Recombinant iron-containing superoxide dismutase (Fe-SOD) from Plasmodium falciparum was produced in a SOD-deficient strain of Escherichia coli, purified and characterised. The enzyme is a dimer, which contains 1.7 Fe equivalents and is sensitive to hydrogen peroxide (H(2)O(2)). Electron paramagnetic resonance (EPR) analysis showed two different signals, reflecting the presence of two different types of high-spin Fe sites with different symmetries. The role of the W71 residue during inactivation by H(2)O(2) of the P. falciparum Fe-SOD was studied by site-directed mutagenesis. First, the W71V mutation led to a change in the relative proportion of the two Fe-based EPR signals. Second, the mutant protein was almost as active as the wild-type (WT) protein but more sensitive to heat inactivation. Third, resistance to H(2)O(2) was only slightly increased indicating that W71 was marginally responsible for the sensitivity of Fe-SOD to H(2)O(2). A molecular model of the subunit was designed to assist in interpretation of the results. The fact that the parasite SOD does not belong to classes of SOD present in humans may provide a novel approach for the design of antimalarial drugs.

The role of endogenous and internalized catalase in the protection of Plasmodium against oxidant stress was studied. Catalase activities were measured in isolated Plasmodium falciparum at different stages of intererythrocytic development. Activities measured at late schizont stages were compared to parasite markers (glutamate dehydrogenase, SOD) and to red blood cell markers (haemoglobin, Cu/Zn‐SOD). The fate of the host cell catalase in the parasite digestive system was studied by immunoelectron microscopy using monoclonal antibodies. The internalized catalase appeared to be dissociated in the digestive system of the parasite and inactivated. To examine the protective role of the endogenous and internalized catalase in the parasite protection against oxidant stress, parasites were cultivated at two oxygen concentrations (5% and 20%) in inhibited catalase red blood cells. These experiments suggested that the catalases present both in red blood cell and parasite are not essential when parasites are cultivated under 5% oxygen, but are necessary to protect the parasite under 20% oxygen. Catalase may not be the main protective enzyme involved in the protection of P. falciparum in standard in vitro culture conditions, but may become critical under the higher oxygen tensions conditions encountered in vivo .

Catalase and glutathione peroxidase (Gpx), two enzymes destroying hydrogen peroxide, were reported in two Babesia species: B. divergens cultivated in vitro and B. hylomysci obtained in vivo. On the use of specific substrate and inhibitor, we confirmed that the Gpx activity detected was selenium-dependent. Moreover, the two Babesia species contain glutamate dehydrogenase activity. This enzyme is capable of providing to the cell the reduced nicotinamide adenine dinucleotide phosphate (NADPH) necessary for regeneration of the reduced glutathione. Gpx activity is weaker in B. divergens than in B. hylomysci and seems to be compensated by higher levels of catalase activity. Such a balance between the two enzymes may depend on the selenium concentration available for the parasite.

Two main superoxide dismutase activities at isoelectric points (pI) 6.2 and 6.8 and two minor at pI 5.6 and 6.4 were found in crude extracts of Plasmodium falciparum. These activities were cyanide-resistant and hydrogen peroxide-sensitive and represented 20-30% of the total SOD activity found in the crude extract. A fragment of 424 bp, amplified from genomic DNA from P. falciparum, was cloned and sequenced. The deduced amino acid sequence identified this fragment as a coding region of an SOD gene. A cDNA corresponding to SOD was then isolated from a P. falciparum cDNA library and sequenced. The deduced amino acid sequence of SOD (197 aa) was compared with 32 known Feor Mn-SODs by the 'DARWIN' system. This analysis showed that the parasitic enzyme was related to typical Fe-SODs. The SOD subunit was purified and the N-terminal sequence, determined up to 29 residues, corresponded to that of cDNA isolated. The iron-dependent SOD activity found in Plasmodium falciparum represents the first level of the antioxidant defence system of the parasite. It is also the first SOD characterized in the parasitic Apicomplexa phylum whose sequence can be compared to equivalent iron-dependent enzymes known in other protozoa and bacteria.

Glutathione peroxidase (GPx), a key enzyme involved in the detoxification of many peroxides, has been investigated in two malaria parasite species: P. yoelii in vivo (murine malaria) and P. falciparum in vitro (human malaria). We demonstrate the presence of an endogenous GPx activity in these two Plasmodia species. Enzymatic assays and the use of specific substrates and inhibitors allowed us to determine that the activity is selenium dependent. As this activity was shown to be lower in P. falciparum than in P. yoelii, and selenium levels were found to be low in culture medium and culture red blood cells, we hypothesized that a severe selenium deficiency could be responsible for this difference. After selenium supplementation, with either sodium selenite or selenocystine, we observed an increase in growth of P. falciparum only in with sodium selenite, whereas higher GPx activities were noted in parasites grown in media supplemented with both. An increase in GPx activities was also observed in parasites that had undergone an experimental oxidative stress with TBOOH. As the erythrocyte is unable to synthesize new proteins, these results provide further evidence for the existence of an endogenous parasitic selenium-dependent glutathione peroxidase.

In order to study the effects of acclimatization of Plasmodium in beta-thalassaemic mice, we used a mouse model of beta-thalassaemia (DBA/2J/beta-thal/beta-thal), similar to that observed in humans. We acclimatized 3 rodent malarias (P. berghei, P. chabaudi and P. yoelii) in DBA/2J and DBA/2J/beta-thal mice lines, by 4 intraperitoneal serial transfers. All 3 rodent malarias developed in red blood cells of beta-thalassaemic mice without losing their virulence. There was no delay in infection and peaks of parasitaemia were similar in beta-thalassaemic and normal mice. The mortality occurred earlier in beta-thalassaemic mice than in control mice for P. berghei and P. chabaudi. The difference was more pronounced for P. yoelii NS where normal mice did not die. These results could be explained by a failure of erythropoiesis in beta-thalassaemic mice, which are unable to compensate for the destruction of red blood cells by the parasites, and the mice died of anaemia. Ultrastructural examination of the rodent malaria parasites in beta-thalassaemic RBC showed a normal development even in the presence of Heinz bodies. In conclusion, no effective protection against malaria was provided by the beta-thalassaemia in this mouse model.

In this paper we report the isolation and the characterization of a gene encoding the antioxidant enzyme glutathione peroxidase from the human malaria parasite Plasmodium falciparum. This gene contains two introns of 208 and 168 bp and is present in a single copy on chromosome 13. The open reading frame encodes a protein with a predicted length of 205 amino acids, which possesses a potential cleavage site between residues 21 and 22 after a hydrophobic region with the characteristics of a signal sequence. Therefore, the mature protein is predicted to be 184 residues long with a molecular mass of 21404 Da. In comparison with other known glutathione peroxidases many amino acid residues implicated in catalysis are conserved in the malarial enzyme. Phylogenetic analysis indicates that the deduced protein sequence is more closely related to plant glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase. A 1.5-kb transcript was identified in asynchronous erythrocytic stages.

Journal of Eukaryotic MicrobiologyVolume 43, Issue 5 p. 26S-26S Manganese-cofactored Superoxide Dismutase Activity in Rat-derived Pneumocystis carinii. CECILE-MARE DENIS, CECILE-MARE DENIS U42 INSERM, BP 39, 59651 Villeneuve d'Ascq F.L.S., Univ. Catholiquc de Lille, FranceSearch for more papers by this authorCARMEN ÖDBERG-FERRAGUT, CARMEN ÖDBERG-FERRAGUT U42 INSERM, BP 39, 59651 Villeneuve d'AscqSearch for more papers by this authorKARINE GUYOT, KARINE GUYOT U42 INSERM, BP 39, 59651 Villeneuve d'AscqSearch for more papers by this authorEDUARDO DEI-CAS, EDUARDO DEI-CAS U42 INSERM, BP 39, 59651 Villeneuve d'Ascq F.L.S., Univ. Catholiquc de Lille, FranceSearch for more papers by this authorDANIEL CAMUS, DANIEL CAMUS U42 INSERM, BP 39, 59651 Villeneuve d'Ascq C.H.U.R., Faculté de Médecine de Lille, France.Search for more papers by this authorDANIEL DIVE, DANIEL DIVE U42 INSERM, BP 39, 59651 Villeneuve d'AscqSearch for more papers by this author CECILE-MARE DENIS, CECILE-MARE DENIS U42 INSERM, BP 39, 59651 Villeneuve d'Ascq F.L.S., Univ. Catholiquc de Lille, FranceSearch for more papers by this authorCARMEN ÖDBERG-FERRAGUT, CARMEN ÖDBERG-FERRAGUT U42 INSERM, BP 39, 59651 Villeneuve d'AscqSearch for more papers by this authorKARINE GUYOT, KARINE GUYOT U42 INSERM, BP 39, 59651 Villeneuve d'AscqSearch for more papers by this authorEDUARDO DEI-CAS, EDUARDO DEI-CAS U42 INSERM, BP 39, 59651 Villeneuve d'Ascq F.L.S., Univ. Catholiquc de Lille, FranceSearch for more papers by this authorDANIEL CAMUS, DANIEL CAMUS U42 INSERM, BP 39, 59651 Villeneuve d'Ascq C.H.U.R., Faculté de Médecine de Lille, France.Search for more papers by this authorDANIEL DIVE, DANIEL DIVE U42 INSERM, BP 39, 59651 Villeneuve d'AscqSearch for more papers by this author First published: September 1996 https://doi.org/10.1111/j.1550-7408.1996.tb04963.xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Aliouat EM, Dei-Cas E., Billaut P., Dujardin L., Camus D. Parasitol Res (1995) 81: 85. 2 Beauchamps C., Fridovitch I. Anal Biochem (1971) 44: 276. 3 Ben Abderrazak S., Guerrini F., Mathieu-Daudé F. et al. Protocols in molecular parasitology. Totowa : Humana Press Inc. (1993) Vol 21. 4 Fletcher LD, Berger LC, Peel SA, Baric RS, Tidwell RR, Dykstra CC. Gene. (1993) 129: 167. 5 Gille G., Sigler K. Folia Microbiol. (1995) 40(2): 131. 6 Maniatis T., Fritsch E., Sambrook J. Molecular cloning. (1989) 3th Ed. 7 Marklund S., Marklund G. Eur J Biochem (1974) 47: 469. 8 Raddin JA, Armstrong MYK, Ullu E., Richards FF. Infect Immun (1989) 57: 2149. 9 Sanger F., Nicklen S., Coulson AR. Proc Natl Acad Sci USA (1977) 74: 5463. Citing Literature Volume43, Issue5September 1996Pages 26S-26S ReferencesRelatedInformation

Babesia hylomysci was found to contain two superoxide dismutase (SOD) isoenzymes with isoelectric points (pI) of 4.9 and 5.2. The two isoenzymes (45 and 47 kDa) were composed of two subunits of 22 kDa. An unique amino terminal sequence was determined up to 34 residues from the pooled isoenzymes and was identified as a sequence of SOD. The comparison of this N-terminal sequence of B. hylomysci SOD with 29 known Fe- or Mn-SODs showed more homologies with Fe-SODs.

This paper presents the bioassay Colpidium campylum, a ciliate protozoan of freshwater. Easy to learn, cost-effective, sensitive, this test appears to be complementary to other aquatic tests such as the Daphnia or the Microtox tests. It is a miniaturized test usable as a substitution method for cellular tissues. Two study methods were developed: measurement of IC50 of products, and polynomial modelling of interactions between components, which allows estimation of the toxicity of an effluent in the receiving medium. This bioassay and the method of study of interactions are two tools which seem to have their place in risk assessment in aquatic medium.

Superoxide dismutase (SOD) was investigated in three species of rodent malaria (Plasmodium berghei, P. yoelii and P. vinckei). The isoelectric points (pI) of isozymes found in purified parasites were identical. SOD activities detected by isoelectrofocusing at pl 5.0, 5.6, and 6.4 were cyanide-sensitive and could be considered as having been adopted by the parasites from the host red blood cell. The three rodent malaria parasites also contained a cyanide-resistant, hydrogen peroxide-sensitive SOD activity not found in the host red blood cell. It is therefore concluded that the three rodent malaria parasites possess an endogenous SOD. Two bands of endogenous SOD were found at pl 6.2 and 6.8 for the three species, and one additional band was detected at pl 5.7 for P. berghei and P. vinckei. This first report in rodent Plasmodium of a cyanide-resistant, hydrogen peroxide-sensitive SOD suggests that these parasites may be capable of at least partly resisting activated oxygen species using an endogenous SOD.

Babesia hylomysci and B. divergens were studied for superoxide dismutase (SOD) activity by enzyme assay and isoelectric focusing (IEF). In the two Babesia species, parasite-associated SOD is cyanide-insensitive and inhibited by H2O2, indicating that iron is the cofactor metal. Measurements of SOD activity from purified parasites show that the SOD activity detected in Babesia is, for the main part, due to an endogenous enzyme.

Various chemicals were tested in Escherichia coli for the ability to modify the cellular growth rate and to induce the synthesis of heat shock and stress proteins. The toxicity of chemicals as observed by modification of the growth rate depended on concentration and duration of treatment, except for thiram. In this last case, no modification was observed up to a concentration of 10 micrograms.ml-1. In contrast, all toxicants tested enhanced the synthesis of heat shock and stress proteins. The stress response was similar but not identical. Heat shock proteins and stress proteins appear to be a more sensitive toxicity marker than growth inhibition. Suggestions for the use of stress proteins as a practical bioassay are made.

A copper-cadmium-nickel-zinc mixture was assessed in seven different river waters to study metal toxicity to the ciliate protozoan Colpidium campylum, the interactions occurring between metals, and the influence of the receiving water on toxicity. In the range of concentrations tested, which are representative of electroplating industry wastes, the main part of the toxicity can be attributed to copper and to cadmium-copper synergy. A classification of waters, based on a principal component analysis (PCA), was used to examine the main parameters of the water, which can affect the toxicity of metal mixtures. It appears that the mineralization of the water, more than the total organic carbon (TOC), is an important parameter for the expression of toxicity. A strategy for the estimation of ecotoxicological hazard assessment, based on a simplified factorial experiment is proposed. It enables one to study, in a two-step bioassay, the toxicity of an effluent, the influence of river water on its toxicity, and the effects of contact time and dilution. By applying PCA to data from very different waters, it may be possible to estimate the ecotoxicological risk associated with the discharge of an effluent, on the basis of the chemistry of the receiving water.

The toxicity of seven dithiocarbamates and interactions occurring with copper were studied with the ciliate protozoan Colpidium campylum. No product was toxic at 0.1 mg liter-1. Thiram and mancozeb are the most toxic products (lethal at 1 mg liter-1) and ziram and propineb the least toxic. Concerning interactions with copper, no or slight interactions were observed with propineb and mancozeb, although a strong synergistic toxic effect was observed in a previous work with two structurally related products, maneb and zineb. On the contrary, the responses observed with alkyl-related dithiocarbamates (ferbam and ziram) and ethylene bis-related dithiocarbamates (nabam, thiram, and metiram) are in correlation with the results known in the literature. The synergistic toxic effect of copper and dithiocarbamates thus seems to be related mainly to the alkyl- or the ethylene bis-dithiocarbamate structure, but is not constant for molecules associated with metals (propineb, mancozeb, maneb, and zineb).

Hazard assessment of hydroquinone has been evaluated from bibliographical and original data on the physicochemical properties, the environmental behavior, and the biological effects of this aromatic compound. Hydroquinone, which is produced in large amounts and widely used, must be considered as an environmental contaminant. However, it is not persistent. The ecotoxicity of this molecule, which must be linked to its physicochemical properties, varies from species to species. Its acute and chronic toxicity toward higher terrestrial organisms is moderate. Hydroquinone is estimated to be nonmutagenic by the Ames test but induces chromosome aberrations or karyotypic effects in eucaryotic cells. Carcinogenic and teratogenic potentials have been at present inadequately studied. The study underlines the complementarity of QSAR models and experimental approaches when an attempt is made to obtain ecotoxicological profiles of pollutants.

Binary mixtures of atrazine (herbicide) and copper were studied in three microorganisms: a bacterium, a protozoan and a microalga. Factorial experiments were carried out to detect interactive effects and toxicity results were interpreted with multiple regression analysis. The levels of concentrations tested on algae were close to those found in aquatic environments.

Interactions between nickel, cadmium, copper, chromium and zinc, and the influence of calcium concentration were studied by factorial experiments and the multiple regression method, using the growth rate of the ciliate protozoan Colpidium campylum as the biological response. The mixtures were representative of electroplating industry wastes. The effects of nickel are additive with those of copper and trivalent chromium. Effects of copper and cadmium are strongly synergistic, but this synergism disappears in the presence of trivalent chromium. A slight synergism was found between nickel and hexavalent chromium. A strong antagonism was observed between nickel and cadmium. The results suggest that chemical analysis of the effluents gives an unrealistic evaluation of the risk of such wastes, which is dependent upon multiple interactions occurring between components. As the residence times of these metallic components in water are different, interactions will be modified after discharge. The influence of calcium, in the range of concentrations tested, is not very significant.

A bioassay method using the ciliate protozoan is presented in a standardized form. The influence of the initial cell concentration on the potassium dichromate EC50 values was determined. Two intercalibration experiments between two laboratories were performed on ten toxicants in two different conditions. The potassium dichromate EC50 determinations performed by eight different people are also presented. All results are discussed in terms of feasibility and reproducibility of the method, fields of application, and limitations.