Monique Vuillaume

Characterization of the Electrochemical Oxidation of Peroxynitrite: Relevance to Oxidative Stress Bursts Measured at the Single Cell Level

The electrochemical signature of peroxynitrite oxidation is reported for the first time, and its mechanism discussed in the light of data obtained by steady-state and transient voltammetry at microelectrodes. Peroxynitrite is an important biological species generated by aerobic cells presumably via the near diffusion-limited coupling of nitric oxide and superoxide ion. Its production by living cells has been previously suspected during cellular oxidative bursts as well as in several human pathologies (arthritis, inflammation, apoptosis, ageing, carcinogenesis, Alzheimer disease, AIDS, etc.). However, this could only be inferred on the basis of characteristic patient metabolites or through indirect detection, or by observation of follow-up species resulting supposedly from its chemical reactions in vivo. In this work, thanks to the independent knowledge of the electrochemical characteristics of ONO2- oxidation, the kinetics and intensity of this species released by single human fibroblasts could be established directly and quantitatively based on the application of the artificial synapse method. It was then observed and established that fibroblasts submitted to mechanical stresses produce oxidative bursts, which involve the release within less than a tenth of a second of a complex cocktail composed of several femtomoles of peroxynitrite, hydrogen peroxide, nitric oxide, and nitrite ions.

Analysis of individual biochemical events based on artificial synapses using ultramicroelectrodes: cellular oxidative burst.

Carbon fiber platinized ultramicroelectrodes placed within micrometres of a single living cell are used to monitor cellular events. This artificial synapse is used here to collect and examine the very nature of the massive oxidative bursts produced by human fibroblasts when their membrane is locally depolarized by a puncture made with a micrometre sized sealed pipette. The electrochemical analysis of the response indicates that oxidative bursts consist of a mixture of a few femtomoles of highly cytotoxic chemicals: hydrogen peroxide, nitrogen monoxide and peroxynitrite, together with nitrite ions, which may result from a partial spontaneous decomposition of peroxynitrite prior to its release by the cell.

Retroviral-mediated correction of DNA repair defect in xeroderma pigmentosum cells is associated with recovery of catalase activity

Xeroderma pigmentosum (XP) is a rare inherited disease associated with photosensitivity, a very high susceptibility to develop neoplasm on sun-exposed skin and neurological abnormalities for some patients. We previously reported that diploid cell lines established from XP skin biopsies present an abnormal low level of catalase activity, which is involved in the defense against oxygen free radicals. This biochemical dysfunction, probably involved in the skin cancer formation, has been difficult to be directly related to the nucleotide excision repair (NER) defect in XP. In this paper we report that the retroviral-mediated transduction of XP diploid cells by the XPC and XPD/ERCC2 cDNAs fully and stably corrects the NER defect in terms of survival and unscheduled DNA synthesis (UDS) after ultraviolet (UV) irradiation. The catalase activity in transduced cells was recovered up to normal levels only in cells transduced with repair genes correcting the repair defect. These results imply that: (i) the reduced catalase activity in XP, which might result from cellular depletion of its NADPH cofactor, is directly related to impaired DNA repair, and (ii) this depletion might be one of the multiple cellular consequences of XP inborn defect.

Phenylarsine oxide inhibits ex vivo HIV-1 expression.

Phenylarsine oxide (PAO), which is described as an inhibitor of tyrosine phosphatase activity, inhibits H2O2 release from human peripheral blood mononuclear cells (PBMCs) as measured by electrochemistry. Since human immunodeficiency virus type 1 (HIV-1) replication is known to be favored under oxidative stress conditions, ex vivo experiments using uninfected PBMCs, primary monocytes or a latently infected promonocytic U1 cell line show that HIV-1 replication and reactivation, monitored by p24 antigen measurement, are inhibited by PAO in a time- and concentration-dependent manner. These observations can be linked with the inhibition of NF-kappa B activation when uninfected monocytes are induced by either tumor necrosis factor alpha (TNF-alpha) phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS).

ATP production and catalatic-type activities in etiolated or green Euglena gracilis

1. 1. Whole Euglena cells, as well as mitochondrial and chloroplastic fractions (12,000–100,000 and 200,000 g supernatants), displayed a H2O2-mediated ATP production with ADP and PO43- as necessary substrates. 2. 2. The adenylate kinase activity could not solely account for such a reaction since AP5A did not affect it. 3. 3. Inhibitors of oxidative phosphorylation did not inhibit but sometimes enhanced the phenomenon. 4. 4. Irradiation of the fractions could produce an ATP increase which suggested the participation of superoxide ions. 5. 5. We conclude that the energy of H2O2, photoproduced or experimentally added, could be converted by a catalatic-type system, normally integrated into organelle membranes, in energy-rich ATP bonds.

Etude photophysique de la ptérobiline

The first results of a photophysical study on pterobilin (biliverdin IX γ, a Lepidopter blue bile pigment) are presented. From the absorption and fluorescence spectra, it is deduced that the low yield of fluorescence indicates a desactivation of the excited singlet state occurring mainly by a non-radiative process. Analyses of the chemical compounds formed after irradiation of pterobilin in methanol show that it is rearranged into a series of new blue pigments among which phorcabilin and sarpedobilin (the two neobiliverdins IX γ isolated from Lepidopters) have been identified.

A new property of catalase: the concerted synthesis of nucleotide triphosphates

Abstract We have observed a new property of catalase: the in vitro concerted synthesis of triphosphate nucleotides (XTP) from the corresponding diphosphate nucleotides (XDP), a reaction which is greatly enhanced in the presence of H 2 O 2 . Kinetic data and inhibition studies showed that the reaction is neither of the adenylate kinase type, nor of the nucleoside-diphosphate kinase type. Although the reaction depends on the presence of phosphate buffer, inorganic phosphate was not used in the phosphorylation reaction. ATP production did not occur with NADPH-depleted catalase, but was restored when the D-catalase was preincubated in the presence of NADPH and H 2 O 2 . These data suggest that the NADPH molecule may be the phosphate donor in the reaction. The possible physiological significance of the reaction is discussed.

Antioxidant activity of hemocyanin; a pulse radiolysis study

In order to determine the reactivity on hemocyanin from Androctonus australis, the reaction of superoxide anion has been investigated using pulse radiolysis. The kinetics of O2- decays have been studied in aqueous buffered media at various basic pH (8, 8.5 and 9), first in the absence and then in the presence of hemocyanin (in oxygenated solutions containing formate anion 0.16 mol.l-1). We have shown that, in the presence of hemocyanin, O2- decay is a first-order process whose apparent rate constant is proportional to protein concentration (10(-7) to 10(-6) mol.l-1) and pH independent between 8 to 9. A second-order rate constant of 3.5 +/- 0.1.10(7) mol-1.l.s-1, has been deduced for the catalytic rate constant of hemocyanin with O2-. Meanwhile, this activity is smaller than that described for free copper, eukaryotic Cu-Zn-SOD or some copper chelates. We have verified that apohemocyanin--the copper deprived protein--does not exhibit such an activity vs. SOD (superoxide dismutase).

Catalatic properties of hemocyanin in helping to account for the Scorpion's radioresistance

1. 1. Scorpions are exceedingly resistant to ionizing irradiations. 2. 2. Hemocyanin, the blue pigment of hemolymph, is undoubtedly an important factor in this resistance because of its copper content and oxyphoric properties, and of its catalatic activity demonstrated here. 3. 3. These characteristics, found in crude or purified hemocyanin and in its heavy dissociated products (i.e. dodecamers and hexamers) made it possible to show that hemocyanin neutralizes the effects of irradiation by disproportionation of the toxic H2O2 product. 4. 4. The finding that catalase is a component of this complex oxyphoric metalloprotein is an exciting discovery that warrants further study, since catalase is considered essential to cell protection against all types of irradiation.

Sarpedobilin: In vitro interactions with ATP and growth inhibiting properties

Abstract 1. 1. As the result of a systematic search for interactions between bile pigments and natural substances of biological significance, an interaction has been found between sarpedobilin 3 and ATP. 2. 2. This interaction has been demonstrated by different methods, thin-layer chromatography, extraction procedure, bioluminescence determinations (luciferin-luciferase reaction) and by the modification of the VIS-spectra. 3. 3. Sarpedobilin has shown inhibiting properties on watercress seed germination and on Artemia salina egg development. 4. 4. The results so far obtained are discussed in relation to the biochemical situation of this butterfly neopterobilin. The possibility for sarpedobilin to control metabolic processes in butterflies is considered as an hypothesis. Sarpedobilin could be included in feedback-like mechanisms in which pterobilin is implied, this pigment 1 being the support of the cyclizations leading to 2 and 3.