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The mammalian intestine is colonized by beneficial commensal bacteria and is a site of infection by pathogens, including helminth parasites. Helminths induce potent immunomodulatory effects, but whether these effects are mediated by direct regulation of host immunity or indirectly through eliciting changes in the microbiota is unknown. We tested this in the context of virus-helminth coinfection. Helminth coinfection resulted in impaired antiviral immunity and was associated with changes in the microbiota and STAT6-dependent helminth-induced alternative activation of macrophages. Notably, helminth-induced impairment of antiviral immunity was evident in germ-free mice, but neutralization of Ym1, a chitinase-like molecule that is associated with alternatively activated macrophages, could partially restore antiviral immunity. These data indicate that helminth-induced immunomodulation occurs independently of changes in the microbiota but is dependent on Ym1.

Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (TH2) effector mechanisms leading to host protection against helminthic parasites remain elusive1. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4+ T cells that induced IL-4 receptorhi (IL-4Rhi) CD206+ alternatively activated macrophages2. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.

We previously reported dipeptidomimetic compounds as inhibitors of neuronal and/or inducible NO synthases (n/iNOS) with significant selectivity against endothelial NOS (eNOS). They were composed of an S-ethylisothiocitrullin-like moiety linked to an extension through a peptide bond or a 1,2,4-oxadiazole link. Here, we developed two further series where the extension size was increased to establish more favorable interactions in the NOS substrate access channel. The extension was introduced on the solid phase by the reductive alkylation of an amino-piperidine moiety or an aminoethyl segment in the case of dipeptide-like and 1,2,4-oxadiazole compounds, respectively, with various benzaldehydes. Compared to the previous series, more potent inhibitors were identified with IC50 in the micromolar to the submicromolar range, with significant selectivity toward nNOS. As expected, most compounds did not inhibit eNOS, and molecular modeling was carried out to characterize the reasons for the selectivity toward nNOS over eNOS. Spectral studies showed that compounds were interacting at the heme active site. Finally, selected inhibitors were found to inhibit intra-cellular iNOS and nNOS expressed in RAW264.7 and INS-1 cells, respectively.

In a guinea pig model of allergic asthma, using perfused tracheal preparations ex vivo, we demonstrated that L-arginine limitation due to increased arginase activity underlies a deficiency of bronchodilating nitric oxide (NO) and airway hyperresponsiveness (AHR) after the allergen-induced early and late asthmatic reaction. Using the same animal model, we investigated the acute effects of the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) and of L-arginine on AHR after the early and late reaction in vivo. In addition, we investigated the protection of allergen-induced asthmatic reactions, AHR, and airway inflammation by pretreatment with the drug. Airway responsiveness to inhaled histamine was measured in permanently instrumented, freely moving guinea pigs sensitized to ovalbumin at 24 hours before allergen challenge and after the allergen-induced early and late asthmatic reactions by assessing histamine PC(100) (provocative concentration causing a 100% increase of pleural pressure) values. Inhaled ABH acutely reversed AHR to histamine after the early reaction from 4.77 +/- 0.56-fold to 2.04 +/- 0.34-fold (P < 0.001), and a tendency to inhibition was observed after the late reaction (from 1.95 +/- 0.56-fold to 1.56 +/- 0.47-fold, P < 0.10). Quantitatively similar results were obtained with inhaled l-arginine. Remarkably, after pretreatment with ABH a 33-fold higher dose of allergen was needed to induce airway obstruction (P < 0.01). Consequently, ABH inhalation 0.5 hour before and 8 hours after allergen challenge protected against the allergen-induced early and late asthmatic reactions, AHR and inflammatory cell infiltration. Inhalation of ABH or l-arginine acutely reverses allergen-induced AHR after the early and late asthmatic reaction, presumably by attenuating arginase-induced substrate deficiency to NO synthase in the airways. Moreover, ABH considerably reduces the airway sensitivity to inhaled allergen and protects against allergen-induced bronchial obstructive reactions, AHR, and airway inflammation. This is the first in vivo study indicating that arginase inhibitors may have therapeutic potential in allergic asthma.

Nitric oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho‐physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast‐kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise FeII‐O2 reaction intermediates in the presence of L‐arginine (Arg), alkyl‐ and aryl‐guanidines. The presence of new reaction intermediates, such as ferric haem‐peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H‐bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (Nω‐hydroxy‐L‐arginine oxidation). The reactive nitrogen species produced by nitric oxide (NO) synthases play crucial pathophysiological roles at the interface of oxidative stress and signaling processes. Using a combination of fast‐kinetics methods, resonance Raman spectroscopy and synthetic analogs of NOS substrate, we investigated the proton transfer processes involved in NOS catalysis. Our results suggest that arginine is not a proton donor, but indirectly intervenes in oxygen activation mechanism.

Neuronal nitric oxide synthase (nNOS) catalyzes single-electron reduction of quinones (Q), nitroaromatic compounds (ArNO2) and aromatic N-oxides (ArN → O), and is partly responsible for their oxidative stress-type cytotoxicity. In order to expand a limited knowledge on the enzymatic mechanisms of these processes, we aimed to disclose the specific features of nNOS in the reduction of such xenobiotics. In the absence or presence of calmodulin (CAM), the reactivity of Q and ArN → O increases with their single-electron reduction midpoint potential (E17). ArNO2 form a series with lower reactivity. The calculations according to an “outer-sphere” electron transfer model show that the binding of CAM decreases the electron transfer distance from FMNH2 to quinone by 1–2 Å. The effects of ionic strength point to the interaction of oxidants with a negatively charged protein domain close to FMN, and to an increase in accessibility of the active center induced by high ionic strength. The multiple turnover experiments of nNOS show that, in parallel with reduced FAD-FMN, duroquinone reoxidizes the reduced heme, in particular its Fe2+-NO form. This finding may help to design the heme-targeted bioreductively activated agents and contribute to the understanding of the role of P-450-type heme proteins in the bioreduction of quinones and other prooxidant xenobiotics.

Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the active‐site residue Val567 (numbering for rat neuronal NOS, nNOS). NOS‐like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO‐binding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UV‐visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme‐FeII‐CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme‐bound gaseous ligands, and potential NO production. To better understand the impact of the active‐site Val567 residue on the heme distal environment and substrate recognition by neuronal NO‐synthase, we used EPR and UV‐visible spectroscopy to investigate the effects of five mutations. Our results demonstrate the important role of Val567 in preserving the integrity of the active site for substrate binding and stability.

Cytochrome P450 2U1 (CYP2U1) identified from the human genome remains poorly known since few data are presently available on its physiological function(s) and substrate(s) specificity. CYP2U1 mutations are associated with complicated forms of hereditary spastic paraplegia, alterations of mitochondrial architecture and bioenergetics. In order to better know the biological roles of CYP2U1, we used a bioinformatics approach. The analysis of the data invited us to focus on leukotriene B4 (LTB4), an important inflammatory mediator. Here, we show that CYP2U1 efficiently catalyzes the hydroxylation of LTB4 predominantly on its ω-position. We also report docking experiments of LTB4 in a 3D model of truncated CYP2U1 that are in agreement with this hydroxylation regioselectivity. The involvement of CYP2U1 in the metabolism of LTB4 could have strong physiological consequences in cerebral pathologies including ischemic stroke because CYP2U1 is predominantly expressed in the brain.

Specific inhibition of NADPH oxidases (NOX) and NO-synthases (NOS), two enzymes associated with redox stress in tumor cells, has aroused great pharmacological interest. Here, we show how these enzymes distinguish between isomeric 2′- and 3′-phosphate derivatives, a difference used to improve the specificity of inhibition by isolated 2′- and 3′-phosphate isomers of our NADPH analogue NS1. Both isomers become fluorescent upon binding to their target proteins as observed by in vitro assay and in vivo imaging. The 2′-phosphate isomer of NS1 exerted more pronounced effects on NOS and NOX-dependent physiological responses than the 3′-phosphate isomer did. Docking and molecular dynamics simulations explain this specificity at the level of the NADPH site of NOX and NOS, where conserved arginine residues distinguished between the 2′-phosphate over the 3′-phosphate group, in favor of the 2′-phosphate.

Cholinergic airway constriction is functionally antagonized by agonist‐induced constitutive nitric oxide synthase (cNOS)‐derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L‐arginine to L‐ornithine and urea, use L‐arginine as a common substrate, competition between both enzymes for the substrate could be involved in the regulation of cholinergic airway reactivity. Using a perfused guinea‐pig tracheal tube preparation, we investigated the modulation of methacholine‐induced airway constriction by the recently developed, potent and specific arginase inhibitor NΩ‐hydroxy‐nor‐L‐arginine (nor‐NOHA). Intraluminal (IL) administration of nor‐NOHA caused a concentration‐dependent inhibition of the maximal effect (Emax) in response to IL methacholine, which was maximal in the presence of 5 μM nor‐NOHA (Emax=31.2±1.6% of extraluminal (EL) 40 mM KCl‐induced constriction versus 51.6±2.1% in controls, P<0.001). In addition, the pEC50 (−log10 EC50) was slightly but significantly reduced in the presence of 5 μM nor‐NOHA. The inhibition of Emax by 5 μM nor‐NOHA was concentration‐dependently reversed by the NOS inhibitor NΩ‐nitro‐L‐arginine methyl ester (L‐NAME), reaching an Emax of 89.4±7.7% in the presence of 0.5 mM L‐NAME (P<0.01). A similar Emax in the presence of 0.5 mM L‐NAME was obtained in control preparations (85.2±9.7%, n.s.). In the presence of excess of exogenously applied L‐arginine (5 mM), 5 μM nor‐NOHA was ineffective (Emax=33.1±5.8 versus 31.1±7.5% in controls, n.s.). The results indicate that endogenous arginase activity potentiates methacholine‐induced airway constriction by inhibition of NO production, presumably by competition with cNOS for the common substrate, L‐arginine. This finding may represent an important novel regulation mechanism of airway reactivity. British Journal of Pharmacology (2000) 130, 1793–1798; doi:10.1038/sj.bjp.0703488