Exogenous CO Research Articles

Circadian dependency of microglial heme oxygenase-1 expression and inflammation determine neuronal injury in hemorrhagic stroke

BackgroundThe heme oxygenase-1 (HO-1) enzyme pathway is of crucial importance in the removal of toxic blood components and regulation of neuroinflammation following hemorrhagic stroke. Although a circadian pattern dependency in the incidence and severity of hemorrhagic stroke exists, it is unknown whether the activity of the HO-1 system in the context of hemorrhagic injury also exhibits circadian dependency. We hypothesized that the circadian regulation of microglial HO-1 would determine the extent of neuroinflammation and neuronal injury in a murine model of subarachnoid hemorrhage (SAH).MethodsIn vitro expression patterns of HO-1 and circadian rhythm genes were analyzed in the microglial BV-2 cell line and primary microglia (PMG) using Western blot and qPCR. PMG isolated from Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl mice were used to evaluate the role of microglial HO-1. We further investigated the in vivo relevance in a murine subarachnoid hemorrhage (SAH) model using Hmox1fl/fl and LyzM-Cre-Hmox1fl/fl mice with myeloid cell HO-1 deficiency, inducing SAH at different zeitgeber (ZT) times and analyzing the expression of HO-1 and the circadian control gene Period-2 (Per-2), respectively. Furthermore, we measured the inflammatory cytokine Monocyte Chemoattractant Protein-1 (MCP-1) in the cerebrospinal fluid of SAH patients in correlation with clinical outcome.ResultsHO-1 baseline expression and response to CO with blood exposure depended on ZT. In vitro expression of circadian control genes was de-synchronized in LyzM-Cre-Hmox1fl/fl PMG and did not respond to exogenous CO exposure. We found that circadian rhythm plays a crucial role in brain damage after SAH. At ZT2, we observed less phagocytic function, more vasospasm and increased microglial activation. CO reduced mortality at ZT12 in HO-1 deficient mice and reduced the difference between ZT2 and ZT12 in the inflammatory response. Induction of MCP-1 in the CSF from SAH patients was time-dependent and correlated with the expression of circadian control genes, SAH severity, functional impairment and delirium.ConclusionsOur data point towards a crucial role for the HO-1 enzyme system and circadian control in neuronal injury after a hemorrhagic stroke.

Carbon Monoxide Releasing Molecule-3 Alleviates Oxidative Stress and Apoptosis in Selenite-Induced Cataract in Rats via Activating Nrf2/HO-1 Pathway

Purpose This study investigated the protective effect of carbon monoxide releasing molecule-3 (CORM-3), the classical donor of carbon monoxide, on selenite-induced cataract in rats and explore its possible mechanism. Methods Sprague-Dawley rat pups treated with sodium selenite (Na2SeO3) were chosen as the cataract model. Fifty rat pups were randomly divided into 5 groups: Control group, Na2SeO3 (3.46 mg/kg) group, low-dose CORM-3 (8 mg/kg/d) + Na2SeO3 group, high-dose CORM-3 (16 mg/kg/d) + Na2SeO3 group, and inactivated CORM-3 (iCORM-3) (8 mg/kg/d) + Na2SeO3 group. The protective effect of CORM-3 was tested by lens opacity scores, hematoxylin and eosin staining, TdT-mediated dUTP nick-end labeling assay, and enzyme-linked immunosorbent assay. Besides, quantitative real-time PCR and western blotting were used for mechanism validation. Results Na2SeO3 induced nuclear cataract rapidly and stably, and the achievement ratio of Na2SeO3 group was 100%. CORM-3 alleviated lens opacity of selenite-induced cataract and attenuated the morphological changes of the rat lens. The levels of antioxidant enzymes GSH and SOD in rat lens were also increased by CORM-3 treatment. CORM-3 significantly reduced the ratio of apoptotic lens epithelial cells, besides, CORM-3 decreased the expression of Cleaved Caspase-3 and Bax induced by selenite and increased the expression of Bcl-2 in rat lens inhibited by selenite. Moreover, Nrf-2 and HO-1 were upregulated and Keap1 was downregulated after CORM-3 treatment. While iCORM-3 did not exert the same effect as CORM-3. Conclusions Exogenous CO released from CORM-3 alleviates oxidative stress and apoptosis in selenite-induced rat cataract via activating Nrf2/HO-1 pathway. CORM-3 may serve as a promising preventive and therapeutic strategy for cataract.

Exogenous ligand binding kinetics of globin X.

Intrinsically hexa-coordinate vertebrate globins have been shown to carry various cellular functions including neuroprotective and antitumor properties but the precise mechanism of their action remains unclear. Here we focus on the molecular mechanism of diatomic ligand interactions with globin X (GbX) which is a hexa-coordinate globin expressed in lower vertebrate such as fish, amphibians, and reptiles. GbX was proposed to have a variety of enzymatic activities including nitrite reductase, and nitric oxide dioxygenase along with the ability to bind several exogeneous ligands such as O2, CO, and CN-. The wide array of enzymatic functions present in GbX along with its low intracellular concentration in vivo and its hexa-coordination has led to the theory that GbX may serve to scavenge reactive oxygen and nitrogen species. Another interesting feature of GbX is the presence of an internal disulfide bond between residues Cys63 and Cys141 and myristoylation of its N-terminus with an as of yet unknown role in the functional properties of GbX. In this study interactions of exogenous ligands, CO and CN- with GbXWT and GbXH90V and GbXC60A mutants were investigated by combining in vitro and in silica approaches. The results of flash photolysis and stop flow experiments demonstrate multiphasic ligand rebinding to all three constructs with the slowest ligand binding to GbXWT. Time-resolved UV-Vis measurements also revealed multiphasic binding of CN- to GbX. The structures of GbX constructs were determined using homology modeling and subsequent MD simulations and the possible ligand migration pathways were probed using Implicit Ligand Sampling.

Water-Soluble Carbon Monoxide-Releasing Molecules (CORMs)

Carbon monoxide-releasing molecules (CORMs) are promising candidates for producing carbon monoxide in the mammalian body for therapeutic purposes. At higher concentrations, CO has a harmful effect on the mammalian organism. However, lower doses at a controlled rate can provide cellular signaling for mandatory pharmacokinetic and pathological activities. To date, exploring the therapeutic implications of CO dose as a prodrug has attracted much attention due to its therapeutic significance. There are two different methods of CO insertion, i.e., indirect and direct exogenous insertion. Indirect exogenous insertion of CO suggests an advantage of reduced toxicity over direct exogenous insertion. For indirect exogenous insertion, researchers are facing the issue of tissue selectivity. To solve this issue, developers have considered the newly produced CORMs. Herein, metal carbonyl complexes (MCCs) are covalently linked with CO molecules to produce different CORMs such as CORM-1, CORM-2, and CORM-3, etc. All these CORMs required exogenous CO insertion to achieve the therapeutic targets at the optimized rate under peculiar conditions or/and triggering. Meanwhile, the metal residue was generated from i-CORMs, which can propagate toxicity. Herein, we explain CO administration, water-soluble CORMs, tissue accumulation, and cytotoxicity of depleted CORMs and the kinetic profile of CO release.

Surface Hydrides on Fe2P Electrocatalyst Reduce CO2 at Low Overpotential: Steering Selectivity to Ethylene Glycol.

Development of efficient electrocatalysts for the CO2 reduction reaction (CO2RR) to multicarbon products has been constrained by high overpotentials and poor selectivity. Here, we introduce iron phosphide (Fe2P) as an earth-abundant catalyst for the CO2RR to mainly C2-C4 products with a total CO2RR Faradaic efficiency of 53% at 0 V vs RHE. Carbon product selectivity is tuned in favor of ethylene glycol formation with increasing negative bias at the expense of C3-C4 products. Both Grand Canonical-DFT (GC-DFT) calculations and experiments reveal that *formate, not *CO, is the initial intermediate formed from surface phosphino-hydrides and that the latter form ionic hydrides at both surface phosphorus atoms (H@Ps) and P-reconstructed Fe3 hollow sites (H@P*). Binding of these surface hydrides weakens with negative bias (reactivity increases), which accounts for both the shift to C2 products over higher C-C coupling products and the increase in the H2 evolution reaction (HER) rate. GC-DFT predicts that phosphino-hydrides convert *formate to *formaldehyde, the key intermediate for C-C coupling, whereas hydrogen atoms on Fe generate tightly bound *CO via sequential PCET reactions to H2O. GC-DFT predicts the peak in CO2RR current density near -0.1 V is due to a local maximum in the binding affinity of *formate and *formaldehyde at this bias, which together with the more labile C2 product affinity, accounts for the shift to ethylene glycol and away from C3-C4 products. Consistent with these predictions, addition of exogenous CO is shown to block all carbon product formation and lower the HER rate. These results demonstrate that the formation of ionic hydrides and their binding affinity, as modulated by the applied potential, controls the carbon product distribution. This knowledge provides new insight into the influence of hydride speciation and applied bias on the chemical reaction mechanism of CO2RR that is relevant to all transition metal phosphides.

Palladium-catalysed carboformylation of alkynes using acid chlorides as a dual carbon monoxide and carbon source.

Hydroformylation, a reaction that installs both a C-H bond and an aldehyde group across an unsaturated substrate, is one of the most important catalytic reactions in both industry and academia. Given the synthetic importance of creating new C-C bonds, the development of carboformylation reactions, wherein a new C-C bond is formed instead of a C-H bond, would bear enormous synthetic potential to rapidly increase molecular complexity in the synthesis of valuable aldehydes. However, the demanding complexity inherent in a four-component reaction, utilizing an exogenous CO source, has made the development of a direct carboformylation reaction a formidable challenge. Here, we describe a palladium-catalysed strategy that uses readily available aroyl chlorides as a carbon electrophile and CO source, in tandem with a sterically congested hydrosilane, to perform a stereoselective carboformylation of alkynes. An extension of this protocol to four chemodivergent carbonylations further highlights the creative opportunity offered by this strategy in carbonylation chemistry.

Carbon Monoxide-Releasing Molecule-2 Ameliorates Particulate Matter-Induced Aorta Inflammation via Toll-Like Receptor/NADPH Oxidase/ROS/NF-κB/IL-6 Inhibition.

Particulate matter (PM), a major air pollutant, may be associated with adverse cardiovascular effects. Reactive oxygen species- (ROS-) dependent proinflammatory cytokine production, such as interleukin-6 (IL-6), is a possible underlying mechanism. Carbon monoxide- (CO-) releasing molecule-2 (CORM-2) which liberates exogenous CO can exert many beneficial effects, particularly anti-inflammation and antioxidant effects. The purpose of this study was to explore the protective effects and underpinning mechanisms of CORM-2 on PM-induced aorta inflammation. Here, human aortic vascular smooth muscle cells (HASMCs) were utilized as in vitro models for the assessment of signaling pathways behind CORM-2 activities against PM-induced inflammatory responses, including Toll-like receptors (TLRs), NADPH oxidase, ROS, nuclear factor-kappa B (NF-κB), and IL-6. The modulation of monocyte adherence and HASMC migration, that are two critical cellular events of inflammatory process, along with their regulators, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) and MMP-9, in response to PM by CORM-2, were further evaluated. Finally, mice experiments under different conditions were conducted for the in vivo evaluation of CORM-2 benefits on the expression of inflammatory molecules including IL-6, ICAM-1, VCAM-1, MMP-2, and MMP-9. Our results found that PM could induce aorta inflammation in vitro and in vivo, as evidenced by the increase of IL-6 expression that was regulated by the TLR2 and TLR4/NADPH oxidase/ROS/NF-κB signaling pathway, thereby promoting ICAM-1- and VCAM-1-dependent monocyte adhesion and MMP-2- and MMP-9-dependent HASMC migration. Importantly, our experimental models demonstrated that CORM-2-liberated CO effectively inhibited the whole identified PM-induced inflammatory cascade in HASMCs and tissues. In conclusion, CORM-2 treatment may elicit multiple beneficial effects on inflammatory responses of aorta due to PM exposure, thereby providing therapeutic value in the context of inflammatory diseases of the cardiovascular system.

CO-Releasing Molecule (CORM)-3 Ameliorates Spinal Cord-Blood Barrier Disruption Following Injury to the Spinal Cord.

Spinal cord injury (SCI) is a clinical tough neurological problem without efficient cure currently. Blood-spinal cord barrier (BSCB) interruption is not only a crucial pathological feature for SCI process but is a possible target for future SCI treatments; however, few treatments have been developed to intervene BSCB. In the present study, we intravenously injected CO-releasing molecule3 (CORM-3), a classical exogenous CO donor, to the rats experiencing SCI and assessed its protection on BSCB integrity in rats. Our results demonstrated that the exogenous increasing of CO by CORM-3 blocked the tight junction (TJ) protein degeneration and neutrophils infiltration, subsequently suppressed the BSCB damage and improved the motor recovery after SCI. And we certified that the CO-induced down-regulation of MMP-9 expression and activity in neutrophil might be associated with the NF-κB signaling. Taken together, our study indicates that CO-releasing molecule (CORM)-3 ameliorates BSCB after spinal cord injury.

Carbon monoxide controls microglial erythrophagocytosis by regulating CD36 surface expression to reduce the severity of hemorrhagic injury.

Microglial erythrophagocytosis is crucial in injury response to hemorrhagic stroke. We hypothesized that regulation of microglial erythrophagocytosis via HO-1/CO depends on a pathway involving reactive oxygen species (ROS) and CD36 surface-expression. The microglial BV-2 cell line and primary microglia (PMG) were incubated +/-blood and +/-CO-exposure. PMG isolated from tissue-specific HO-1-deficient (LyzM-Cre-Hmox1 fl/fl ) and CD36 -/- mice or siRNA against AMPK (AMP-activated protein kinase) were used to test our hypothesis. In a murine subarachnoid hemorrhage (SAH) model, we compared neuronal injury in wild-type and CD36 -/- mice. Readouts included vasospasm, microglia activation, neuronal apoptosis, and spatial memory. We observed increased microglial HO-1-expression after blood-exposure. A burst in ROS-production was seen after CO-exposure, which led to increased amounts of phosphorylated AMPK with subsequently enhanced CD36 surface-expression. Naïve PMG from LyzM-Cre-Hmox1 fl/fl mice showed reduced ROS-production and CD36 surface-expression and failed to respond to CO with increased CD36 surface-expression. Lack of HO-1 and CD36 resulted in reduced erythrophagocytosis that could not be rescued with CO. Erythrophagocytosis was enhanced in BV-2 cells in the presence of exogenous CO, which was abolished in cells treated with siRNA to AMPK. CD36 -/- mice subjected to SAH showed enhanced neuronal cell death, which resulted in impaired spatial memory function. We demonstrate that microglial phagocytic function partly depends on a pathway involving HO-1 with changes in ROS-production, phosphorylated AMPK, and surface expression of CD36. CD36 was identified as a crucial component in blood clearance after hemorrhage that ultimately determines neuronal outcome. These results demand further investigations studying the potential neuroprotective properties of CO.

Catalytic M Center of Copper Monooxygenases Probed by Rational Design. Effects of Selenomethionine and Histidine Substitution on Structure and Reactivity.

The M centers of the mononuclear monooxygenases peptidylglycine monooxygenase (PHM) and dopamine β-monooxygenase bind and activate dioxygen en route to substrate hydroxylation. Recently, we reported the rational design of a protein-based model in which the CusF metallochaperone was repurposed via a His to Met mutation to act as a structural and spectroscopic biomimic. The PHM M site exhibits a number of unusual attributes, including a His2Met ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N3-, and complete coupling of oxygen reduction to substrate hydroxylation even at extremely low turnover rates. In particular, mutation of the Met ligand to His completely eliminates the catalytic activity despite the propensity of CuI-His3 centers to bind and activate dioxygen in other metalloenzyme systems. Here, we further develop the CusF-based model to explore methionine variants in which Met is replaced by selenomethionine (SeM) and histidine. We examine the effects on coordinate structure and exogenous ligand binding via X-ray absorption spectroscopy and electron paramagnetic resonance and probe the consequences of mutations on redox chemistry via studies of the reduction by ascorbate and oxidation via molecular oxygen. The M-site model is three-coordinate in the Cu(I) state and binds CO to form a four-coordinate carbonyl. In the oxidized forms, the coordination changes to tetragonal five-coordinate with a long axial Met ligand that like the enzymes is undetectable at either the Cu or Se K edges. The EXAFS data at the Se K edge of the SeM variant provide unique information about the nature of the Cu-methionine bond that is likewise weak and fluxional. Kinetic studies document the sluggish reactivity of the Cu(I) complexes with molecular oxygen and rapid rates of reduction of the Cu(II) complexes by ascorbate, indicating a remarkable stability of the Cu(I) state in all three derivatives. The results show little difference between the Met ligand and its SeM and His congeners and suggest that the Met contributes to catalysis in ways that are more complex than simple perturbation of the redox chemistry. Overall, the results stimulate a critical re-examination of the canonical reaction mechanisms of the mononuclear copper monooxygenases.

A Fluorescent ESIPT Probe for Imaging CO-Releasing Molecule-3 in Living Systems.

CO-releasing molecule-3 (CORM-3) has been widely used recently as a convenient and safe CO donor to release exogenous CO in living cells and to study the effects of CO on cellular systems. Accordingly, development of effective methods for detecting and tracking CORM-3 in living systems is of great significance. In this work, a readily available fluorescent probe for detection of CORM-3 was reported for the first time. This probe is based on an excited-state intramolecular proton transfer (ESIPT) dye phthalimide and uses the reducing ability of CORM-3 to convert a nitro group to an amino group, and more importantly, it can be used for rapid, highly selective, and sensitive detection of CORM-3 with a distinct turn-on green fluorescence change in aqueous solution, living cells, and animals, thus providing a useful tool for studying CORM-3 in living systems.

Cloning and expression of a recombinant tumor necrosis factor-α (TNF-α) protein

Introduction: Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease and in many patients can lead to joint destruction and loss of function. The expression of the proinflammatory cytokine (TNF-a) is widely related to the pathogenesis of RA. Recent evidence suggests that providing exogenous CO suppresses the inflammatory response associated with various disease states this is one of the mechanisms involved in the regulation of inflammation based on the inhibition of pro-inflammatory cytokine production such as TNF-α, CO might be a novel and important molecule in the treatment of RA. However, inhalation of gaseous CO presents serious intrinsic limitations as a drug due to the potent scavenging effect of hemoglobin, to circumvent these problems, CO Releasing Molecules (CORMs) have been designed to act as pro-drugs, releasing CO in vivo and eliciting its therapeutic action. In order to developed ligands (monoclonal and recombinant antibodies and recombinant peptides) to CORMS for direct delivery to inflammatory sites, there was the need to produced human recombinant TNF-a protein. Materials and methods: A synthetic recombinant tnf-a (rTNF-a), was cloned in the vector pNZY29V (NZYTECH), with the codon usage for expression in E.coli. This synthetic gene was then subcloned by PCR ligation in pHTP1 expression vector (NZYEasy Cloning & Expression System, NZYTECH). The protein rTNF-a was expressed in E.coli XJB (DES3) bacteria (Zymo Research, arabinose lysis dependent) by IPTG (1mM, 3h at 37ºC). The purification process occurred by affinity chromatography with immobilized nickel ions (IMAC, Fast-Flow GE Biosciences). The eluted samples were analyzed by reading the absorbance at 280 nm, ELISA, SDS-PAGE electrophoresis and western-blot, using commercial anti-TNF-a and anti-HIS Tag antibodies. Results: The cloning and expression system used was a directional cloning of PCR-generated fragments or synthetic gene, a DNA ligase free, with this system we have achieve a high cloning efficiency (80-100%). The protein was ready expressed, and the lysis was very efficient after arabinose-induced expression of the bacteriophage λ endolysin protein, coupled to a single freeze-thaw cycle. After IMAC chromatography the purification was between 90 to 95% (gel quantification). The rTNF-a expression was ready detected after IPTG induction using ELISA and wester-blot assays. Discussion and conclusions: We succeeded in producing a rTNF-a protein, capable of functioning as a antigen for the production of monoclonal and recombinant antiboides and recombinant peptide ligands.

The impact of exogenous CO releasing molecule CORM‑2 on inflammation and signaling of orthotopic lung cancer

The present study aimed to evaluate the therapeutic effect of CO-releasing molecule-2 (CORM-2) in an established mouse orthotopic lung cancer model and investigate the underlying mechanism associated with inflammation pathway. A total of 80 mice were randomly divided into two groups with 20 serving as a normal control and 60 used for the orthotopic lung cancer model. The tumor group was either untreated, or administrated with DMSO or CORM-2. The mice were sacrificed at day 7 and 14 post-treatment, and the body weight, and thymus and spleen indices were determined. Pathological analysis was performed with hematoxylin and eosin (HE) staining. Serous inflammatory factors were measured using an ELISA. The expression levels of eukaryotic translation initiation factor 4E, p70S6K and toll-like receptor-4 (TLR4) were quantified by reverse transcription-polymerase chain reaction. The effects of CORM-2 on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), TLR4/nuclear factor (NF)-κB signaling pathways were determined by western blotting. The body weight increased over time in the control group, while it significantly declined in tumor-bearing mice (P<0.05). CORM-2 treatment significantly increased body weight in comparison with the model and DSMO treatment groups (P<0.05). The thymus and spleen indices both reduced in the model and DMSO treatment groups, which was significantly rescued with CORM-2 administration (P<0.05). The HE staining results demonstrated few nodule formations, fibrous hyperplasia and extensive necrosis, which suggested overt inhibitory effects against cancer of CORM-2. The serous contents of tumor necrosis factor-α, interleukin (IL)-1β and IL-6 in the CORM-2 group was significantly lower compared with that in the model and DMSO groups (P<0.05). The ratio of phosphorylated (p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, p-NF-κB-p65/NF-κB-p65 and expression of TLR4 significantly decreased in the CORM-2 group compared with the model and DMSO groups (P<0.05). To the best of our knowledge, the data in the present study demonstrated in vivo for the first time, the therapeutic potential of the CORM complex, which is associated with suppression of inflammation and general protein synthesis.

Mitochondria-targeted near-infrared fluorescent probe for the detection of carbon monoxide in vivo

Carbon monoxide is a critical gasotransmitter in the body and related with mitochondrial respiration. To date, various fluorescent probes for CO have been well proposed, but two main problems remain. One is that most of the probes are not mitochondria-targeting, even if the probes claim to be able to detect CO in living cells. The other is that the probes for CO display excitation and emission within the ultraviolet or visible range, which hinders their applications in vivo. Herein, a hemicyanine-based near-infrared (NIR) fluorescent probe named CyAPC is first synthesized and used to detect mitochondrial CO. The characteristics of probe CyAPC are as follows: (1) The fluorescence emission of the sensing system is at 736 nm belonging to NIR region, which is suitable for bioimaging in vivo. (2) CyAPC, a positively charged molecule, would have a high tendency to localize in mitochondria of cells. (3) The fluorescence change of the probe is attributed to the fact that CO with Pd2+ induced cleavage of the allyl formate group from the probe and CyAPC (fluorescence off) is transformed into CyOH (fluorescence on), which is proved by HPLC, MS and DFT calculation. (4) The NIR fluorescent probe is applied for the detection of exogenous and endogenous CO in various biological samples such as cell, tissue and in vivo with satisfactory results.

Importance of the Active Site "Canopy" Residues in an O2-Tolerant [NiFe]-Hydrogenase.

The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a "canopy" above the bimetallic center, closest to the site at which exogenous agents CO and O2 interact, substrate H2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N-metal distance of 4.4 Å): its replacement with lysine lowers the H2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H2/D2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O2 tolerance by stabilizing the oxidized resting NiIII-OH state ("Ni-B"). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.

Reactivity of the Excited States of the H-Cluster of FeFe Hydrogenases.

FeFe hydrogenases catalyze H2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe2(CO)3(CN)2(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the use of hydrogenase for the photoproduction of H2. Here, we explore in great detail the reactivity of the excited states of the H-cluster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This strategy could be used for probing other aspects of the photoreactivity of the H-cluster.

Suppressive effect of exogenous carbon monoxide on endotoxin-stimulated platelet over-activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway

Platelet activation is an important event involved in the pathophysiological processes of the coagulation system. Clinical evidence has shown that platelets undergo distinctive pathological processes during sepsis. Unfortunately, how platelets physiologically respond to inflammation or sepsis is not well understood. In this study, we used a lipopolysaccharide (LPS)-stimulated platelet model to systemically investigate alterations in membrane glycoprotein expression, molecular signaling, morphology and critical functions of platelets. We found that platelet adhesion, aggregation, secretion, and spreading on immobilized fibrinogen and the expression of platelet membrane glycoproteins were significantly increased by LPS stimulation, and these changes were accompanied by a significant decrease in cGMP levels and an abnormal distribution of platelet α-granules. Exogenous CO reversed these alterations. Profound morphological changes in LPS-stimulated platelets were observed using atomic force microscopy and phase microscopy. Furthermore, the elevated activities of PI3Ks, AKt and GSK-3β were effectively suppressed by exogenous CO, leading to the improvement of platelet function. Together, these results provide evidence that platelet over-activation persists under LPS-stimulation and that exogenous CO plays an important role in suppressing platelet activation via the glycoprotein-mediated PI3K-Akt-GSK3β pathway.

Exogenous CO2in South American sparkling wine

The sparkling wine production and consumption have increased significantly in the last years. With the increased demand appear the necessity to check the sparkling wine authenticity, because the practice of adding CO2 in sparkling wine is not allow. A way to control the carbonation process is through the determination of CO2 δ13 C, because the sugar added during the second fermentation define the CO2 isotopic value, according to elaboration process. For this reason, the aim of this study was to evaluate the relationship between values of δ13 C from still wines and sparkling wines, in order to set up limit values to exogenous carbonation control. Thirty-eight still wines elaborated by microvinification and 59 samples of commercial sparkling wines were analyzed, using an isotope ratio mass spectrometer (IRMS). The most negative value of natural δ13 C from still wine found was − 24.7‰, it can be to estimate that lowest values are an indicative of industrial CO2 addition. Among the commercial sparkling wine from South America evaluated in this study, 10% from the samples showed signs of carbonation. Through this research was possible to establish limits of isotopic values to determine the presence of exogenous CO2 .

Isolation and characterization of a new [FeFe]-hydrogenase from Clostridium perfringens.

This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron-sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645±16s(-1) . The optimal conditions for catalysis are in the pH range 6.5-8.0 and at the temperature of 50°C. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO-inhibited enzyme has a spectrum coherent with the Hox -CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox ; upon reduction with H2 , vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox -CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster.

Regulation of microglial migration, phagocytosis, and neurite outgrowth by HO-1/CO signaling.

Clearance of infected and apoptotic neuronal corpses during inflammatory conditions is a fundamental process to create a favorable environment for neuronal recovery. Microglia are the resident immune cells and the predominant phagocytic cells of the CNS, showing a multitude of cellular responses upon activation. Here, we investigated in functional assays how the CO generating enzyme heme oxygenase 1 (HO-1) influences BV-2 microglial migration, clearance of debris, and neurite outgrowth of human NT2 neurons. Stimulation of HO-1 activity attenuated microglial migration in a scratch wound assay, and phagocytosis in a cell culture model of acute inflammation comprising lipopolysaccharide (LPS)-activated microglia and apoptosis-induced neurons. Application of a CO donor prevented the production of NO during LPS stimulation, and reduced microglial migration and engulfment of neuronal debris. LPS-activated microglia inhibited neurite elongation of human neurons without requiring direct cell-cell surface contact. The inhibition of neurite outgrowth was totally reversed by application of exogenous CO or increased internal CO production through supply of the substrate hemin to HO. Our results point towards a vital cytoprotective role of HO-1/CO signaling after microglial activation. In addition, they support a therapeutic potential of CO releasing chemical agents in the treatment of excessive inflammatory conditions in the CNS.