1 The relative contributions of superoxide anion (O 2 7 ) and peroxynitrite (PN) were evaluated in the pathogenesis of intestinal microvascular damage caused by the intravenous injection of E. coli lipopolysaccharide (LPS) in rats. The superoxide dismutase mimetic (SODm) SC-55858 and the active peroxynitrite decomposition catalysts 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulphonatophenyl)-porphyrinato iron (III) and 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)-porphyrinato iron (III) (FeTMPS, FeTMPyP respectively) were used to assess the roles of O 2 7 and PN respectively. 2 The intravenous injection of LPS elicited an in¯ammatory response that was characterized by a time-dependent in®ltration of neutrophils, lipid peroxidation, microvascular leakage (indicative of microvascular damage), and epithelial cell injury in both the duodenum and jejunum. 3 Administration of the SODm SC-55858, FeTMPS or FeTMPyP at 3 h post LPS reduced the subsequent increase in microvascular leakage, lipid peroxidation and epithelial cell injury. Inactive peroxynitrite decomposition catalysts exhibited no protective e ects. Only, SC-55858 inhibited neutrophil in®ltration. 4 Our results suggest that O 2 7 and peroxynitrite play a signi®cant role in the pathogenesis of duodenal and intestinal injury during endotoxaemia and that their removal by SODm and peroxynitrite decomposition catalysts o ers a novel approach to the treatment of septic shock or clinical conditions of gastrointestinal in¯ammation. Furthermore, the remarkable protection of the intestinal epithelium by these agents suggests their use during chemo-and radiation therapy, cancer treatments characterized by gastrointestinal damage. Potential mechanisms through which these radicals evoke damage are discussed.
Superoxide radical anion has been demonstrated to be a mediator of many disease states, including inflammatory, autoimmune, and cancerous diseases. As a consequence, we have developed a program to design, synthesize, and test synthetic low-molecular weight superoxide dismutase (SOD) mimics as potential pharmaceutical agents. A critical feature of the design of metal-based drugs is not only high activity as an enzyme mimic but also chemical stability. In this report we describe the synthesis and characterization of a series of C-substituted 1,4,7,-10,13-pentaazacyclopentadecane, [15]aneN 5 , ligands, 1, and their corresponding Mn(II) complexes, 2 and 3, as their dichloro complexes, [Mn([15]aneN 5 )Cl 2 ]. The purpose of the work is to probe the role that substituent (methyl and fused cycloalkyl) groups exert on the catalytic activity and stability of the complexes. All of the 18 new complexes described here are catalysts for the dismutation of superoxide, and substantial substituent effects are observed with k cat values at pH ) 7.4 varying in the range (1.4-9.1) × 10 7 M -1 s -1 . The kinetic and thermodynamic stabilities of these new complexes were studied by a Cu(II) ion competition spectrophotometric assay and by potentiometric titrations, respectively. It is observed that increasing the number of carbon substituents increases the kinetic stability of the complexes, while the thermodynamic stability increases, but less dramatically. The crystal structure for one of these new complexes, [Mn(2,2,3,3-Me 4 [15]aneN 5 )Cl 2 ], 2i, was determined, and reveals that the Mn(II) ion is coordinated by a pentagonal bipyramid array of five macrocyclic derived nitrogen atoms in a plane with the Mn(II) center and capped by trans-dichloro ligands. Crystal data for MnC 14 H 33 N 5 -Cl 2 ‚CH 3 CH 2 OH: triclinic, P1 h-C i 1 (#2), a ) 9.941(2) Å, b ) 11.190(2) Å, c ) 11.613(2) Å, V ) 1125.4(4) Å 3 , Z ) 2, T ) 20 ( 1°C, for 3357 independent absorption-corrected reflections having 2θ(Cu KR) < 120.0°and I > 3σ(Ι), with R int ) 0.027.
Two new isomeric bis(trans-fused cyclohexano) substituted 1,4,7,10,13-pentaazacyclopentadecane ligands and their Mn(II) complexes, 3 and 4, have been synthesized, and their activity as superoxide dismutase (SOD) catalysts has been studied. Complex 3 is an excellent SOD catalyst with a second-order rate constant at pH = 7.4 of 1.2 × 10+8 M-1 s-1. In contrast, the isomeric complex 4 has virtually no detectable catalytic SOD activity, implying the need to understand the effect that the position, number, and stereochemistry of substituents exert on the catalytic rate. The crystal structure of the complex 4 was determined and reveals that the Mn(II) ion is coordinated in a pentagonal bipyramid array of the five nitrogens of the macrocyclic ligand and capped by two trans-chloro ligands. Crystal data for MnC18H37Cl2N5 are as follows: triclinic at 20 °C, space group P 1--C i 2 (no. 2); a = 9.746(3) Å, b = 12.631(6) Å, c = 11.311(5) Å; α = 73.14(4)°, β = 76.39(3)°, γ = 79.98(3)°, V = 1287(1) Å3, and Z = 2 (ρcalc = 1.279 g/cm3; μa Mo Kα = 6.23 mm-1). Mechanistic studies with the complex 3 and the pentamethyl susbstituted complex, 5, including D2O rate studies, are reported and are consistent with the existence of two pathways for the rate-determining electron-transfer from Mn(II) to superoxide: (1) hydrogen atom transfer from a bound water on Mn(II) to HO2 • to yield a Mn(III) hydroxo intermediate and (2) the dissociative pathway in which superoxide anion binds to a vacant coordination site on Mn(II) followed by protonation/oxidation to yield a Mn(III)hydroperoxo species. Subsequent reduction of the intermediate Mn(III) with superoxide anion completes the catalytic cycle. Substituent effects on the rates and relative contribution of the two pathways to the overall rate of SOD activity is ascribed to the propensity of the ligand to fold around Mn(II) forming a pseudo-octahedral complex similar in geometry to the oxidized Mn(III) complex. Folding of the pentaaza macrocyclic ligand is confirmed as a relevant structural motif for this series of Mn(II) complexes by the X-ray structure determination of the bis(nitrate) derivative of 1, [Mn(C10H25N5)NO3]NO3, which reveals a six-coordinate structure with a folded conformation of the macrocyclic ligand. Crystal data for [Mn(C10H25N5)NO3]NO3: orthorhombic at −100 °C, space group P212121; a = 9.457(2) Å, b = 12.758(2) Å, c = 13.834(2) Å, V = 1669.1(5) Å3, and Z = 4 (ρcalc = 1.549 g/cm3).
Fusion of intracellular membrane-bound compartments is a common step in the transport of macromolecules along the endocytic and secretory pathways. A large number of factors active in the fusion process or its regulation have been identified; however, the actual sequence of events leading to membrane fusion is still unknown. In this study, we have assessed a possible role for PLA2 in endosome fusion by using an in vitro reconstitution assay and by examining endocytosis in intact cells. Several PLA2 inhibitors blocked endosome fusion in a broken-cell preparation. Inhibition was reversed by addition of arachidonic acid. At the electron microscope level, endosome clusters were observed even in the presence of inhibitors; however, actual fusion between endosomes was largely reduced. Fusion frequency increased upon the addition of arachidonic acid. A membrane-permeable PLA2 inhibitor blocked mixing of ligands internalized sequentially but did not affect internalization. The results indicate that vesicle fusion along the endocytic pathway requires a PLA2 activity. The effect of this activity would be, at least in part, mediated by arachidonic acid release.
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