Low influenza vaccination rate among patients with systemic sclerosis.

OBJECTIVE: To evaluate the influenza vaccination rate and factors influencing it in patients with SSc. METHODS: A total of 177 SSc patients fulfilling the ACR and/or LeRoy and Medsger criteria were evaluated during annual meetings of the French patient association in 2006 (n = 71) and 2007 (n = 70) or during hospitalization in the Internal Medicine Department of Cochin Hospital in 2007 (n = 36). Information on influenza vaccination was collected by a standardized form. RESULTS: Mean (s.d.) age and disease duration were 58.7 (12.6) and 10.5 (9.5) years, respectively. Overall, 69 (39%) patients received an influenza vaccination during the previous year. Among the 108 patients who were not vaccinated, 78 (72.2%) presented at least one indication for vaccination. The most frequent reasons for non-vaccination were absence of physician recommendation and fear of side effects. Patients who were and were not vaccinated did not differ in anxiety, depression, global disability or quality of life. Vaccination rate was significantly higher (59%) for patients who remembered receiving a letter from the French National Health Insurance Agency encouraging vaccination than among those who did not (26%, P = 0.0001). Multivariate analysis revealed the year of the last vaccination and age as two independent parameters associated with vaccination. CONCLUSIONS: Influenza vaccination coverage is low in SSc patients. Lack of information and fear of adverse effects are the most common reasons for non-vaccination. Efforts are needed to increase the influenza vaccination coverage in this population.

The crystal structures of oxidized forms of human peroxiredoxin 5 with an intramolecular disulfide bond confirm the proposed enzymatic mechanism for atypical 2-Cys peroxiredoxins.

Peroxiredoxin 5 (PRDX5) belongs to the PRDX superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides and peroxynitrite. PRDX5 is classified in the atypical 2-Cys subfamily of PRDXs. In this subfamily, the oxidized form of the enzyme is characterized by the presence of an intramolecular disulfide bridge between the peroxidatic and the resolving cysteine residues. We report here three crystal forms in which this intramolecular disulfide bond is indeed observed. The structures are characterized by the expected local unfolding of the peroxidatic loop, but also by the unfolding of the resolving loop. A new type of interface between PRDX molecules is described. The three crystal forms were not oxidized in the same way and the influence of the oxidizing conditions is discussed.

Flagellar length depends on LdARL-3A GTP/GDP unaltered cycling in Leishmania amazonensis.

We have shown previously that expression of the GTP-blocked form of the small G protein LdARL-3A/Q70L led to a marked shortening of Leishmania promastigotes flagella. In contrast, there was no effect with the T30N mutant, thought to represent the GDP-blocked form. However, recent data, obtained with human ARF-6, a member of the same family of G proteins, revealed that the corresponding mutant T27N was nucleotide-free and that the GDP-blocked form was the T44N mutant. When expressed in Leishmania, the corresponding new mutant, LdARL-3A/T47N, provoked also flagellum shortening. Then, it is the interruption of the cycling of LdARL-3A between a GDP- and a GTP-bound form which leads to the reduction of the flagellar length. This findings change significantly the understanding and the approaches for studying the mode of action and the role of LdARL-3A.

Crystal structures of oxidized and reduced forms of human mitochondrial thioredoxin 2.

Mammalian thioredoxin 2 is a mitochondrial isoform of highly evolutionary conserved thioredoxins. Thioredoxins are small ubiquitous protein-disulfide oxidoreductases implicated in a large variety of biological functions. In mammals, thioredoxin 2 is encoded by a nuclear gene and is targeted to mitochondria by a N-terminal mitochondrial presequence. Recently, mitochondrial thioredoxin 2 was shown to interact with components of the mitochondrial respiratory chain and to play a role in the control of mitochondrial membrane potential, regulating mitochondrial apoptosis signaling pathway. Here we report the first crystal structures of a mammalian mitochondrial thioredoxin 2. Crystal forms of reduced and oxidized human thioredoxin 2 are described at 2.0 and 1.8 A resolution. Though the folding is rather similar to that of human cytosolic/nuclear thioredoxin 1, important differences are observed during the transition between the oxidized and the reduced states of human thioredoxin 2, compared with human thioredoxin 1. In spite of the absence of the Cys residue implicated in dimer formation in human thioredoxin 1, dimerization still occurs in the crystal structure of human thioredoxin 2, mainly mediated by hydrophobic contacts, and the dimers are associated to form two-dimensional polymers. Interestingly, the structure of human thioredoxin 2 reveals possible interaction domains with human peroxiredoxin 5, a substrate protein of human thioredoxin 2 in mitochondria.

Impacts of antibiotics on in vitro UVA-susceptibility of human skin fibroblasts.

Many studies of UVA-induced cell damage use skin cells obtained during plastic surgery. As the skin is contaminated by micro-organisms, antibiotics need to be added to primary skin cell culture media. This study analysed the impact of the most widely used agents, penicillin, streptomycin, and amphotericin B deoxycholate (amB), on UVA-irradiated human skin fibroblasts. The results show that the presence of amB in cell culture media increases the susceptibility of fibroblasts to UVA and the intracellular level of reactive oxygen species, even when cells are irradiated in amB-free saline. This photosensitising effect of amB can be prevented if the antifungal agent is removed from the culture medium at least 24 hours before irradiation. Moreover, the use of streptomycin during cell culture partly protects cells against the UVA-induced mortality linked to amB. Acellular tests on lipid micelles suggest that this protective effect could result from an inhibition of lipid peroxidation by the antibacterial agent. In conclusion, antibiotics should be used with care in cell culture media if the cells are to be used in physiological studies of fine mechanisms in UVA-susceptibility of skin cells. In other cases, cells should be maintained in antibiotic-free media for 24 hours before irradiation.

Human mitochondrial peroxiredoxin 5 protects from mitochondrial DNA damages induced by hydrogen peroxide.

Peroxiredoxin 5 is a thioredoxin peroxidase ubiquitously expressed in mammalian tissues. Peroxiredoxin 5 can be addressed intracellularly to mitochondria, peroxisomes, the cytosol and the nucleus. Here, we show that mitochondrial human peroxiredoxin 5 protects mitochondrial DNA (mtDNA) from oxidative attacks. In an acellular assay, recombinant peroxiredoxin 5 was shown to protect plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. In Chinese hamster ovary cells, overexpression of mitochondrial peroxiredoxin 5 significantly decreased mtDNA damages caused by exogenously added hydrogen peroxide. Altogether our results suggest that mitochondrial peroxiredoxin 5 may play an important role in mitochondrial genome stability.

Fire fly luciferin as antioxidant and light emitter: the evolution of insect bioluminescence.

Insects are the main group with luminescent species among terrestrial animals. In this paper, we report that fire fly luciferin is endowed with antioxidant properties against oxidative and nitrosative stress. The luciferin reduces linoleate peroxidation in acellular tests and increases the viability of mammalian cells exposed to the oxidant tert-butyl hydroperoxide. Dehydrorhodamine-based tests indicate that fire fly luciferin also scavenges peroxynitrite, whereas parallel tests on cells showed a marked protection of cells subjected to the peroxynitrite generator SIN-1. Together, these results suggest that fire fly luciferin's antioxidant properties could help photocytes coping with the hyperoxidant conditions to which they are submitted during luminous emissions. These data could also suggest that the evolutionary foundation of the bioluminescent system could have been the luciferin, and not the luciferase, first serving as a scavenger of oxidants toxic to the cells, then as a light emitting substrate for luciferase precursors. Similarities with the evolutionary scenario proposed for marine bioluminescent organisms relying on coelenterazine suggest that the surprisingly high success rate observed in the independent emergence of bioluminescent animals could reflect the ease of transformation of antioxidant mechanisms into light-producing systems.

Crystal structure of a dimeric oxidized form of human peroxiredoxin 5.

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified as an atypical 2-Cys peroxiredoxin due to the presence of a conserved peroxidatic N-terminal cysteine (Cys47) and an unconserved resolving C-terminal cysteine residue (Cys151) forming an intramolecular disulfide intermediate in the oxidized enzyme. We have recently reported the crystal structure of human peroxiredoxin 5 in its reduced form. Here, a new crystal form of human peroxiredoxin 5 is described at 2.0 A resolution. The asymmetric unit contains three polypeptide chains. Surprisingly, beside two reduced chains, the third one is oxidized although the enzyme was crystallized under initial reducing conditions in the presence of 1 mM 1,4-dithio-dl-threitol. The oxidized polypeptide chain forms an homodimer with a symmetry-related one through intermolecular disulfide bonds between Cys47 and Cys151. The formation of these disulfide bonds is accompanied by the partial unwinding of the N-terminal parts of the alpha2 helix, which, in the reduced form, contains the peroxidatic Cys47 and the alpha6 helix, which is sequentially close to the resolving residue Cys151. In each monomer of the oxidized chain, the C-terminal part including the alpha6 helix is completely reorganized and is isolated from the rest of the protein on an extended arm. In the oxidized dimer, the arm belonging to the first monomer now appears at the surface of the second subunit and vice versa.

Overexpression of human peroxiredoxin 5 in subcellular compartments of Chinese hamster ovary cells: effects on cytotoxicity and DNA damage caused by peroxides.

Peroxiredoxin 5 is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Peroxiredoxin 5 can be intracellularly localized to mitochondria, peroxisomes, the cytosol, and, to a lesser extent, the nucleus. This remarkably wide subcellular distribution compared with the five other mammalian peroxiredoxins prompted us to further investigate the antioxidant protective function of peroxiredoxin 5 in mammalian cells according to its subcellular localization. Chinese hamster ovary cells overexpressing human peroxiredoxin 5 in the cytosol, in mitochondria, or in the nucleus were established by stable transfection. Cells overexpressing peroxiredoxin 5 were exposed for 1 h to low or acute oxidative stress with exogenously added hydrogen peroxide or tert-butylhydroperoxide. Cell protection conferred by peroxiredoxin 5 was evaluated by clonogenicity and lactate dehydrogenase assays. Overexpressing peroxiredoxin 5 in either the cytosolic, mitochondrial, or nuclear compartment significantly reduced cell death, with more effective protection with overexpression of peroxiredoxin 5 in mitochondria, confirming that this organelle is a major target of peroxides. Moreover, we evaluated, with the comet assay, nuclear DNA damage induced by hydrogen peroxide or tert-butylhydroperoxide. Overexpression of peroxiredoxin 5 in the nucleus significantly decreased DNA damage induced by both peroxides. In conclusion, the present study suggests that multiple subcellular targeting of peroxiredoxin 5 in mammalian cells can be implicated in antioxidant protective mechanisms under nonpathological conditions but also during acute oxidative stress caused by peroxides occurring in pathophysiological situations.

Protective effect of imidazolopyrazinone antioxidants on ischemia/reperfusion injury.

A series of 2-substituted 3,7-dihydroimidazolo[1,2-a]pyrazine-3-ones has been synthesized and evaluated for their antioxidant activity. Compounds 1-8 are inhibitors of AAPH-induced lipid peroxidation (in vitro) and excellent protectors against microvascular damages in ischemia/reperfusion (in vivo). Hence, the bicyclic structure typical of coelenterazine (luciferin) could be considered as a useful lead in medicinal chemistry.

Synthesis, structure-activity relationship and in vitro evaluation of coelenterazine and coelenteramine derivatives as inhibitors of lipid peroxidation.

Coelenterazine (2-p-hydroxybenzyl-6-(3'-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazolo[1,2-a]pyrazin-3-one, CLZn) and coelenteramine (2-amino-3-benzyl-5-(4'-hydroxyphenyl)-1,4-pyrazine, CLM), first described as luciferin and etioluciferin, respectively, of bioluminescent systems in marine organisms are endowed with antioxidant properties. This study was aimed at understanding the structural basis of their chain-breaking properties and at designing new compounds with improved antioxidative properties. For this, a series of 2-amino-1,4-pyrazine derivatives and their related imidazolopyrazinones were synthesised and examined for their capacity to inhibit lipid peroxidation in linoleate micelles subjected to the peroxidizing action of AAPH. Structure-activity relationship studies indicated that the reduction of the peroxidation rate by CLM is mainly determined by the concomitant presence of 5-p-hydroxyphenyl and 2-amino groups in para position. The lipophilic character of substituents also affected this effect. All imidazolopyrazinones induced a lag-time before the onset of the peroxidation process. The hetero-bicyclic imidazolopyrazinone moiety appears as the main contributor to this activity while phenol groups play little role in it. On the other hand, phenol groups were required for the reduction of the peroxidation rate after the lag-phase. The introduction of a supplementary p-hydroxyphenyl substituent at C8 position did not increase chain-breaking properties. The substitution of the C5-p-hydroxyphenyl with a catechol moiety or the introduction of a second amino group on the pyrazine ring yielded the most active compounds, superior to imidazolopyrazinones and reference antioxidants like epigallocatechin gallate, vitamin E and trolox. The strong antioxidant properties of 2,6-diaminopyrazines are not dependent on the presence of hydroxyl groups indicating that their reaction mechanism differs from that of 2-amino-1,4-pyrazine derivatives.