The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra-and extracellular compartments. Peroxynitrite anion (ONOO ؊ ) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO 2 ؊ ), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO 3 . ). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6 -9. The radical was unambiguously identified by its EPR parameters (g ‫؍‬ 2.0113; line width ‫؍‬ 5.5 G) and by experiments with bicarbonate labeled with 13 C. In this case, the singlet EPR signal obtained with 12 C bicarbonate splits into the expected doublet because of 13 C (a( 13 C)‫؍‬ 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO 3 . ).Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.Peroxynitrite 1 is formed from the very fast reaction between nitric oxide and superoxide anion (k ϭ (6.7 Ϫ 19) ϫ 10 9 M Ϫ1 s Ϫ1 ) (see Reaction 1) (1, 2). The compound is a potent oxidant that has been receiving increasing attention as a potential pathogenic mediator in human diseases and as a cellular toxin in host defense mechanisms against invading microorganisms (3-6). At present, a significant part of the biological reactivity of peroxynitrite is ascribed to the adduct produced by its reaction with carbon dioxide (7-13). The peroxynitrite anion (ONOO Ϫ ), which is the predominant form at physiological pHs (pK a ϭ 6.8) (see reaction 2, Table II) (2, 3), reacts fast with carbon dioxide (pH-independent k ϭ 5.8 ϫ 10 4 M Ϫ1 ⅐ s Ϫ1 at 37°C) (11), producing an adduct whose structure is proposed to be ([ONOOCO 2 ] Ϫ , nitrosoperoxocarboxylate) (see reaction 3, Table II) (7). Taking into account the concentrations of carbon dioxide in equilibrium with bicarbonate present in physiological fluids, model calculations have suggested that most of the peroxynitrite that might be formed in these fluids will produce the carbon dioxide adduct before reacting with other biological targets (5, 13).Carbon dioxide modulates the reactivity of peroxynitrite by altering reaction rates, product yields, and product distribution (7-13). In these reactions, formation of the adduct nitrosoperoxocarboxylate is rate-limiting, as first proposed by Lymar and Hurst (7). This suggestion was confirmed by other authors (8 -13), and the current proposal is that in the absence of substrates, the carbon dioxide adduct decomposes to nitrate and carbon dioxide, but in ...

The larval stage of the cestode parasite Echinococcus granulosus causes hydatid disease in humans and livestock. This infection is characterized by the growth in internal organ parenchymae of fluid-filled structures (hydatids) that elicit surprisingly little inflammation in spite of their massive size and persistence. Hydatids are protected by a millimeter-thick layer of mucin-based extracellular matrix, termed the laminated layer (LL), which is thought to be a major factor determining the host response to the infection. Host cells can interact both with the LL surface and with materials that are shed from it to allow parasite growth. In this work, we analyzed the response of dendritic cells (DCs) to microscopic pieces of the native mucin-based gel of the LL (pLL). In vitro, this material induced an unusual activation state characterized by upregulation of CD86 without concomitant upregulation of CD40 or secretion of cytokines (interleukin 12 [IL-12], IL-10, tumor necrosis factor alpha [TNF-α], and IL-6). When added to Toll-like receptor (TLR) agonists, pLL-potentiated CD86 upregulation and IL-10 secretion while inhibiting CD40 upregulation and IL-12 secretion. In vivo, pLL also caused upregulation of CD86 and inhibited CD40 upregulation in DCs. Contrary to expectations, oxidation of the mucin glycans in pLL with periodate did not abrogate the effects on cells. Reduction of disulfide bonds, which are known to be important for LL structure, strongly diminished the impact of pLL on DCs without altering the particulate nature of the material. In summary, DCs respond to the LL mucin meshwork with a “semimature” activation phenotype, both in vitro and in vivo.

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This paper reports on the synthesis and characterization of two new ternary copper(II) complexes: [Cu(doxycycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (1) and [Cu(tetracycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (2). These compounds exhibit a distorted tetragonal geometry around copper, which is coordinated to two bidentate ligands, 1,10-phenanthroline and tetracycline or doxycyline, a water molecule, and a perchlorate ion weakly bonded in the axial positions. In both compounds, copper(II) binds to tetracyclines via the oxygen of the hydroxyl group and oxygen of the amide group at ring A and to 1,10-phenanthroline via its two heterocyclic nitrogens. We have evaluated the binding of the new complexes to DNA, their capacity to cleave it, their cytotoxic activity, and uptake in tumoral cells. The complexes bind to DNA preferentially by the major groove, and then cleave its strands by an oxidative mechanism involving the generation of ROS. The cleavage of DNA was inhibited by radical inhibitors and/or trappers such as superoxide dismutase, DMSO, and the copper(I) chelator bathocuproine. The enzyme T4 DNA ligase was not able to relegate the products of DNA cleavage, which indicates that the cleavage does not occur via a hydrolytic mechanism. Both complexes present an expressive plasmid DNA cleavage activity generating single- and double-strand breaks, under mild reaction conditions, and even in the absence of any additional oxidant or reducing agent. In the same experimental conditions, [Cu(phen)(2)](2+) is approximately 100-fold less active than our complexes. These complexes are among the most potent DNA cleavage agents reported so far. Both complexes inhibit the growth of K562 cells with the IC(50) values of 1.93 and 2.59 μmol L(-1) for compounds 1 and 2, respectively. The complexes are more active than the free ligands, and their cytotoxic activity correlates with intracellular copper concentration and the number of Cu-DNA adducts formed inside cells.

Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40–50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.