The kinetics of the gas-phase reactions of selected monoterpenes and cyclo-alkenes with ozone and the NO3 radical

The relative rate method has been used to measure the room-temperature rate constants for the gasphase reactions of ozone and NO3 with selected monoterpenes and cyclo-alkenes with structural similarities to monoterpenes. Measurements were carried out at 298 ! 2 K and 760 ! 10 Torr. The following rate constants (in units of 10"18 cm3 molecule"1 s"1) were obtained for the reaction with ozone: methyl cyclohexene (132 ! 17), terpinolene (1290 ! 360), ethylidene cyclohexane (223 ! 57), norbornene (860 ! 240), t-butyl isopropylidene cyclohexane (1500 ! 460), cyclopentene (543 ! 94), cyclohexene (81 ! 18), cyclooctene (451 ! 66), dicyclopentadiene (1460 ! 170) and a-pinene (107 ! 13). For the reaction with NO3 the rate constants obtained (in units of 10"12 cm3 molecule"1 s"1) were: methyl cyclohexene (7.92 ! 0.95), terpinolene (47.9 ! 4.0), ethylidene cyclohexane (4.30 ! 0.24), norbornene (0.266 ! 0.029), cyclohexene (0.540 ! 0.017), cyclooctene (0.513 ! 0.029), dicyclopentadiene (1.20 ! 0.10) and a-pinene (5.17 ! 0.62). Errors are quoted as the root mean square of the statistical error (95% con!dence) and the quoted error in the rate constant for the reference compound. Combining these results with previous studies, new recommendations for the rate constants are presented. Molecular orbital energies were calculated for each alkene and the kinetic data are discussed in terms of the deviation from the structureeactivity relationship obtained from the rate constants for a series of simple alkenes. Lifetimes with respect to key initiators of atmospheric oxidation have been calculated suggesting that the studied reactions play dominant roles in the night-time removal of these compounds from the atmosphere.

Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides

Chemotaxis is one of the best characterised signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli. We have characterised the properties of the chemosensory response of Rhodobacter sphaeroides, an -proteobacterium with multiple chemotaxis pathways, under two growth conditions allowing the effects of protein expression levels and cell architecture to be investigated. Using tethered cell assays we measured the responses of the system to step changes in concentration of the attractant propionate and show that, independently of the growth conditions, R. sphaeroides is chemotactic over at least five orders of magnitude and has a sensing profile following Weber’s law. Mathematical modelling also shows that, like E. coli, R. sphaeroides is capable of showing Fold-Change Detection (FCD). Our results indicate that general features of bacterial chemotaxis such as the range and sensitivity of detection, adaptation times, adherence to Weber’s law and the presence of FCD may be integral features of chemotaxis systems in general, regardless of network complexity, protein expression levels and cellular architecture across different species.

The kinetics of αIIbβ3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy.

Two major pathways contribute to Ras-proximate-1-mediated integrin activation in stimulated platelets. Calcium and diacyglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI, RasGRP2) mediates the rapid but reversible activation of integrin αIIbβ3, while the adenosine diphosphate receptor P2Y12, the target for antiplatelet drugs like clopidogrel, facilitates delayed but sustained integrin activation. To establish CalDAG-GEFI as a target for antiplatelet therapy, we compared how each pathway contributes to thrombosis and hemostasis in mice. Ex vivo, thrombus formation at arterial or venous shear rates was markedly reduced in CalDAG-GEFI(-/-) blood, even in the presence of exogenous adenosine diphosphate and thromboxane A(2). In vivo, thrombosis was virtually abolished in arterioles and arteries of CalDAG-GEFI(-/-) mice, while small, hemostatically active thrombi formed in venules. Specific deletion of the C1-like domain of CalDAG-GEFI in circulating platelets also led to protection from thrombus formation at arterial flow conditions, while it only marginally increased blood loss in mice. In comparison, thrombi in the micro- and macrovasculature of clopidogrel-treated wild-type mice grew rapidly and frequently embolized but were hemostatically inactive. Together, these data suggest that inhibition of the catalytic or the C1 regulatory domain in CalDAG-GEFI will provide strong protection from athero-thrombotic complications while maintaining a better safety profile than P2Y12 inhibitors like clopidogrel.

Hippurate: the natural history of a mammalian-microbial co-metabolite

Hippurate, the glycine conjugate of benzoic acid, is a normal constituent of the endogenous urinary metabolite profile and has long been associated with the microbial degradation of certain dietary components, hepatic function and toluene exposure, and is also commonly used as a measure of renal clearance. Here we discuss the potential relevance of hippurate excretion with regards to normal endogenous metabolism and trends in excretion relating to gender, age, and the intestinal microbiota. Additionally, the significance of hippurate excretion with regards to disease states including obesity, diabetes, gastrointestinal diseases, impaired renal function, psychological disorders and autism, as well as toxicity and parasitic infection, are considered.

Ozonolysis of methyl oleate monolayers at the air–water interface: oxidation kinetics, reaction products and atmospheric implications

Ozonolysis of methyl oleate monolayers at the air–water interface results in surprisingly rapid loss of material through cleavage of the C[double bond, length as m-dash]C bond and evaporation/dissolution of reaction products. We determine using neutron reflectometry a rate coefficient of (5.7 ± 0.9) × 10−10 cm2 molecule−1 s−1 and an uptake coefficient of [similar]3 × 10−5 for the oxidation of a methyl ester monolayer: the atmospheric lifetime is [similar]10 min. We obtained direct experimental evidence that <2% of organic material remains at the surface on atmospheric timescales. Therefore known long atmospheric residence times of unsaturated fatty acids suggest that these molecules cannot be present at the interface throughout their ageing cycle, i.e. the reported atmospheric longevity is likely to be attributed to presence in the bulk and viscosity-limited reactive loss. Possible reaction products were characterized by ellipsometry and uncertainties in the atmospheric fate of organic surfactants such as oleic acid and its methyl ester are discussed. Our results suggest that a minor change to the structure of the molecule (fatty acid vs. its methyl ester) considerably impacts on reactivity and fate of the organic film.

Dominant components of the Thoroughbred metabolome characterised by 1H‐NMR spectroscopy: a metabolite atlas of common biofluids

Summary Reasons for performing study: Metabonomics is emerging as a powerful tool for disease screening and investigating mammalian metabolism. This study aims to create a metabolic framework by producing a preliminary reference guide for the normal equine metabolic milieu. Objectives: To metabolically profile plasma, urine and faecal water from healthy racehorses using high resolution 1H-NMR spectroscopy and to provide a list of dominant metabolites present in each biofluid for the benefit of future research in this area. Study design: This study was performed using seven Thoroughbreds in race training at a single time-point. Urine and faecal samples were collected non-invasively and plasma was obtained from samples taken for routine clinical chemistry purposes. Methods: Biofluids were analysed using 1H-NMR spectroscopy. Metabolite assignment was achieved via a range of 1D and 2D experiments. Results: A total of 102 metabolites were assigned across the three biological matrices. A core metabonome of 14 metabolites was ubiquitous across all biofluids. All biological matrices provided a unique window on different aspects of systematic metabolism. Urine was the most populated metabolite matrix with 65 identified metabolites, 39 of which were unique to this biological compartment. A number of these were related to gut microbial host co-metabolism. Faecal samples were the most metabolically variable between animals; acetate was responsible for the majority (28%) of this variation. Short chain fatty acids were the predominant features identified within this biofluid by 1H-NMR spectroscopy. Conclusions: Metabonomics provides a platform for investigating complex and dynamic interactions between the host and its consortium of gut microbes and has the potential to uncover markers for health and disease in a variety of biofluids. Inherent variation in faecal extracts along with the relative abundance of microbial-mammalian metabolites in urine and invasive nature of plasma sampling, infers that urine is the most appropriate biofluid for the purposes of metabonomic analysis.

Temperature and oxygen dependent metabolite utilization by Salmonella enterica Serovars Derby and Mbandaka

Salmonella enterica is a zoonotic pathogen of clinical and veterinary significance, with over 2500 serovars. In previous work we compared two serovars displaying host associations inferred from isolation statistics. Here, to validate genome sequence data and to expand on the role of environmental metabolite constitution in host range determination we use a phenotypic microarray approach to assess the ability of these serovars to metabolise ~500 substrates at 25°C with oxygen (aerobic conditions) to represent the ex vivo environment and at 37°C with and without oxygen (aerobic/anaerobic conditions) to represent the in vivo environment. A total of 26 substrates elicited a significant difference in the rate of metabolism of which only one, D-galactonic acid-g-lactone, could be explained by the presence (S. Mbandaka) or the absence (S. Derby) of metabolic genes. We find that S. Mbandaka respires more efficiently at ambient temperatures and under aerobic conditions on 18 substrates including: glucosominic acid, saccharic acid, trehalose, fumaric acid, maltotriose, N-acetyl-D-glucosamine, N-acetyl-beta-D-mannosamine, fucose, L-serine and dihydroxy-acetone; whereas S. Derby is more metabolically competent anaerobically at 37°C for dipeptides, glutamine-glutamine, alanine-lysine, asparagine-glutamine and nitrogen sources glycine and nitrite. We conclude that the specific phenotype cannot be reliably predicted from the presence of metabolic genes directly relating to the metabolic pathways under study.

Acute benefits of the microbial-derived isoflavone metabolite equol on arterial stiffness in men prospectively recruited according to equol producer phenotype: a double-blind randomized controlled trial

There is much speculation with regard to the potential cardioprotective benefits of equol, a microbial-derived metabolite of the isoflavone daidzein, which is produced in the large intestine after soy intake in 30% of Western populations. Although cross-sectional and retrospective data support favorable associations between the equol producer (EP) phenotype and cardiometabolic health, few studies have prospectively recruited EPs to confirm this association. The aim was to determine whether the acute vascular benefits of isoflavones differ according to EP phenotype and subsequently investigate the effect of providing commercially produced S-(–)equol to non-EPs. We prospectively recruited male EPs and non-EPs (n = 14/ group) at moderate cardiovascular risk into a double-blind, placebocontrolled crossover study to examine the acute effects of soy isoflavones (80-mg aglycone equivalents) on arterial stiffness [carotid-femoral pulse-wave velocity (cfPWV)], blood pressure, endothelial function (measured by using the EndoPAT 2000; Itamar Medical), and nitric oxide at baseline (0 h) and 6 and 24 h after intake. In a separate assessment, non-EPs consumed 40 mg S-(–)equol with identical vascular measurements performed 2 h after intake. After soy intake, cfPWV significantly improved in EPs at 24 h (cfPWV change from 0 h: isoflavone, 20.2 6 0.2 m/s; placebo, 0.6 6 0.2 m/s; P , 0.01), which was significantly associated with plasma equol concentrations (R = 20.36, P = 0.01). No vascular effects were observed in EPs at 6 h or in non-EPs at any time point. Similarly, no benefit of commercially produced S-(–)equol was observed in non-EPs despite mean plasma equol concentrations reaching 3.2 mmol/L. Acute soy intake improved cfPWV in EPs, equating to an 11–12% reduced risk of cardiovascular disease if sustained. However, a single dose of commercially produced equol had no cardiovascular benefits in non-EPs. These data suggest that the EP phenotype is critical in unlocking the vascular benefits of equol in men, and long-term trials should focus on confirming the implications of EP phenotype on cardiovascular health. This trial was registered at clinicaltrials.gov as NCT01530893. Am J Clin Nutr doi: 10.3945/ajcn.115.125690.

Kinetics analysis and automated online screening of aminocarbonylation of aryl halides in flow

Temperature, pressure, gas stoichiometry, and residence time were varied to control the yield and product distribution of the palladium-catalyzed aminocarbonylation of aromatic bromides in both a silicon microreactor and a packed-bed tubular reactor. Automation of the system set points and product sampling enabled facile and repeatable reaction analysis with minimal operator supervision. It was observed that the reaction was divided into two temperature regimes. An automated system was used to screen steady-state conditions for offline analysis by gas chromatography to fit a reaction rate model. Additionally, a transient temperature ramp method utilizing online infrared analysis was used, leading to more rapid determination of the reaction activation energy of the lower temperature regimes. The entire reaction spanning both regimes was modeled in good agreement with the experimental data.

Study of the pozzolanic reaction kinetics in sugar cane bagasse-clay ash/calcium hydroxide system: kinetic parameters and pozzolanic activity

This paper presents a study of the pozzolanic reaction kinetics between calcium hydroxide and a mixture of sugar cane bagasse with 20 and 30% of clay, burned at 800 and 1000 degrees C (SCBCA) by electrical conductivity measurements. A kinetic-diffusive model produced in previous studies by some of the authors was used. The model was fitted to the experimental data, which allowed the computation of the kinetic parameters of the pozzolanic reaction (reaction rate constant and free energy of activation) that rigorously characterised the pozzolanic activity of the materials. The results show that SCBCA demonstrated reactivity and good pozzolanic qualities in the range 800-1000 degrees C.

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.

Effect of antidepressants on melatonin metabolite in depressed patients

Antidepressants increase melatonin levels, but it is still unclear whether this effect is related to the improvement of depressive symptoms or to unrelated pharmacological action of antidepressants. To answer this question, the effect of antidepressants on 6-sulphatoxymelatonin (aMT6s), the main melatonin urinary metabolite, was examined in drug-free depressed patients - most of them antidepressant-naive. aMT6s was evaluated in 34 depressed patients, before and after 8 weeks of placebo (n = 12) or antidepressant (n = 22; fluoxetine, duloxetine or Hypericum perforatum). Both groups showed an improvement of depressive symptoms after treatment compared to baseline (Hamilton Depression scores): 17.0 +/- 1.4 vs. 9.0 +/- 2.8, P = 0.007 for placebo, and 18.6 +/- 1.1 vs. 11.8 +/- 1.6, P < 0.001 for antidepressants). After treatment, aMT6s levels increased after antidepressants (P < 0.01), but not after placebo (P > 0.05). As depressive symptoms improved both in patients taking antidepressant and in those taking placebo, but an effect of antidepressants could only be seen in those taking antidepressants, we suggest that melatonin changes after antidepressants are more likely due to a pharmacological action of these drugs on melatonin secretion.

Adsorption kinetics and charge inversion in layer-by-layer films from nickel tetrasulfonated phthalocyanine and poly(allylamine hydrochloride)

Layer-by-layer (LBL) films of nickel tetrasulfonated phthalocyanine (NiTsPc) alternated with poly(allylamine hydrochloride) (PAH) have been prepared, whose surface charge has been evaluated using surface potential measurements. From adsorption kinetics results, we obtained the immersion time of similar to 40 s, which was used to assemble layers of NiTsPc. The effect of gold (Au) and aluminum (Al) electrodes on the charge behavior was examined. We found that the surface potential (i.e. surface charge) was inverted each time a layer of PAH was alternated with another of NiTsPc molecules for the two types of electrodes, which was attributed to charge overcompensation between positive charges of PAH molecules, and negative charges from NiTsPc molecules. (C) 2009 Elsevier B.V. All rights reserved.

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics, molecular modeling, toxicity and effect on growth

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world`s population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes p stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents. (C) 2010 Elsevier Ltd. All rights reserved.

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.