No abstract

SUMMARY:There is increasing evidence that antiproteases are able to affect the inflammatory response. To further examine this question, we administered human ␣-1-antitrypsin (␣1AT) or a synthetic metalloprotease inhibitor (RS113456) to C57 mice followed by a single intratracheal dose of quartz, a dust that evokes a marked, lasting, polymorphonuclear leukocyte (PMN) infiltrate. At 2 hours after dust administration, both antiproteases completely suppressed silica-induced PMN influx into the lung and macrophage inflammatory protein-2 (MIP-2)/monocyte chemotactic protein-1 (MCP-1) (neutrophil/macrophage chemoattractant) gene expression, partially suppressed nuclear transcription factor B (NF-B) translocation, and increased inhibitor of NF-B (IB) levels. By 24 hours, PMN influx and connective tissue breakdown measured as lavage desmosine or hydroxyproline were still at, or close to, control levels after antiprotease treatment, and increases in NF-B translocation and MIP-2/MCP-1 gene expression were variably suppressed. At both time points, neither agent prevented silica-induced increases in amount of whole lung MIP-2 or MCP-1 protein, but both did prevent increases in whole lung intercellular adhesion molecule-1 (ICAM-1) at 24 hours. Inactivating the ␣1AT by oxidation to the point that it no longer possessed antiproteolytic properties did not affect its ability to suppress inflammation. Both antiproteases also prevented the silica-induced acute inflammatory response in mice with knocked out genes for macrophage metalloelastase (MME Ϫ/Ϫ), mice that develop inflammation, but not connective tissue breakdown, and the pattern of ␣1AT breakdown fragments was identical in control and MME Ϫ/Ϫ animals. These findings suggest that, in this model of acute PMN mediated inflammation, a serine protease inhibitor and a metalloprotease inhibitor have similar anti-inflammatory properties, that inflammation is not mediated by proteolysis with generation of chemotactic matrix fragments, and that classic antiproteolysis (complexing of protease to antiprotease) probably does not play a role in suppression of inflammation. The antiproteolytic effects of these agents do not seem to be mediated by protection of endogenous ␣1AT. (Lab Invest 2001, 81:1119 -1131.

We report the observation of a covalently bound species, formic sulfuric anhydride (FSA), that is produced from formic acid and sulfur trioxide under supersonic jet conditions. FSA has been structurally characterized by means of microwave spectroscopy and further investigated by using density functional theory and ab initio calculations. Theory indicates that a π2 + π2 + σ2 cycloaddition reaction between SO3 and HCOOH is a plausible pathway to FSA formation and that such a mechanism would be effectively barrierless. We speculate on the possible role that FSA may play in the Earth's atmosphere.

Aqueous pyridine plays an important role in a variety of catalytic processes aimed at harnessing solar energy. In this work, the pyridine-water interaction is studied by microwave spectroscopy and density functional theory calculations. Water forms a hydrogen bond to the nitrogen with the oxygen tilted slightly toward either of the ortho-hydrogens of the pyridine, and a tunneling motion involving in-plane rocking of the water interconverts the resulting equivalent structures. A pair of tunneling states with severely perturbed rotational spectra is identified and their energy separation, ΔE, is inferred from the perturbations and confirmed by direct measurement. Curiously, values of ΔE are 10404.45 and 13566.94 MHz for the HO and DO complexes, respectively, revealing an inverted isotope effect upon deuteration. Small splittings in some transitions suggest an additional internal motion making this complex an interesting challenge for theoretical treatments of large amplitude motion. The results underscore the significant effect of the ortho-hydrogens on the intermolecular interaction of pyridine.

Acetic sulfuric anhydride, CHCOOSOOH, was produced by the reaction of SO and CHCOOH in a supersonic jet. Four isotopologues were observed by microwave spectroscopy. Spectra of both A and E internal rotor states were observed and analyzed, yielding a value of 241.093(30) cm for the methyl group internal rotation barrier of the parent species. Similar values were obtained for the other isotopologues studied. M06-2X/6-311++G(3df,3pd) calculations indicate that the formation of the anhydride proceeds via a π + π + σ cycloaddition reaction within the CHCOOH-SO complex. The equilibrium orientation of the methyl group relative to the O═C-C plane is different in the anhydride and in the CHCOOH-SO complex, indicating that the -CH internal rotation accompanies the cycloaddition reaction. The energies of key points on the potential energy surface were calculated using CCSD(T)/complete basis set with double and triple extrapolation [CBS/(D-T)], and the transformation from the CHCOOH-SO complex to CHCOOSOOH is shown to be nearly barrierless regardless of the orientation of the methyl group. This study provides the second experimental observation of the reaction between a carboxylic acid and SO to form a carboxylic sulfuric anhydride in the gas phase. Possible connections to atmospheric aerosol formation are discussed.

Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase-or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors which were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP-and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the loss of dynamic compression and shear moduli was delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

The rotational spectrum of acrylic sulfuric anhydride (CH═CHCOOSOOH, AcrSA) has been observed using pulsed-nozzle Fourier transform microwave spectroscopy. The species was produced from the reaction between acrylic acid and sulfur trioxide in a supersonic jet. Spectroscopic constants are reported for both the s-cis- and s-trans-AcrSA conformers of the parent and monodeuterated (OD) isotopologues. Geometries were optimized for both conformers using M06-2X/6-311++G(3df,3pd) methods. Single-point energy calculations at the M06-2X geometries were calculated using the CCSD(T)/complete basis set method with double and triple extrapolation [CBS(D-T)]. Further calculations indicate that the anhydride results from a π + π + σ cycloaddition reaction within the acrylic acid-SO complex. Because the C═O double bond of the acrylic acid migrates from one of the COOH oxygens to the other during the reaction, the s-cis form of acrylic acid leads to the s-trans form of the anhydride and vice versa. With zero-point energy corrections applied to the CCSD(T) energies, the s-cis and s-trans forms of CH═CHCOOSOOH are 19.0 and 18.8 kcal/mol lower in energy than that of SO + their corresponding CH═CHCOOH precursor conformation. The zero-point-corrected transition state energies for formation of the s-trans and s-cis anhydrides are 0.22 and 0.33 kcal/mol lower than those of the complexes of SO with s-cis and s-trans acrylic acid, respectively, indicating that the reaction is essentially barrierless. This system adds to a growing body of examples demonstrating that carboxylic acids readily add to SO in the gas phase to produce the corresponding carboxylic sulfuric anhydride.

Trifluoroacetic sulfuric anhydride (CF 3 COOSO 2 OH, TFASA) and its deuterated isotopologue have been observed by pulsed-nozzle Fourier transform microwave spectroscopy. TFASA was generated in situ in a supersonic expansion from the reaction of CF 3 COOH or CF 3 COOD with SO 3 . The spectrum, which was notably weaker than those of previously studied carboxylic sulfuric anhydrides, is that of a simple asymmetric rotor with no evidence of internal rotation of the CF 3 group. Calculations at the M06-2X/6-311+ +G(3df,3pd) level indicate that the title compound is produced via a mechanism involving a concerted cycloaddition, analogous to that found for other carboxylic sulfuric anhydrides. The calculations further show that the equilibrium orientation of CF 3 relative to the CO bond changes upon formation of the anhydride, indicating that any path connecting the equilibrium structures of CF 3 COOH and CF 3 COOSO 2 OH necessarily includes both cycloaddition and internal rotation. CCSD(T)/complete basis set with double and triple extrapolation [CBS(D-T)] single-point energy calculations at key points on the potential surface indicate that the barrier to form TFASA from a putative CF 3 COOH•••SO 3 complex is about 1.2 kcal/mol after zero-point energy corrections. This value is significantly larger than the near-zero or slightly negative barriers previously reported for the reactions of SO 3 with nonfluorinated carboxylic acids and likely accounts, at least in part, for the reduced spectral intensity. Thus, TFASA is a somewhat unique addition to the series of carboxylic sulfuric anhydrides studied to date. Theoretical values of certain structural parameters, atomic charges, and vibrational frequencies also support this point of view. Despite the differences, however, this work clearly demonstrates that the reaction RCOOH + SO 3 → RCOOSO 2 OH readily occurs in the gas phase and is not restricted to acids with hydrocarbon R groups.