurtimox anion radical is mediated through NADPH-cytochrome P-450 (c) reductase and not by the cytochrome P-450 system. In accordance with this interpretation, a model system consisting of NADPH and FMN-reduced nifurtimox to the nitro anion-free radical. Nifurtimox anion radical generation is significantly stimulated by rat brain and testes homogenates. The enhanced free radical formation may be the basic cause of nifurtimox toxicity in mammals.     

Revisiting the interaction of the radical anion metabolite of nitrofurantoin with glutathione.

There have been several conflicting reports as to the scavenging nature of glutathione toward the nitro radical anion of the drug nitrofurantoin. We produced the radical anion enzymatically using the xanthine oxidase/hypoxanthine system at pH 7.4 and pH 9.0 in the presence of various levels of glutathione from 10 to 100 mM and monitored any changes in the radical concentration via electron spin resonance spectroscopy. Independent of glutathione concentration, there was no decrease in the steady-state concentration of the radical. In fact, there was an average 30% increase in the concentration of the radical anion, which suggests enhanced enzyme activity in the presence of glutathione (GSH). These results, together with observations of the effects of glutathione on the stability of the radical anion generated by radiolysis or dithionite, rule out any detectable reaction between the nitrofurantoin radical anion and GSH under physiologically relevant conditions.     

Solvent free triglyceride synthesis using lipozymeTM IM-20

Summary The synthesis of triglycerides using Lipozyme^TM IM-20 (Mucor miehei lipase immobilized on weak anion exchange resins by Novo) is described. Use of pure substrates in stoichiometric amounts in the absence of any organic solvent enables high conversion to be obtained with the addition of molecular sieves to remove water produced by the reaction.     

Hodgkin-Huxley analysis of a GCAC1 anion channel in the plasma membrane of guard cells

A quantitative analysis of the time- and voltage-dependent kinetics of the guard cell anion channel (GCAC1) current in guard cell protoplasts from Vicia faba was analyzed using the whole-cell patch clamp technique. The voltage-dependent steady-state activation of GCAC1 current followed a Boltzmann distribution. For the corresponding steady-state value of the activation variable a power of two was derived which yielded suitable fits of the time course of voltage-dependent current activation. The GCAC1 mediated chloride current could successfully be described in terms of the Hodgkin-Huxley equations commonly evoked for the Na channel in nerve. After step depolarizations from a potential in the range of the resting potential to potentials above the equilibrium potential for chloride an activation and also an inactivation could be described. The gating of both processes exhibited an inverse relationship on the polarity of the applied step potentials in the order of milliseconds. Deactivating tail currents decline exponentially. The presented analysis contributes to the understanding of the rising phase of the observed action potentials in guard cells of V. faba. Evidence is presented that the voltage-dependent kinetic properties of the GCAC1 current are different from those properties described for the excitable anion currents in the plasmalemma of Chara corallina (Beilby & Coster, 1979a).The authors gratefully acknowledge the encouragement of Dr. David Colquhoun to apply the Hodgkin-Huxley model to the GCAC1 channel. The work was in part supported by a grant of the Deutsche Forschungsgemeinschaft to R.H. and a grant of the Herman and Lilly Schilling Stiftung to H.-A.K.     

Repair effect of thymine radical anion by echinocoside using pulse radiolysis

Repair activities of thymine radical anion by echinocoside, isolated from Pedicularis plicata. were studied using pulse radiolysis technique. The thymine radical anion was produced by the reaction of hydrated electron with thymine. Echinocoside. one of the polyphenols of phenylpropanoid glycoside, was added to the thymine aqueous solution saturated with N2. Kinetic analysis by transient absorption spectrum showed that thymine radical anion was formed at first, and then after several decades of microseconds of pulse radiolysis. the spectrum of thymine radical anion was changed to that of echinocoside radical anion. The evidence indicated that thymine radical anion was repaired through one-electron-transfer between the DNA base radical anion and echinocoside. The rate constant of electron transfer by echinocoside was 1.45× 109 dm3 · mol1 · s 1.