The antioxidant effects of natural estrogens (estrone, E1; 17 beta-estradiol), synthetic estrogens (17 alpha-ethynylestradiol, EE2; mestranol, MES; diethylstilbestrol, DES) and catecholestrogens (2-hydroxyestradiol; 4-hydroxyestradiol, 4-OHE2) on lipid peroxidation induced by different means in rat liver microsomes were investigated. The extent of lipid peroxidation was determined by measuring thiobarbituric acid reactive substances. Prooxidants included Fe3+/ADP/reduced NADPH, Fe2+/ascorbate, tert-butyl hydroperoxide (t-BOOH) and 2,2'-azobis(2-amidinopropane) (AAPH). Estrogens and catecholestrogens decreased lipid peroxidation in all four systems tested. In the iron/ascorbate model it was shown that (i) 4-OHE2 and DES had analogous patterns of inhibition, irrespective of the presence of NADPH or the functional integrity of the microsomes, and (ii) the antioxidant activities of E1, EE2 and MES were dependent on the assay conditions with the activity being markedly higher when estrogen metabolism was favored. When peroxidation was initiated by the peroxyl radical generator AAPH, the inhibitory effects observed were least pronounced. Our data also showed that, in each of the systems, all inhibitors displayed the same order of inhibitory potency with DES and catecholestrogens being the most potent antioxidants under all experimental conditions used. The present results confirm earlier findings and point toward a link between estrogen metabolism and estrogen antioxidant activity. The data also indicate that estrogens and catecholestrogens interact with the peroxidative process at different levels with their interactions with iron or the metal-derived species being the most important modes of inhibition.
Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium responsible for whooping cough. The 1,706-residue protein can enter eukaryotic cells, where, upon activation by endogenous calmodulin, it increases the intracellular levels of cyclic AMP, leading to severe alterations in cellular physiology, often referred to as intoxication (see reference 28 for a review). ACT belongs to the so-called RTX (repeats in toxin) family of proteins, characterized by a Ca 2ϩ -binding nonapeptide repeated in tandem several times, up to 30 to 38 repeats in the case of ACT, depending on the stringency of repeat definition. This toxin represents the most evolutionarily divergent example of the family (for reviews of RTX proteins, see references 40 and 41). Unlike most other members of the family, ACT remains associated with the bacterial surface after secretion, apparently associated with filamentous hemagglutinin (42).In common with other members of the RTX family, and apart from its unique adenylate cyclase activity, ACT has a capacity to induce cell lysis, usually demonstrated as hemolysis. ACT-induced hemolysis requires higher toxin concentrations (by more than 1 order of magnitude) and occurs more slowly than intoxication (17). Active ACT is acylated at two positions inside the chain, and the acylation pattern appears to affect hemolysis, rather than intoxication (19). Moreover, dose-response experiments suggest that intoxication can be triggered by ACT monomers, while hemolysis is a more cooperative event, mediated by at least trimers (5, 17, 32). These and other observations have led to the conclusion that hemolysis and intoxication occur through separate mechanisms (17,28,32,34).Unlike intoxication, ACT-induced cell lysis has received relatively little attention. Benz et al. (4) and Szabo et al. (39), using planar lipid bilayers, demonstrated that ACT increased membrane conductance, giving rise to small, transient, cationselective channels. These authors also found that ACT was less active in this respect than ␣-hemolysin (HlyA), another member of the RTX family, secreted by Escherichia coli (4). In general, the mechanism of HlyA-induced hemolysis has been studied in more detail (see references 16 and 40 for reviews). In particular, studies in one of our laboratories have examined the capacity of HlyA to destroy the permeability barrier of pure-lipid vesicl...
Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis. Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT-PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT-PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT-PLA activity thus emerges as novel target for therapeutic control of the disease.bacterial toxins | RTX toxin family | protein translocation | biological membranes | membrane remodeling
p.Leu167del mutation in APOE gene is the cause of hypercholesterolemia in the 3.1% of our ADH subjects without LDLR, APOB, and PCSK9 mutations. The mechanism by which this mutation is associated to ADH is that VLDL carrying the mutant apo E produces LDLR down-regulation, thereby raising plasma low-density lipoprotein cholesterol levels.
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