Activation/deactivation of acetylcholinesterase by H2O2: more evidence for oxidative stress in vitiligo

Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo.     

Regulation of tyrosinase by tetrahydropteridines and H2O2

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.     

Polyamines,indole-3-acetic acid and abscisic acid in rice phloem sap

Putrescine, spermidine, spermine and cadaverine have been identified and quantified in rice phloem sap and shoot extracts by HPLC. It is suggested that diamines, putrescine and cadaverine, easily migrate into the phloem, while movement of a triamine, spermidine, and a tetramine, spermine, tend to be restricted. Spermine especially seems to be the most immobile among polyamines. Thus it is indicated that movement of polyamines into phloem is decreased with increasing number of amino groups. Indole-3-acetic acid and abscisic acid in rice phloem sap were also analyzed by HPLC and it is suggested that indole-3-acetic acid is transported freely into phloem, while abscisic acid is much more actively exuded into phloem.     

Multiple reactions in vanadyl-V(IV) oxidation by H2O2

Oxidation of vanadyl sulfate by H₂O₂ involves multiple reactions at neutral pH conditions. The primary reaction was found to be oxidation of V(IV) to V(V) using 0.5 equivalent of H₂O₂, based on the loss of blue color and the visible spectrum. The loss of V(IV) and formation V(V) compounds were confirmed by ESR and⁵¹V-NMR spectra, respectively. In the presence of excess H₂O₂ (more than two equivalents), the V(V) was converted into diperoxovanadate, the major end-product of these reactions, identified by changes in absorbance in ultraviolet region and by the specific chemical shift in NMR spectrum. The stoichiometric studies on the H₂O₂ consumed in this reaction support the occurrence of reactions of two-electron oxidation followed by complexing two molecules of H₂O₂. Addition of a variety of compounds—Tris, ethanol, mannitol, benzoate, formate (hydroxyl radical quenching), histidine, imidazole (singlet oxygen quenching), and citrate—stimulated a secondary reaction of oxygen-consumption that also used V(IV) as the reducing source. This reaction requires concomitant oxidation of vanadyl by H₂O₂, favoured at low H₂O₂:V(IV) ratio. Another secondary reaction of oxygen release was found to occur during vanadyl oxidation by H₂O₂ in acidic medium in which the end-product was not diperoxovanadate but appears to be a mixture of VO ₃ ⁺ (–546 ppm), VO³⁺ (–531 ppm) and VO ₂ ⁺ (–512 ppm), as shown by the⁵¹V-NMR spectrum. This reaction also occurred in phosphate-buffered medium but only on second addition of vanadyl. The compounds that stimulated the oxygen-consumption reaction were found to inhibit the oxygen-release reaction. A combination of these reactions occur depending on the proportion of the reactants (vanadyl and H₂O₂), the pH of the medium and the presence of some compounds that affect the secondary reactions.     

Erythroid precursors cultured from adult mice are sensitive to H2O2 toxicity

Summary The growth of late erythroid precursors (CFU-Es) from adult bone marrow is inhibited when Iscove's modified Dulbecco's medium supplied in liquid form is used. Catalase and other H₂O₂ destroying compounds restore the capacity of culture medium to support colony development. However early precursors from adult bone marrow and fetal liver CFU-Es were resistant to H₂O₂. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and the Fédération Nationale des Centres de Lutte contre le Cancer.     

Mechanisms of cytotoxicity induced by horseradish peroxidase/indole-3-acetic acid gene therapy

We have previously proposed the horseradish peroxidase (HRP) and the non-toxic plant hormone indole-3-acetic acid (IAA) as a novel system for gene-directed enzyme/prodrug therapy (GDEPT). The cytotoxic potential of HRP/IAA GDEPT and the induction of a bystander effect were demonstrated in vitro under normoxic as well as hypoxic tumour conditions. To date, the chemical agents and the cellular targets involved in HRP/IAA-mediated toxicity have not been identified. In the present work, some of the molecular and morphological features of the cells treated with HRP/IAA gene therapy were analysed. Human T24 bladder carcinoma cells transiently transfected with the HRP cDNA and exposed to the prodrug IAA showed chromatin condensation, formation of apoptotic bodies, DNA fragmentation, and Annexin V binding. Similar effects were observed when the cells were incubated with the apoptotic agent cisplatin. Caspases appeared to be involved as effectors in HRP/IAA-mediated apoptosis, since treatment with a general caspase inhibitor decreased the fraction of cells with micronuclei (MN) by 30%, with fragmented DNA by 50%, and with condensed chromatin by 60%. However, very little degradation of one of the downstream targets of caspase-3, PARP, could be detected, and apoptosis alone did not appear to account for the killing levels measured with a clonogenic assay. The effect of HRP/IAA treatment on cell cycle progression was also investigated, and a rapid cytostatic effect, equally affecting all phases of the division cycle, was observed.     

H2O2 resistance is linked to the degree of fatty acid unsaturation in Streptomyces coelicolor A3(2)

In Streptomyces coelicolor A3(2), as the content of palmitoleic acid increased with decreasing growth temperature, H₂O₂ resistance decreased. Production of thiobarbituric acid-reactive substances upon H₂O₂ treatment was increased by supplementing unsaturated fatty acids. Therefore, the content of palmitoleic acid is a determining factor for the survival of Streptomyces coelicolor A3(2) subjected to H₂O₂ stress.     

Regulation of the activity of glyceraldehyde 3-phosphate dehydrogenase by glutathione and H2O2

The activity lost during storage of a solution of muscle glyceraldehyde 3-phosphate dehydrogenase was rapidly restored on adding a thiol compound, but not arsenite or azide. On treatment with H₂O₂, the enzyme was partially inactivated and complete loss of activity occurred in the presence of glutathione. Samples of the enzyme pretreated with glutathione followed by removal of the thiol compound by filtration on a Sephadex column showed both full activity and its complete loss on adding H₂O₂, in the absence of added glutathione. Most of the activity was restored when the H₂O₂-inactivated enzyme was incubated with glutathione (25mM) or dithiothreitol (5mM) whereas arsenite or azide were partly effective and ascorbate was ineffective. The need for incubation for a long time with a strong reducing agent for restoration of activity suggests that the oxidized group (disulfide or sulfenate) must be in a masked state in the H₂O₂-inactivated enzyme. Analysis by SDS-PAGE gave evidence for the formation of a small quantity of glutathione-reversible disulfide-form of the enzyme. Circular dichroic spectra indicated a decrease in -helical content in the inactivated form of the enzyme. The evidence suggest that glutathione and H₂O₂ can regulate the active state of this enzyme.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Suppressive effect of H2O2 on increase in activities of acid invertases in rye roots

Although various effects of H₂O₂ on plant cellular functions have been reported, the effects of H₂O₂ on glycolytic enzymes remain unknown. It was shown that H₂O₂ has a suppressive effect on increase in activities of soluble and insoluble acid invertases among a number of glycolytic enzymes during a growth stage of rye roots.     

Regulation of enzymic oxidation of indole-3-acetic acid by phenols: Structure-activity relationships

Mono- and diphenols were tested for their effects on the decarboxylation of [1-¹⁴C]IAA catalysed by purified horseradish peroxidase (EC 1.11.1.7) in the presence or absence of 2,4-dichlorophenol (DCP). The number of hydroxyl groups and their position relative to each other and the nature and position of other substituents on the aromatic ring were found to affect the activity. Although the effects were complex, the following generalizations may be made. (1) Monophenols produce activation when no other cofactor is present. p-Substituted monophenols are more active than o- or m-compounds. In the presence of DCP, the activity varies from slight activation to strong inhibition. (2) m-Diphenols also produce activation in the absence of other cofactors while o- and p-diphenols, with the exception of 3,4-dihydroxyacetophenone and 3,4-dihydroxypropiophenone, produce strong inhibition in the presence or absence of DCP. The o-diphenolsare degraded in the IAA-oxidizing enzyme system and thus produce only a temporary inhibition. (3) m-Diphenols and 3,4-dihydroxyacetophenone produce a sustained inhibition in the presence of DCP. (4) Substitution at position 2 significantly alters the activity of m-diphenols. (5) O-Methylation alters the activity of most o-diphenols. In the absence of DCP, o-methoxyphenols and certain other phenols such as 3,4-dihydroxyacetophenone and 2,6-dihydroxyacetophenone either promote or inhibit IAA oxidation depending on concentration.     

Effect of selenium in recovery of immunity damaged by H2O2 and60Co radiation

Con A stimulated lymphocytes proliferation was measured as [³H]thymidine incorporation and IgG was quantified by single radial immunodiffusion to study recovering or protecting effect of selenium (Se) on immunity attacked by exogenous active oxygen species, H₂O₂ and⁶⁰Co-radiation, respectively. Lipid peroxidation was also determined to observe the relation between antioxidation ability and protecting ability of Se. It was found that H₂O₂ injured lymphocytes immunocompetence deeply and⁶⁰Co-radiation decreased immune response capacity greatly, but that administration of Se counteracts this damage. The antioxidative ability of Se was correlated with its protecting ability.     

Acetic acid from H2 and CO2

Cell extracts of a nonsporeforming strictly anaerobic bacterium, Acetobacterium woodii produced acetate in N-tris(Hydroxymethyl)methyl-2-aminoethane sulfonic acid or phosphate buffers from hydrogen and carbon dioxide. The formation of acetate was not dependent on the presence of ATP in the reaction mixture; ADP also did not influence the acetate production. Since acetic acid is the main fermentation product during growth of A. woodii with H₂ and CO₂, ATP must be synthesized in the course of acetate formation. The possible sites of ATP synthesis are discussed.     

The biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Oxidative stress in vitiligo: photo-oxidation of pterins produces H(2)O(2) and pterin-6-carboxylic acid

Patients with vitiligo accumulate millimolar levels of H(2)O(2) in their epidermis. The recycling process of (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin in these patients is disrupted due to deactivation of 4a-OH-BH(4) dehydratase by H(2)O(2). The H(2)O(2) oxidation products 6- and 7-biopterin lead to the characteristic fluorescence of the affected skin upon Wood's light examination (UVA 351 nm). Here we report for the first time the presence and accumulation of pterin-6-carboxylic acid (P-6-COOH) in the epidermis of these patients. Exploring potential sources for P-6-COOH revealed that sepiapterin and 6-biopterin are readily photo-oxidised to P-6-COOH by UVA/UVB irradiation. Photolysis of sepiapterin and 6-biopterin produces stoichiometric H(2)O(2) under aerobic conditions, where O(2) is the electron acceptor, thus identifying an additional source for H(2)O(2) generation in vitiligo. A detailed analysis utilising UV/visible spectrophotometry, HPLC, TLC, and mass spectroscopy showed for sepiapterin direct oxidation to P-6-COOH, whereas 6-biopterin formed the intermediate 6-formylpterin (P-6-CHO) which is then further photo-oxidised to P-6-COOH.     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

The bound auxins: Protection of indole-3-acetic acid from peroxidase-catalyzed oxidation

Indole-3-acetic acid (IAA) was oxidized by horseradish peroxidase, but ester and amide conjugates of IAA were not degraded. Addition of indoleacetyl-myo-inositol, indoleacetyl-L-aspartate, indoleacetylglycine, indoleacetyl-L-alanine, indoleacetyl-D-alanine, or indoleacetyl--alanine did not affect the rate of oxidation of IAA by horseradish peroxidase. Peroxidase preparations from Pisum sativum L. and Zea mays L. behaved similarly in that they rapidly oxidized IAA, but not conjugates found in the plant from which the peroxidase was prepared. These results indicate that conjugation could affect the stability of IAA in vivo.Abbreviation IAA Indole-3-acetic acid     

Involvement of calcium in the in vitro sensitivity change of the plasma membrane H+ ATPase to indole-3-acetic acid

The proton pumping activity of phase-partitioning purified plasma membrane fraction from spinach leaves was tested in vitro in the presence of exogenous indole-3-acetic acid. The sensitivity of the H+ pumping activity to the auxin was changed after flowering induction. We investigated the effect of whole spinach leaf treatments with substances affecting the phosphatidylinositol diphosphate transduction pathway on the in vitro sensitivity modification by photoperiodic induction. A role of calcium ions was supported by studies on leaves treated with a specific Ca²⁺ chelator (EGTA), a synthetic Ca²⁺ ionophore (A23187) or with calcium channel blokers (verapamil, lanthan chloride). An experiment using the transduction pathway inhibitor, lithium chloride, indicated that the intracellular concentration of Ca²⁺ was increased by inositol triphosphate.     

H2O2-Induced Cell Death in Human Glioma Cells: Role of Lipid Peroxidation and PARP Activation

underlying mechanism of ROS-induced cell injury remains to be defined. This study was undertaken to examine the role of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in H₂O₂-induced cell death in A172 cells, a human glioma cell line. H₂O₂ induced a dose- and time-dependent cell death. The cell death was prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), H₂O₂ scavengers (catalase and pyruvate), and a hydroxyl radical scavenger (dimethylthiourea). Antioxidants N,N-diphenyl-p-phenylenediamine (DPPD) and Trolox had no effect on the H₂O₂-induced cell death. Lipid peroxidation did not increase in human glioma cells exposed to H₂O₂. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H₂O₂. The PARP activity was increased by H₂O₂ and the H₂O₂ effect was prevented by 3-aminobenzamide, dithiothreitol, and phenanthroline. The ATP depletion induced by H₂O₂ was prevented by catalase, dithiothreitol, phenanthroline, and 3-aminobenzamide, but not by DPPD. These results indicate that the H₂O₂-induced cell death is mediated by PARP activation but not by lipid peroxidation in human glioma cells.     

Rapid detection of cytotoxicity of food additives and contaminants by a novel cytotoxicity test, menadione-catalyzed H2O2 production assay

Menadione-catalyzed H₂O₂ production by viable animal cells was proportional to the viable cell number, and H₂O₂ production decreased with increasing cytotoxic effects after the incubation of cells with cytotoxic compounds. The cytotoxic effects of food additives, pesticides, antibiotics, heavy metals, phytotoxins, mycotoxins, and marine toxins were estimated using the above test employingNIH/3T3 and Neuro-2a cells. Synergistic effects of the toxin mixture were observed and acute cytotoxicity detected 1 h after the incubation of cells with toxins. This menadione-catalyzed H₂O₂production assay is rapid and simple compared to other popular cytotoxicity tests such as the MTT reduction assay and Neutral red inclusion test, requiring4 h. The menadione-catalyzed H₂O₂ production assay is expected to be a useful food safety test for rapidly detecting toxic compounds having a basic cytotoxic effect on common animal cells. This revised version was published online in August 2006 with corrections to the Cover Date.