Connective tissue mast cells are the target of formaldehyde to induce tracheal hyperresponsiveness in rats: Putative role of leukotriene B(4) and nitric oxide

Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbecco`s modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6 h, and 24-72 h, the rings were suspended in an organ bath and dose-response curve to methacholine (MCh) were determined. incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB(4) concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB(4)-receptor antagonist MK886 (1 mu M) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with L-arginine (1 mu M). We suggest that airway connective tissue mast cells constitute the target and may provide the increased LTB(4) generation as well as an elevated consumption of NO leading to tracheal hyperresponsiveness to MCh. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

O-GlcNAcylation Contributes to Augmented Vascular Reactivity Induced by Endothelin 1

O-GlcNAcylation augments vascular contractile responses, and O-GlcNAc-proteins are increased in the vasculature of deoxycorticosterone-acetate salt rats. Because endothelin 1 (ET-1) plays a major role in vascular dysfunction associated with salt-sensitive forms of hypertension, we hypothesized that ET-1-induced changes in vascular contractile responses are mediated by O-GlcNAc modification of proteins. Incubation of rat aortas with ET-1 (0.1 mu mol/L) produced a time-dependent increase in O-GlcNAc levels and decreased expression of O-GlcNAc transferase and beta-N-acetylglucosaminidase, key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine vasoconstriction (maximal effect [in moles]: 19 +/- 5 versus 11 +/- 2 vehicle). ET-1 effects were not observed when vessels were previously instilled with anti-O-GlcNAc transferase antibody or after incubation with an O-GlcNAc transferase inhibitor (3-[2-adamantanylethyl]-2-[{4-chlorophenyl}azamethylene]-4-oxo-1,3-thiazaperhyd roine-6-carboxylic acid; 100 mu mol/L). Aortas from deoxycorticosterone-acetate salt rats, which exhibit increased prepro-ET-1, displayed increased contractions to phenylephrine and augmented levels of O-GlcNAc proteins. Treatment of deoxycorticosterone-acetate salt rats with an endothelin A antagonist abrogated augmented vascular levels of O-GlcNAc and prevented increased phenylephrine vasoconstriction. Aortas from rats chronically infused with low doses of ET-1 (2 pmol/kg per minute) exhibited increased O-GlcNAc proteins and enhanced phenylephrine responses (maximal effect [in moles]: 18 +/- 2 versus 10 +/- 3 control). These changes are similar to those induced by O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate, an inhibitor of beta-N-acetylglucosaminidase. Systolic blood pressure (in millimeters of mercury) was similar between control and ET-1-infused rats (117 +/- 3 versus 123 +/- 4 mm Hg; respectively). We conclude that ET-1 indeed augments O-GlcNAc levels and that this modification contributes to the vascular changes induced by this peptide. Increased vascular O-GlcNAcylation by ET-1 may represent a mechanism for hypertension-associated vascular dysfunction or other pathological conditions associated with increased levels of ET-1. (Hypertension. 2010; 55: 180-188.)

Experimental evidence of the spin-glass transition in the diluted magnetic semiconductor Zn(1-x)Mn(x)In(2)Se(4)

This work reports on magnetic measurements of the quasi-two-dimensional (quasi-2D) system Zn(1-x)Mn(x)In(2)Se(4), with 0.01 <= x <= 1.00. For x > 0.67, the quasi-2D system seems to develop a spin-glass behaviour. Evidence of a true phase transition phenomenon is provided by the steep increase of the nonlinear susceptibility chi(nl) when approaching T(C) from above. The static scaling of chi(nl) data yields critical exponents delta = 4.0 +/- 0.2, phi = 4.37 +/- 0.17 and TC = 3.4 +/- 0.1 K for the sample with x = 1.00 and similar values for the sample with x = 0.87. These critical exponents are in good agreement with values reported for other spin-glass systems with short-range interactions.

Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen`s antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Leukotrienes Produced in Allergic Lung Inflammation Activate Alveolar Macrophages

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance Fc gamma R-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated Fc gamma R-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50: 1) or IgG-opsonized K. pneumoniae (30: 1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via Fc gamma R (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via Fc gamma R. Copyright (C) 2010 S. Karger AG, Basel

Electronic structure and optical properties of BaMoO(4) powders

Barium molybdate (BaMoO(4)) powders were synthesized by the co-precipitation method and processed in microwave-hydrothermal at 140 degrees C for different times. These powders were characterized by X-ray diffraction (XRD), Fourier transform Raman (FT-Raman), Fourier transform infrared (FT-IR), ultraviolet-visible (UV-vis) absorption spectroscopies and photoluminescence (PL) measurements. XRD patterns and FT-Raman spectra showed that these powders present a scheelite-type tetragonal structure without the presence of deleterious phases. FT-IR spectra exhibited a large absorption band situated at around 850.4 cm(-1), which is associated to the Mo-O antisymmetric stretching vibrations into the [MoO(4)] clusters. UV-vis absorption spectra indicated a reduction in the intermediary energy levels within band gap with the processing time evolution. First-principles quantum mechanical calculations based on the density functional theory were employed in order to understand the electronic structure (band structure and density of states) of this material. The powders when excited with different wavelengths (350 nm and 488 nm) presented variations. This phenomenon was explained through a model based in the presence of intermediary energy levels (deep and shallow holes) within the band gap. (C) 2009 Elsevier B.V. All rights reserved.

Reactions of 1,2.,5-thiadiazole 1,1-dioxide derivatives with nitrogen nucleophiles. Part IV. Addition of alpha-diamines

alpha-diamines, such as ethylendiamine and o-phenylendiamine, add to 3,4-aryl-disubstituted 1,2,5-thiadiazole 1,1-dioxides to give dihydropyrazines or quinoxalines, respectively and sulfamide. The new compound acenaphtho [5,6-b]-2,3-dihydropyrazine was synthesized and characterized. The addition of ethylendiamine to 3,4-diphenyl-1,2,5-thiadiazoline 1,1-dioxide gives 3,4-disubstituted thiadiazoildine 1,1-dioxide, dihydropyrazines, or pyrazines, depending on the reaction condition used. The reactions were followed by cyclic voltammetry and NMR spectroscopy which, in some cases, allowed the detection of the thiadiazolidine intermediate. Copyright (c) 2008 John Wiley & Sons, Ltd.

Inhibition of myeloperoxidase-mediated protein nitration by tempol: Kinetics, mechanism, and implications

Despite the therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetra-methyl-1-piperidinyloxy) and related nitroxides as antioxidants, their effects on peroxidase-mediated protein tyrosine nitration remain unexplored. This posttranslational protein modification is a biomarker of nitric oxide-derived oxidants, and, relevantly, it parallels tissue injury in animal models of inflammation and is attenuated by tempol treatment. Here, we examine tempol effects on ribonuclease (RNase) nitration mediated by myeloperoxidase (MPO), a mammalian enzyme that plays a central role in various inflammatory processes.. Some experiments were also performed with horseradish peroxidase (HRP). We show that tempol efficiently inhibits peroxidase-mediated RNase nitration. For instance, 10 mu M tempol was able to inhibit by 90% the yield of 290 mu M 3-nitrotyrosine produced from 370 mu M RNase. The effect of tempol was not completely catalytic because part of it was consumed by recombination with RNase-tyrosyl radicals. The second-order rate constant of the reaction of tempol with MPO compound I and 11 were determined by stopped-flow kinetics as 3.3 x 10(6) and 2.6 x 10(4) M-1 s(-1), respectively (pH 7.4, 25 degrees C); the corresponding HRP constants were orders of magnitude smaller. Time-dependent hydrogen peroxide and nitrite consumption and oxygen production in the incubations were quantified experimentally and modeled by kinetic simulations. The results indicate that tempol inhibits peroxidase-mediated RNase nitration mainly because of its reaction with nitrogen dioxide to produce the oxammonium cation, which, in turn, recycles back to tempol by reacting with hydrogen peroxide and superoxide radical to produce oxygen and regenerate nitrite. The implications for nitroxide antioxidant mechanisms are discussed.

Radical acetylation of 2`-deoxyguanosine and L-histidine coupled to the reaction of diacetyl with peroxynitrite in aerated medium

Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant (k(2) = 1.0 X 10(4) M-1 s(-1)) when compared with monocarbonyl substrates (k(2) < 10(3) M-1 s(-1)). Phosphate ions (100-500 MM) do not affect the rate of spontaneous peroxynitrite decay, but the H2PO4- anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum (a(N) = a(H) = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either L-histidine or 2`-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS"". These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.

Inhibition Mechanism of Rat alpha(3)beta(4) Nicotinic Acetylcholine Receptor by the Alzheimer Therapeutic Tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer`s disease, on currents elicited by activation of rat alpha(3)beta(4) nAChR heterologously expressed in KX alpha(3)beta(4)R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 +/- 0.2 mu M and 4.3 +/- 1.3 for the channel opening equilibrium constant, Phi(-1). Experiments were performed to investigate whether tacrine is able to activate the alpha(3)beta(4) nAChR. Tacrine did not activate whole-cell currents in KX alpha(3)beta(4)R2 cells but inhibited receptor activity at submicromolar concentration. Dose response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 mu M. The increase of Phi(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.

Acetyl Radical Production by the Methylglyoxal-Peroxynitrite System: A Possible Route for L-Lysine Acetylation

Methylglyoxal is an a-oxoaldehyde putatively produced in excess from triose phosphates, aminoacetone, and acetone in some disorders, particularly in diabetes. Here, we investigate the nucleophilic addition of ONOO(-), known as a potent oxidant and nucleophile, to methylglyoxal, yielding an acetyl radical intermediate and ultimately formate and acetate ions. The rate of ONOO(-) decay in the presence of methylglyoxal [k(2,app) = (1.0 +/- 0.1) x 10(3) M(-1) s(-1); k(2) approximate to 1.0 x 10(5) M(-1) s(-1)] at pH 7.2 and 25 degrees C was found to be faster than that reported with monocarbonyl substrates (k(2) < 10(3) M(-1) diacetyl (k(2) = 1.0 x 10(4) M(-1) s(-1)), or CO(2) (k(2) = 3-6 x 10(4) M(-1) s(-1)). The pH profile of the methylglyoxal peroxynitrite reaction describes an ascendant curve with an inflection around pH 7.2, which roughly coincides with the pK(a) values of both ONOOH and H(2)PO(4)(-) ion. Electron paramagnetic resonance spin trapping experiments with 2-methyl-2-nitrosopropane revealed concentration-dependent formation of an adduct that can be attributed to 2-methyl-2-nitrosopropane-CH(3)CO(center dot) (a(N) = 0.83 mT). Spin trapping with 3,5-dibromo-4-nitrosobenzene sulfonate gave a signal that could be assigned to a methyl radical adduct [a(N) = 1.41 mT; a(H) = 1.35 mT; a(H(m)) = 0.08 mT]. The 2-methyl-2-nitrosopropane-CH(3)CO(center dot) adduct could also be observed by replacement of ONOO(-) with H(2)O(2), although at much lower yields. Acetyl radicals could be also trapped by added L-lysine as indicated by the presence of W-acetyl-L-lysine in the spent reaction mixture. This raises the hypothesis that ONOO(-)/H(2)O(2) in the presence of methylglyoxal is endowed with the potential to acetylate proteins in post-translational processes.

Spectroscopic and theoretical studies of some N-methoxy-N-methyl-2-[(4 `-substituted) phenylsulfonyl]propanamides

The analysis of the IR carbonyl band of the N-methoxy-N-methyl-2-[(4`-substituted)phenyisulfonyI]propanamides Y-PhSO(2)CH(Me)C(O)N(OMe)Me (Y = OMe 1, Me 2, H 3, Cl 4, NO(2) 5), supported by HF/6-31G(d,p) calculations of 3,, indicated the existence of two gauche conformers (g(1) and g(2)), the g, conformer being the most stable and the least polar one (in gas phase and in solution). Both conformers are present in solution of the non polar solvent (CCl(4)) for 1-5 and in solution of the more polar solvents (CHCl(3)) for 1. 4, 5 and (CH(2)Cl(2)) for 5, while only the g(1) conformer is present in solution of the most polar solvent (CH(3)CN) for 1-5. The g, and g2 conformers correspond to the enantiomeric pairs of diastereomers (diast(1) and diast(2)) whose relative configurations are [C(3)(R)N(R)]/[C(3)(s)N(s)] and [C(3)(R)N(s)]/[C(3)(s)N(R)], respectively. The computed carbonyl frequencies for g(1) (diast(1)) and g(2) (diast(2)) stereoisomers of3 match well the experimental values. The NBO analysis, for 3 shows the important role of the orbital interactions in conformer stabilization and the overall balance of these interactions corroborates that the g, conformer is more stable than the 92 one. The observed abnormal solvent effect on the relative intensities of the carbonyl doublet components is attributed to the molecular crowding in the g2 conformer which hinders its solvation in comparison to the g, conformer (diast(1)). X-ray single crystal analysis performed for 3 shows the existence Of two 92, and g(1b) conformers of diastereomers (diast2, and diast(1b)) whose absolute configurations are [C(3)(R)N(s)] and [C(3)(R)N(R)], respectively. The larger population and. thus, the larger stabilization of the g(2), conformer over the gib form in the crystals may be associated with a larger energy gain deriving from dipole moment coupling in the former conformer along with a series of C-H center dot center dot center dot O electrostatic and hydrogen bond interactions, (C) 2009 Elsevier B.V. All rights reserved.