Simulation chamber studies of the atmospheric degradation of naphthalene, 1-nitronaphthalene and phthaldialdehyde

A detailed series of simulation chamber experiments has been performed on the atmospheric degradation pathways of the primary air pollutant naphthalene and two of its photooxidation products, phthaldialdehyde and 1-nitronaphthalene. The measured yields of secondary organic aerosol (SOA) arising from the photooxidation of naphthalene varied from 6-20%, depending on the concentrations of naphthalene and nitrogen oxides as well as relative humidity. A range of carbonyls, nitro-compounds, phenols and carboxylic acids were identified among the gas- and particle-phase products. On-line analysis of the chemical composition of naphthalene SOA was performed using aerosol time-of-flight mass spectrometry (ATOFMS) for the first time. The results indicate that enhanced formation of carboxylic acids may contribute to the observed increase in SOA yields at higher relative humidity. The photolysis of phthaldialdehyde and 1-nitronaphthalene was investigated using natural light at the European Photoreactor (EUPHORE) in Valencia, Spain. The photolysis rate coefficients were measured directly and used to confirm that photolysis is the major atmospheric loss process for these compounds. For phthaldialdehyde, the main gas-phase products were phthalide and phthalic anhydride. SOA yields in the range 2-11% were observed, with phthalic acid and dihydroxyphthalic acid identified among the particle phase products. The photolysis of 1-nitronaphthalene yielded nitric oxide and a naphthoxy radical which reacted to form several products. SOA yields in the range 57-71% were observed, with 1,4-naphthoquinone, 1-naphthol and 1,4-naphthalenediol identified in the particle phase. On-line analysis of the SOA generated in an indoor chamber using ATOFMS provided evidence for the formation of high-molecular-weight products. Further investigations revealed that these products are oxygenated polycyclic compounds most likely produced from the dimerization of naphthoxy radicals. These results of this work indicate that naphthalene is a potentially large source of SOA in urban areas and should be included in atmospheric models. The kinetic and mechanistic information could be combined with existing literature data to produce an overall degradation mechanism for naphthalene suitable for inclusion in photochemical models that are used to predict the effect of emissions on air quality.

EPR spectroscopy of nitrite complexes of methemoglobin.

The chemical interplay of nitrogen oxides (NO's) with hemoglobin (Hb) has attracted considerable recent attention because of its potential significance in the mechanism of NO-related vasoactivity regulated by Hb. An important theme of this interplay-redox coupling in adducts of heme iron and NO's-has sparked renewed interest in fundamental studies of FeNO(x) coordination complexes. In this Article, we report combined UV-vis and comprehensive electron paramagnetic resonance (EPR) spectroscopic studies that address intriguing questions raised in recent studies of the structure and affinity of the nitrite ligand in complexes with Fe(III) in methemoglobin (metHb). EPR spectra of metHb/NO(2)(-) are found to exhibit a characteristic doubling in their sharper spectral features. Comparative EPR measurements at X- and S-band frequencies, and in D(2)O versus H(2)O, argue against the assignment of this splitting as hyperfine structure. Correlated changes in the EPR spectra with pH enable complete assignment of the spectrum as deriving from the overlap of two low-spin species with g values of 3.018, 2.122, 1.45 and 2.870, 2.304, 1.45 (values for samples at 20 K and pH 7.4 in phosphate-buffered saline). These g values are typical of g values found for other heme proteins with N-coordinated ligands in the binding pocket and are thus suggestive of N-nitro versus O-nitrito coordination. The positions and shapes of the spectral lines vary only slightly with temperature until motional averaging ensues at approximately 150 K. The pattern of motional averaging in the variable-temperature EPR spectra and EPR studies of Fe(III)NO(2)(-)/Fe(II)NO hybrids suggest that one of two species is present in both of the alpha and beta subunits, while the other is exclusive to the beta subunit. Our results also reconfirm that the affinity of nitrite for metHb is of millimolar magnitude, thereby making a direct role for nitrite in physiological hypoxic vasodilation difficult to justify.

The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery

Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.

Development of a rapid colorimetric time-kill assay for determining the in vitro activity of ceftazidime and tobramycin in combination against Pseudomonas aeruginosa

It is standard clinical practice to use a combination of two or more antimicrobial agents to treat an infection caused by Pseudonionas aeruginosa. The antibiotic combinations are usually selected empirically with methods to determine the antimicrobial effect of the combination such as the time-kill assay rarely used as they are time-consuming and labour intensive to perforin. Here, we report a modified time-kill assay, based on the reduction of the tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfopheny1]-2H-tetrazolium-5-carboxanilide (XTT), that allows simple, inexpensive and more rapid determination of the in vitro activity of antibiotic combinations against P aeruginosa. The assay was used to determine the in vitro activity of ceftazidime and tobramycin in combination against P. aertiginosa isolates from cystic fibrosis patients and the results obtained compared with those from conventional viable count time-kill assays. There was good agreement in interpretation of results obtained by the XTT and conventional viable count assays, with similar growth curves apparent and the most effective concentration combinations determined by both methods identical for all isolates tested. The XTT assay clearly indicated whether an antibiotic combination had a synergistic, indifferent or antagonistic effect and could, therefore, provide a useful method for rapidly determining the activity of a large number of antibiotic combinations against clinical isolates. (C) 2004 Elsevier B.V. All rights reserved.

Upregulation of adrenomedullin and its receptor components during cardiomyocyte hypertrophy induced by chronic inhibition of nitric oxide synthesis in rats

Adrenomedullin may provide a compensatory mechanism to attenuate left ventricular hypertrophy (LVH). Nitric oxide synthase inhibition, induced by chronic administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats, induces cardiac hypertrophy in some, but not all cases; there are few reports of direct assessment of cardiomyocyte parameters. The objective was to characterize hypertrophic parameters in left (LV) and right ventricular (RV) cardiomyocytes after administration of L-NAME to rats for 8 wk and to determine whether adrenomedullin and its receptor components were upregulated. After treatment with L-NAME (20 and 50 mg x kg(-1) x day(-1)), compared with nontreated animals, 1) systolic blood pressure increased (by 34.2 and 104.9 mmHg), 2) heart weight-to-body wt ratio increased 24.1% at the higher dose (P

Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension.

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

Synthesis and characterisation of polymerisable photochromic spiropyrans: towards photomechanical biomaterials

A methodology for the synthesis of novel polymerisable spiropyrans with photomechanical properties suitable for subsequent copolymerisation with either vinyl or acrylate-based biomaterials is described. UV-vis spectroscopic characterisation of photoisomerism shows that photochromic behaviour with respect to related non-polymerisable compounds is retained and is solvent dependent. In acetone, conventional spiropyran-merocyanine photochromism is observed for nitro-spiropyran derivatives, whereas in dichloromethane both nitro-spiropyrans and spiropyrans isomerise to merocyanines which rapidly form H-aggregates. The monomers were designed such that an alkyl spacer of variable length, both electronically and sterically, separates the polymerisable moiety from the photochromic core and allows steric aspects of the resulting photomechanical behaviour to be explored. (c) 2006 Elsevier Ltd. All rights reserved.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Influence of atenolol and nifedipine on nitric oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

Functional domains of the human epididymal protease inhibitor, eppin

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin’s domains are involved in the protein’s antibacterial activity, only the Kunitz domain is required for selective protease inhibition.

Hydrogenation/hydrogenolysis of disulfides using sulfided Ni/Mo catalysts

The mechanism of the hydrogenation/hydrogenolysis of dinitrodiphenyldisulfides using sulfided NiMo/ gamma Al2O3 catalysts has been examined in detail. Although two routes are possible, the major pathway involves an initial S-S bond cleavage followed by reduction of the nitro group. Importantly, the disulfide hydrogenolysis occurs in the absence of the catalyst with the role of the catalyst thought to be to activate the hydrogen and trap the cleaved intermediate as well as facilitate the reduction of the nitro group. Monitoring the mass balance throughout the reaction demonstrates the difficulty in measuring intrinsic kinetics for gas-liquid-solid reactions. Although the mass balance is restored at the end of the reaction, up to 45% of the substrate/products is found to be adsorbed on the catalyst during the reaction. (c) 2008 Elsevier B.V. All rights reserved.

A review of the selective reduction of NOx, with hydrocarbons under lean-burn conditions with non-zeolitic oxide and platinum group metal catalysts

Research on the selective reduction of NOx with hydrocarbons under lean-burn conditions using non-zeolitic oxides and platinum group metal (PGM) catalysts has been critically reviewed. Alumina and silver-promoted alumina catalysts have been described in detail with particular emphasis on an analysis of the various reaction mechanisms that have been put forward in the literature. The influence of the nature of the reducing agent, and the preparation and structure of the catalysts have also been discussed and rationalised for several other oxide systems. It is concluded for non-zeolitic oxides that species that are strongly adsorbed on the surface, such as nitrates/nitrites and acetates, could be key intermediates in the formation of various reduced and oxidised species of nitrogen, the further reaction of which leads eventually to the formation of molecular nitrogen. For the platinum group metal catalysts, the different mechanisms that have been proposed in the literature have been critically assessed. It is concluded that although there is indirect, mainly spectroscopic, evidence for various reaction intermediates on the catalyst surface, it is difficult to confirm that any of these are involved in a critical mechanistic step because of a lack of a direct quantitative correlation between infrared and kinetic measurements. A simple mechanism which involves the dissociation of NO on a reduced metal surface to give N(ads) and O(ads), with subsequent desorption of N-2 and N2O and removal of O(ads) by the reductant can explain many of the results with the platinum group metal catalysts, although an additional contribution from organo-nitro-type species may contribute to the overall NOx reduction activity with these catalysts.

Transient receptor potential melastatin 8 (TRPM8) channel involvement in the regulation of vascular tone

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 microM). However, both menthol and another agonist icilin (50 microM) caused relaxation of vessels precontracted with KCl (60 mM) or the alpha-adrenoceptor agonist phenylephrine (2 microM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca(2+)-independent phospholipase A(2) activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 microM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 microA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms.

In search of ionic liquids incorporating azolate anions

Twenty-eight novel salts with tetramethyl-, tetraethyl-, and tetrabutylammonium and 1-butyl-3-methylimidazolium cations paired with 3,5-dinitro-1,2,4-triazolate, 4-nitro-1,2,3-triazolate, 2,4-dinitroimidazolate, 4,5-dinitroimidazolate, 4,5-dicyanoimidazolate, 4-nitroimidazolate, and tetrazolate anions have been prepared and characterized by using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and single-crystal Xray crystallography. The effects of cation and anion type and structure on the physicochemical properties of the resulting salts, including several ionic liquids, have been examined and discussed. Ionic liquids (defined as having m.p. <100 degrees C) were obtained with all combinations of the 1-butyl-3-methylimidazolium cation ([C(4)mim](+)) and the heterocyclic azolate anions studied, and with several combinations of tetraethyl or tetrabutylammonium cations and the azolate anions. The [C(4)mim](+) azolates were liquid at room temperature exhibiting large liquid ranges and forming glasses on cooling with glasstransition temperatures in the range of -53 to -82 degrees C (except for the 3,5-dinitro-1,2,4-triazolate salt with m.p. 33 degrees C). Six crystal structures of the corresponding tetraalkylammonium salts were determined and the effects of changes to the cations and anions on the packing of the structure have been investigated.

Prediction of the formation and stabilities of energetic salts and ionic liquids based on ab initio electronic structure calculations

A computational approach to predict the thermodynamics for forming a variety of imidazolium-based salts and ionic liquids from typical starting materials is described. The gas-phase proton and methyl cation acidities of several protonating and methylating agents, as well as the proton and methyl cation affinities of many important methyl-, nitro-, and cyano- substituted imidazoles, have been calculated reliably by using the computationally feasible DFT (B3LYP) and MP2 (extrapolated to the complete basis set limit) methods. These accurately calculated proton and methyl cation affinities of neutrals and anions are used in conjunction with an empirical approach based on molecular volumes to estimate the lattice enthalpies and entropies of ionic liquids, organic solids, and organic liquids. These quantities were used to construct a thermodynamic cycle for salt formation to reliably predict the ability to synthesize a variety of salts including ones with potentially high energetic densities. An adjustment of the gas phase thermodynamic cycle to account for solid- and liquid-phase chemistries provides the best overall assessment of salt formation and stability. This has been applied to imidazoles (the cation to be formed) with alkyl, nitro, and cyano substituents. The proton and methyl cation donors studied were as follows: HCl, HBr, HI, (HO)(2)SO2, HSO3CF3 (TfOH), and HSO3(C6H4)CH3 (TsOH); CH3Cl, CH3Br, CH3I, (CH3O)(2)SO2, CH3SO3CF3 (TfOCH3) and CH3SO3(C6H4)CH3 (TsOCH3). As substitution of the cation with electron-withdrawing groups increases, the triflate reagents appear to be the best overall choice as protonating and methylating agents. Even stronger alkylating agents should be considered to enhance the chances of synthetic success. When using the enthalpies of reaction for the gas-phase reactants (eq 6) to form a salt, a cutoff value of - 13 kcal mol(-1) or lower (more negative) should be used as the minimum value for predicting whether a salt can be synthesized.