Carbon Monoxide Inhibition Research Articles

Mutational Analysis of the Nitrogenase Carbon Monoxide Protective Protein CowN Reveals That a Conserved C-Terminal Glutamic Acid Residue Is Necessary for Its Activity.

Nitrogenase is the only enzyme that catalyzes the reduction of nitrogen gas into ammonia. Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Many nitrogen fixing bacteria protect nitrogenase from CO inhibition using the protective protein CowN. This work demonstrates that a conserved glutamic acid residue near the C-terminus of Gluconacetobacter diazotrophicus CowN is necessary for its function. Mutation of the glutamic acid residue abolishes both CowN's protection against CO inhibition and the ability of CowN to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection by CowN. Overall, this work uncovers structural features in CowN that are required for its function and provides new insights into its nitrogenase binding and CO protection mechanism.

Structural and spectroscopic characterization of CO inhibition of [NiFe]-hydrogenase from Citrobacter sp. S-77.

Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) isknown to be a competitive inhibitor of O2-sensitive [NiFe]-hydrogenases. Although the activities of some O2-tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O2-tolerant [NiFe]-hydrogenase from Citrobacter sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni-Fe active site of S77-HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]-hydrogenases.

Individual red blood cell nitric oxide production in sickle cell anemia: Nitric oxide production is increased and sickle shaped cells have unique morphologic change compared to discoid cells

Sickle cell anemia (SCA) is characterized by decreased red blood cell (RBC) deformability due to polymerization of deoxygenated hemoglobin, leading to abnormal mechanical properties of RBC, increased cellular adhesion, and microcirculatory obstruction. Prior work has demonstrated that NO• influences RBC hydration and deformability and is produced at a basal rate that increases under shear stress in normal RBC. Nevertheless, the origin and physiological relevance of nitric oxide (NO•) production and scavenging in RBC remains unclear. We aimed to assess the basal and shear-mediated production of NO• in RBC from SCA patients and control (CTRL) subjects. RBCs loaded with a fluorescent NO• detector, DAF-FM (4-Amino-5-methylamino- 2′,7′-difluorofluorescein diacetate), were imaged in microflow channels over 30-min without shear stress, followed by a 30-min period under 0.5Pa shear stress. We utilized non-specific nitric oxide synthase (NOS) blockade and carbon monoxide (CO) saturation of hemoglobin to assess the contribution of NOS and hemoglobin, respectively, to NO• production. Quantification of DAF-FM fluorescence intensity in individual RBC showed an increase in NO• in SCA RBC at the start of the basal period; however, both SCA and CTRL RBC increased NO• by a similar quantity under shear. A subpopulation of sickle-shaped RBC exhibited lower basal NO• production compared to discoid RBC from SCA group, and under shear became more circular in the direction of shear when compared to discoid RBC from SCA and CTRL, which elongated. Both CO and NOS inhibition caused a decrease in basal NO• production. Shear-mediated NO• production was decreased by CO in all RBC, but was decreased by NOS blockade only in SCA. In conclusion, total NO• production is increased and shear-mediated NO• production is preserved in SCA RBC in a NOS-dependent manner. Sickle shaped RBC with inclusions have higher NO• production and they become more circular rather than elongated with shear.

The effect of carbon monoxide on meiotic maturation of porcine oocytes.

Oxidative stress impairs the correct course of meiotic maturation, and it is known that the oocytes are exposed to increased oxidative stress during meiotic maturation in in vitro conditions. Thus, reduction of oxidative stress can lead to improved quality of cultured oocytes. The gasotransmitter carbon monoxide (CO) has a cytoprotective effect in somatic cells. The CO is produced in cells by the enzyme heme oxygenase (HO) and the heme oxygenase/carbon monoxide (HO/CO) pathway has been shown to have an antioxidant effect in somatic cells. It has not yet been investigated whether the CO has an antioxidant effect in oocytes as well. We assessed the level of expression of HO mRNA, using reverse transcription polymerase chain reaction. The HO protein localization was evaluated by the immunocytochemical method. The influence of CO or HO inhibition on meiotic maturation was evaluated in oocytes cultured in a culture medium containing CO donor (CORM-2 or CORM-A1) or HO inhibitor Zn-protoporphyrin IX (Zn-PP IX). Detection of reactive oxygen species (ROS) was performed using the oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate. We demonstrated the expression of mRNA and proteins of both HO isoforms in porcine oocytes during meiotic maturation. The inhibition of HO enzymes by Zn-PP IX did not affect meiotic maturation. CO delivered by CORM-2 or CORM-A1 donors led to a reduction in the level of ROS in the oocytes during meiotic maturation. However, exogenously delivered CO also inhibited meiotic maturation, especially at higher concentrations. In summary, the CO signaling molecule has antioxidant properties in porcine oocytes and may also be involved in the regulation of meiotic maturation.

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

CO as a substrate and inhibitor of H+ reduction for the Mo-, V-, and Fe-nitrogenase isozymes

Three known nitrogenase isozymes, Mo-, V-, and Fe-, catalyze biological reduction of dinitrogen (N2) to ammonia (NH3). All three utilize the same reductive elimination mechanism: an intermediate with two metal-bound hydrides reductively-eliminates hydrogen gas (H2) in a reaction coupled to binding and activation of N2. Nonetheless, the three isozymes show dramatically different relative rates of H2 formation and N2 reduction, revealing important differences in reactivity with substrates. Carbon monoxide (CO) has been characterized as both an inhibitor and substrate for Mo- and V‑nitrogenases, but not for the Fe‑nitrogenase. Here, we present a comparative study of the reactivity of the three isozymes with CO, examining CO both as a substrate and as an inhibitor of proton (H+) reduction under steady-state conditions. For Mo‑nitrogenase, there is neither detectable reduction of CO nor inhibition of H+ reduction. Fe- and V‑nitrogenase show CO reduction and inhibition of H+ reduction that depends on the CO partial pressure. For V‑nitrogenase, ethylene (C2H4) is the major reduction product with a maximum specific activity of ~7.5 nmol C2H4/nmol VFe protein/min at 1 atm CO. The major product of CO reduction for Fe‑nitrogenase is methane (CH4) with a maximum specific activity of ~4.8 nmol CH4/nmol FeFe protein/min at 0.05 atm CO. The rate of CH4 production by Fe‑nitrogenase progressively increases to a maximum at 0.05 atm CO and then declines by ~90% with increasing CO partial pressure up to 1 atm. CO does not inhibit proton reduction in Mo‑nitrogenase but shows 16% inhibition for V‑nitrogenase and 35% for Fe‑nitrogenase.

Biophysical Characterization of CowN from Gluconacetobacter diazotrophicus

Gluconacetobacter diazotrophicus is a nitrogen fixing bacterium that associates with plants and plays a crucial part in providing fixed nitrogen to many crops such as sugar cane. The enzyme responsible for reducing atmospheric nitrogen to ammonia is nitrogenase. The presence of carbon monoxide gas will inhibit nitrogen fixation by nitrogenase. Many diazotrophs have a mechanism of protecting nitrogenase in vivo. In G. diazotrophicus, a protein called CowN protects nitrogenase from inhibition by carbon monoxide. Here, we present structural and biophysical characterization of CowN. Following heterologous expression of CowN in Escherichia coli and purification by affinity and size exclusion chromatography, CowN is found in two different oligomeric states; monomer and large soluble aggregate. Dynamic light scattering indicates the monomer has a radius of 1.9 nm, whereas the aggregate is larger than 5 nm. While the monomer protects nitrogenase from carbon monoxide inhibition, the aggregate does not. To determine what causes CowN aggregation and if it represents a physiologically relevant process, we investigated the mechanism of CowN aggregation. We discovered that high salt and protein concentration increase the propensity of CowN to aggregate. We further determined that under reducing conditions the monomeric state is preferred, suggesting that oxidation of CowN’s single cysteine residue plays a role in aggregation. Together, the data suggests that CowN may be most active under oxidizing conditions and, further, that aggregation might represent a mechanism to inactivate the enzyme at high concentration.Support or Funding InformationThis research was supported by the Chapman Center for Undergraduate Excellence, USDA‐NIFA (Grant no. 2015‐67012‐22895) and the National Science Foundation (Grant no. 1905399).

Cytochrome P450. The Dioxygen-Activating Heme Thiolate.

Cytochromes P450 (CYPs) are heme b-binding enzymes and belong to Nature's most versatile catalysts. They participate in countless essential life processes, and exist in all domains of life, Bacteria, Archaea, and Eukarya, and in viruses. CYPs attract the interest of researchers active in fields as diverse as biochemistry, chemistry, biophysics, molecular biology, pharmacology, and toxicology. CYPs fight chemicals such as drugs, poisonous compounds in plants, carcinogens formed during cooking, and environmental pollutants. They represent the first line of defense to detoxify and solubilize poisonous substances by modifying them with dioxygen. The heme iron is proximally coordinated by a thiolate residue, and this ligation state represents the active form of the enzyme. The Fe(III) center displays characteristic UV/Vis and EPR spectra (Soret maximum at 418 nm; g-values at 2.41, 2.26, 1.91). The Fe(II) state binds the inhibitor carbon monoxide (CO) to produce a Fe(II)-CO complex, with the major absorption maximum at 450 nm, hence, its name P450. CYPs are flexible proteins in order to allow a vast range of substrates to enter and products to leave. Two extreme forms exist: substrate-bound (closed) and substrate-free (open). CYPs share a sophisticated catalytic cycle that involves a series of consecutive transformations of the heme thiolate active site, with the strong oxidants compound I and II as key intermediates. Each of these high-valent Fe(IV) species has its characteristic features and chemical properties, crucial for the activation of dioxygen and cleavage of strong C-H bonds.

Determination of the Intrinsic Gasification Kinetics of a Bituminous Coal Including Product Gas Inhibition and Char Deactivation Under Entrained Flow Conditions

Abstract This work provides experimentally obtained data concerning the gasification of bituminous coal under entrained-flow conditions. The main focus lies on the determination of reaction kinetics with and without product gas inhibition as well as on thermal deactivation behavior. For this reason, experiments were carried out in a pressurized high-temperature entrained-flow reactor. The reactor is designed for temperatures of up to 1800 °C and pressures of up to 50 bar. In this study, char samples with different residence times at temperatures between 1200 °C and 1600 °C and a pressure of 10 bar were obtained. Pyrolysis experiments were performed in pure nitrogen, while an O/C ratio of one was selected for the gasification. In addition to ultimate, proximate, and structural analyses of the char samples, e.g., to calculate conversion according to the ash tracer method, intrinsic reaction kinetics of the pyrolysis chars with carbon dioxide and steam were determined in a high-pressure thermogravimetric analyzer. The influence of carbon monoxide inhibition on carbon dioxide gasification and of hydrogen on steam gasification was quantified using the Langmuir–Hinshelwood equation. Further, the deactivation behavior of the pyrolysis chars was analyzed by measuring their reactivities under constant reaction conditions and plotting them as a function of residence time. The presented results give an overview about factors like temperature, pressure, gas composition, and residence time affecting fuel conversion. Furthermore, constants describing the reaction behavior of the fuel were determined, which can be used for future simulation of gasification processes.

In the respiratory chain of Escherichia coli cytochromes bd-I and bd-II are more sensitive to carbon monoxide inhibition than cytochrome bo3

Bacteria can not only encounter carbon monoxide (CO) in their habitats but also produce the gas endogenously. Bacterial respiratory oxidases, thus, represent possible targets for CO. Accordingly, host macrophages were proposed to produce CO and release it into the surrounding microenvironment to sense viable bacteria through a mechanism that in Escherichia (E.) coli was suggested to involve the targeting of a bd-type respiratory oxidase by CO. The aerobic respiratory chain of E. coli possesses three terminal quinol:O2-oxidoreductases: the heme-copper oxidase bo3 and two copper-lacking bd-type oxidases, bd-I and bd-II. Heme-copper and bd-type oxidases differ in the mechanism and efficiency of proton motive force generation and in resistance to oxidative and nitrosative stress, cyanide and hydrogen sulfide. Here, we investigated at varied O2 concentrations the effect of CO gas on the O2 reductase activity of the purified cytochromes bo3, bd-I and bd-II of E. coli. We found that CO, in competition with O2, reversibly inhibits the three enzymes. The inhibition constants Ki for the bo3, bd-I and bd-II oxidases are 2.4 ± 0.3, 0.04 ± 0.01 and 0.2 ± 0.1 μM CO, respectively. Thus, in E. coli, bd-type oxidases are more sensitive to CO inhibition than the heme-copper cytochrome bo3. The possible physiological consequences of this finding are discussed.

Geometry of the Catalytic Active Site in [FeFe]-Hydrogenase Is Determined by Hydrogen Bonding and Proton Transfer

[FeFe]-hydrogenases are efficient metalloenzymes that catalyze the oxidation and evolution of molecular hydrogen, H2. They serve as a blueprint for the design of synthetic H2-forming catalysts. [FeFe]-hydrogenases harbor a six-iron cofactor that comprises a [4Fe-4S] cluster and a unique diiron site with cyanide, carbonyl, and hydride ligands. To address the ligand dynamics in catalytic turnover and upon carbon monoxide (CO) inhibition, we replaced the native aminodithiolate group of the diiron site by synthetic dithiolates, inserted into wild-type and amino acid variants of the [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. The reactivity with H2 and CO was characterized using in situ and transient infrared spectroscopy, protein crystallography, quantum chemical calculations, and kinetic simulations. All cofactor variants adopted characteristic populations of reduced species in the presence of H2 and showed significant changes in CO inhibition and reactivation kinetics. Differences were attribut...

The kallikrein-like activity of Heloderma venom is inhibited by carbon monoxide.

Lizards in the genus Heloderma are the most ancient venomous reptiles, with a traceable lineage nearly 100 million years old. The proteome of the venom of three of the remaining species (Heloderma suspectum, H. exasperatum, H. horridum) are very conserved, with kallikrein-like activity present to cause critical hypotension to immobilize and outright kill prey. Kallikrein-like activity would be expected to activate the contact protein pathway of coagulation, which would be detectable with thrombelastography in human plasma. Thus, it was proposed to determine if kallikrein-like activity could be detected with thrombelastography, and if this activity could be inhibited by carbon monoxide (CO) via a putative heme-based mechanism. Procoagulant activity of each venom was assessed via thrombelastography with normal plasma, and kallikrein-like activity confirmed with FX-depleted plasma. Venom was then exposed to carbon monoxide releasing molecule-2 (CORM-2) or its inactive releasing molecule to assess CO inhibition. All three venoms demonstrated kallikrein-like activity with the same potency and inhibition of activity by CO. In conclusion, the present work documented that procoagulant, kallikrein-like activity containing venoms of the oldest species of venomous reptiles was inhibited by CO, potentially via heme modulation. This is also the first identification and characterization of a kallikrein-like enzyme utilizing coagulation factor-depleted plasma to assess venom that inflicts hypotension. Future investigations will continue to define the vulnerability of venom enzymatic activities to CO.

Effects of Heme Modulation on Ovophis and Trimeresurus Venom Activity in Human Plasma

Geographic isolation and other factors result in evolution-driven diversity of the enzymatic composition of venom of pit vipers in the same genus. The present investigation sought to characterize venoms obtained from such genetically diverse Ovophis and Trimeresurus pit vipers utilizing thrombelastographic coagulation kinetic analyses. The coagulation kinetics of human plasma were assessed after exposure to venom obtained from two Ovophis and three Trimeresurus species. The potency of each venom was defined (µg/mL required to equivalently change coagulation); additionally, venoms were exposed to carbon monoxide (CO) or a metheme-inducing agent to modulate any enzyme-associated heme. All venoms had fibrinogenolytic activity, with four being CO-inhibitable. While Ovophis venoms had similar potency, one demonstrated the presence of a thrombin-like activity, whereas the other demonstrated a thrombin-generating activity. There was a 10-fold difference in potency and 10-fold different vulnerability to CO inhibition between the Trimeresurus species. Metheme formation enhanced fibrinogenolytic-like activity in both Ovophis species venoms, whereas the three Trimeresurus species venoms had fibrinogenolytic-like activity enhanced, inhibited, or not changed. This novel “venom kinetomic” approach has potential to identify clinically relevant enzymatic activity and assess efficacy of antivenoms between genetically and geographically diverse species.

Spectroscopical Investigations on the Redox Chemistry of [FeFe]-Hydrogenases in the Presence of Carbon Monoxide.

[FeFe]-hydrogenases efficiently catalyzes hydrogen conversion at a unique [4Fe–4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Fe–4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CN−) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the Fe–Fe bridging position (µCO). In the presence of exogenous CO, A fourth CO ligand binds at the diiron site to form the oxidized, CO-inhibited H-cluster (Hox-CO). We investigated the reduced, CO-inhibited H-cluster (Hred´-CO) in this work. The stretching vibrations of the diatomic ligands were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). Density functional theory (DFT) at the TPSSh/TZVP level was employed to analyze the cofactor geometry, as well as the redox and protonation state of the H-cluster. Selective 13CO isotope editing, spectro-electrochemistry, and correlation analysis of IR data identified a one-electron reduced, protonated [4Fe–4S] cluster and an apical CN− ligand at the diiron site in Hred´-CO. The reduced, CO-inhibited H-cluster forms independently of the sequence of CO binding and cofactor reduction, which implies that the ligand rearrangement at the diiron site upon CO inhibition is independent of the redox and protonation state of the [4Fe–4S] cluster. The relation of coordination dynamics to cofactor redox and protonation changes in hydrogen conversion catalysis and inhibition is discussed.

P-376 - Crosstalk between gasotransmitters at the H2S-synthesizing human cystathionine β-synthase: heme-based regulation by CO and NO

Cystathionine β-synthase (CBS) plays a central role in hydrogen sulfide (H2S) metabolism in human (patho) physiology. Each CBS monomer is composed of a central catalytic domain containing a pyridoxal 5’-phosphate active site flanked by two regulatory domains: an N-terminal domain harboring a b-type heme, and a C-terminal S-adenosyl-L-methionine (AdoMet)-binding domain. CBS is intricately regulated at different levels, its function depending on the redox state and availability of exogenous ligands, such as AdoMet and the gasotransmitters nitric oxide (NO) and carbon monoxide (CO). Herein, we employed static and stopped-flow absorption spectroscopy to investigate the kinetics of CO and NO binding to the CBS heme. NO binds faster and with higher affinity than CO, each gasotransmitter initially binding to opposite sides of the heme plane. AdoMet surprisingly enhances CO inhibition of H2S production, as well as CO and NO binding. In the clinically relevant p.P49L CBS variant, we observed much higher CO affinity with respect to WT CBS, likely resulting from increased flexibility of the heme surroundings. Overall, the binding kinetics attest the in vivo relevance of CO and NO inhibition of CBS-catalyzed H2S production, an underlying mechanism in the crosstalk between gasotransmitters.

Reactive oxygen radicals and gaseous transmitters in carotid body activation by intermittent hypoxia.

Sleep apnea is a prevalent respiratory disease characterized by periodic cessation of breathing during sleep causing intermittent hypoxia (IH). Sleep apnea patients and rodents exposed to IH exhibit elevated sympathetic nerve activity and hypertension. A heightened carotid body (CB) chemoreflex has been implicated in causing autonomic abnormalities in IH-treated rodents and in sleep apnea patients. The purpose of this article is to review the emerging evidence showing that interactions between reactive oxygen species (ROS) and gaseous transmitters as a mechanism cause hyperactive CB by IH. Rodents treated with IH exhibit markedly elevated ROS in the CB, which is due to transcriptional upregulation of pro-oxidant enzymes by hypoxia-inducible factor (HIF)-1 and insufficient transcriptional regulation of anti-oxidant enzymes by HIF-2. ROS, in turn, increases cystathionine γ-lyase (CSE)-dependent H2S production in the CB. Blockade of H2S synthesis prevents IH-evoked CB activation. However, the effects of ROS on H2S production are not due to direct effects on CSE enzyme activity but rather due to inactivation of heme oxygenase-2 (HO-2), a carbon monoxide (CO) producing enzyme. CO inhibits H2S production through inactivation of CSE by PKG-dependent phosphorylation. During IH, reduced CO production resulting from inactivation of HO-2 by ROS releases the inhibition of CO on CSE thereby increasing H2S. Inhibiting H2S synthesis prevented IH-evoked sympathetic activation and hypertension.

Multiple mechanisms mediating carbon monoxide inhibition of the voltage-gated K+ channel Kv1.5

The voltage-gated K+ channel has key roles in the vasculature and in atrial excitability and contributes to apoptosis in various tissues. In this study, we have explored its regulation by carbon monoxide (CO), a product of the cytoprotective heme oxygenase enzymes, and a recognized toxin. CO inhibited recombinant Kv1.5 expressed in HEK293 cells in a concentration-dependent manner that involved multiple signalling pathways. CO inhibition was partially reversed by superoxide dismutase mimetics and by suppression of mitochondrial reactive oxygen species. CO also elevated intracellular nitric oxide (NO) levels. Prevention of NO formation also partially reversed CO inhibition of Kv1.5, as did inhibition of soluble guanylyl cyclase. CO also elevated intracellular peroxynitrite levels, and a peroxynitrite scavenger markedly attenuated the ability of CO to inhibit Kv1.5. CO caused nitrosylation of Kv1.5, an effect that was also observed in C331A and C346A mutant forms of the channel, which had previously been suggested as nitrosylation sites within Kv1.5. Augmentation of Kv1.5 via exposure to hydrogen peroxide was fully reversed by CO. Native Kv1.5 recorded in HL-1 murine atrial cells was also inhibited by CO. Action potentials recorded in HL-1 cells were increased in amplitude and duration by CO, an effect mimicked and occluded by pharmacological inhibition of Kv1.5. Our data indicate that Kv1.5 is a target for modulation by CO via multiple mechanisms. This regulation has important implications for diverse cellular functions, including excitability, contractility and apoptosis.

Crotalus atrox Venom Exposed to Carbon Monoxide Has Decreased Fibrinogenolytic Activity In Vivo in Rabbits.

Envenomation by haemotoxic enzymes remains a significant source of human morbidity and mortality worldwide, with administration of long-acting or multiple doses of antivenom first-line therapy. However, coagulopathy can still occur and recur. Of interest, it has been recently demonstrated that direct, isolated exposure of snake venom enzymes with fibrinogenolytic activity to carbon monoxide (CO) abrogates venom-mediated loss of coagulation in human plasma. These observations of CO inhibition of venom fibrinogenolytic activity were subsequently repeated in rabbit whole blood. This study sought to translate these findings in an in vivo rabbit model of envenomation with fibrinogenolytic Crotalus atrox venom. Sedated rabbits were intravenously administered C. atrox venom (400 μg/kg) pre-exposed to 0 or 1000 μM carbon monoxide-releasing molecule-2 (CORM-2) in vitro. Arterial whole-blood samples demonstrated that compared to pre-envenomation values, the CORM-2-naïve venom significantly prolonged the onset of coagulation, decreased the velocity of clot growth and decreased clot strength as determined by thromboelastography an hour after venom injection. In contrast, CORM-2 pre-exposure prevented or attenuated C. atrox venom effects on coagulation kinetics. Future studies to determine whether rabbits injected with such venom subcutaneously/intramuscularly can have consequent coagulopathy abrogated by injection of carbon monoxide-releasing molecules into the 'bite site' are justified.

Biosynthesis and biotransformation of bile acids

Bile acids are steroidal compounds, which contain 24 carbon atoms. They can be classified into two major groups: primary and secondary. The most abundant bile acids: The primary bile acids include cholic acid and chenodeoxycholic acid, while the major secondary bile acids are deoxycholic acid and litocholic acid. Bile acids are important physiological agents for intestinal absorption of nutrients and are used for biliary lipid secretion, toxic metabolites and xenobiotics. The aim of this paper is to analyze biosynthesis and biotransformation of bile acids, as preparation for practical usage in laboratory and clinical conditions. Topic: Biosynthesis and biotransformation of bile acids: The biosynthesis of bile acids is the dominant metabolic pathway for catabolism of cholesterol in humans. The classical route of biosynthesis of bile acids is embarking on the conversion of cholesterol into 7α-hydroxycholesterol using enzyme 7α-cholesterol hydroxylase (CYP7A1). This enzyme is one of the microsomal cytochrome P450 enzyme is localized exclusively in the liver. Classical road is the main road in the biosynthesis of bile acids, and its total contribution amounts to 90% for people, and 75% in mice. CYP 7A1 enzyme is considered to be sensitive to the inhibition of carbon monoxide, and the condition for the effect of NADPH, the oxygen, lecithin, and the NADPH-cytochrome P450 reductase. Bile acids are important signaling molecules and metabolic controls which activate the nuclear receptor and the G protein-coupled receptors (GPCR), a signaling lipid regulation of the liver, glucose and energy homeostasis. Also, bile acids maintain metabolic homeostasis. Biotransformation of bile acids: The conversion of cholesterol into bile acids just important for maintenance of cholesterol homeostasis, but also to prevent the accumulation of cholesterol, triglycerides and toxic metabolites as well as violations of the liver and other organs. Enterohepatic circulation of bile acids from the liver to the intestine and back to the liver occupies the most important role in the processes of absorption and distribution, as well as in metabolic regulation and homeostasis. Conclusions: This physiological process is complicated and regulates the membrane transport system in the liver and intestine by means of nuclear receptors. It is very dangerous fact that toxic bile acids may be causes of inflammation, apoptosis and cell death. On the other hand activated GPCR signaling and nuclear bile acid protects against inflammation of the liver, intestine and macrophages. Bile acid metabolism disorders cause cholestatic liver disease, dyslipidemia, fatty liver disease, cardiovascular disease and diabetes.

Carbon monoxide: a critical quantitative analysis and review of the extent and limitations of its second messenger function.

Endogenously produced carbon monoxide (CO) is commonly believed to be a ubiquitous second messenger involved in a wide range of physiological and pathological responses. The major evidence supporting this concept is that CO is produced endogenously via heme oxygenase-catalyzed breakdown of heme and that experimental exposure to CO alters tissue function. However, it remains to be conclusively demonstrated that there are specific receptors for CO and that endogenous CO production is sufficient to alter tissue function. Unlike other signaling molecules, CO is not significantly metabolized, and it is removed from cells solely via rapid diffusion into blood, which serves as a near infinite sink. This non-metabolizable nature of CO renders the physiology of this gas uniquely susceptible to quantitative modeling. This review analyzes each of the steps involved in CO signaling: 1) the background CO partial pressure (PCO) and the blood and tissue CO binding; 2) the affinity of the putative CO receptors; 3) the rate of endogenous tissue CO production; and 4) the tissue PCO that results from the balance between this endogenous CO production and diffusion to the blood sink. Because existing data demonstrate that virtually all endogenous CO production results from the routine “housekeeping” turnover of heme, only a small fraction can play a signaling role. The novel aspect of the present report is to demonstrate via physiological modeling that this small fraction of CO production is seemingly insufficient to raise intracellular PCO to the levels required for the conventional, specific messenger receptor activation. It is concluded that the many physiological alterations observed with exogenous CO administration are probably produced by the non-specific CO inhibition of cytochrome C oxidase activity, with release of reactive oxygen species (ROS) and that this ROS signaling pathway is a potential effector mechanism for endogenously produced CO.