Nitric Oxide Adduct Research Articles

Uncovering Nitrosyl Reactivity at N-Heterocyclic Carbene Center.

N-heterocyclic carbenes (NHCs) have garnered much attention due to their unique properties, such as strong σ-donating and π-accepting abilities, as well as their transition-metal-like reactivity toward small molecules. In 2015, we discovered that NHCs can react with nitric oxide (NO) gas to form radical adducts that resemble transition metal nitrosyl complexes. To elucidate the analogy between NHC and transition metal NO adducts, here we have undertaken a systematic investigation of the electron- and proton-transfer chemistry of [NHC-NO]• (N-heterocyclic carbene nitric oxide radical) compounds. We have accessed a suite of compounds, comprised of [NHC-NO]+, [NHC-NO]-, [NHC-NOH]0, and [NHC-NHOH]+ species. In particular, [NHC-NO]- was isolated as potassium and lithium ion adducts. Most interestingly, a monomeric potassium [NHC-NO]- compound was isolated with the assistance of 18-crown-6, which is the first instance of a monomeric alkali N-oxyl compound to the best of our knowledge. Our results demonstrate that [NHC-NO]• exhibits redox behavior broadly similar to metal nitrosyl complexes, which opens up more possibilities for utilizing NHCs to build on the known reactivity of metal complexes.

Protection of Pacific white shrimp (Penaeus vannamei) against white spot syndrome virus infection by nitric oxide-generating compound S-nitrosoglutathione

Nitric oxide (NO), a highly reactive free radical generated endogenously by nitric oxide synthase (NOS), plays crucial roles in immune defense against a broad range of pathogens including viruses, bacteria, fungi, and protozoans. However, the regulation and effect of NO during viral infection in invertebrates remain largely unexplored. White spot syndrome virus (WSSV) is one of the most severe pathogens affecting the global shrimp farming industry. Our study shows that NO production in Pacific white shrimp Penaeus vannamei decreases during WSSV infection. Reducing NO production using the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly promotes WSSV replication in primary culture of hemocytes isolated from P. vannamei. This effect is partically reversed by L-arginine, a native substrate for NOS, indicating that L-NAME's immunity-reducing action is NO-dependent. In vitro studies also reveal inhibition of WSSV replication in the presence of three NO donors: S-nitrosoglutathione (GSNO), S-nitro-N-acetylpenicillamine (SNAP), and diethylenetriamine nitric oxide adduct (DETA/NO). The involvement of NO in anti-WSSV protection is further demonstrated by an in vivo study where dietary supplementation with GSNO at non-toxic doses increases NO levels and reduces viral load and mortality in WSSV-challenged shrimp. Our results contribute to understanding the antiviral activity of NO in invertebrates and provide a theoretical basis for developing NO-based therapeutic strategies to control WSSV infection.

A physiologically relevant role for NO stored in vascular smooth muscle cells: A novel theory of vascular NO signaling

S-nitrosothiols (SNO), dinitrosyl iron complexes (DNIC), and nitroglycerine (NTG) dilate vessels via activation of soluble guanylyl cyclase (sGC) in vascular smooth muscle cells. Although these compounds are often considered to be nitric oxide (NO) donors, attempts to ascribe their vasodilatory activity to NO-donating properties have failed. Even more puzzling, many of these compounds have vasodilatory potency comparable to or even greater than that of NO itself, despite low membrane permeability. This raises the question: How do these NO adducts activate cytosolic sGC when their NO moiety is still outside the cell? In this review, we classify these compounds as ‘nitrodilators’, defined by their potent NO-mimetic vasoactivities despite not releasing requisite amounts of free NO. We propose that nitrodilators activate sGC via a preformed nitrodilator-activated NO store (NANOS) found within the vascular smooth muscle cell. We reinterpret vascular NO handling in the framework of this NANOS paradigm, and describe the knowledge gaps and perspectives of this novel model.

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells.

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.

S-nitrosothiols signaling in cystic fibrosis airways

S-nitrosothiols (SNOs) are small naturally occurring thiol and nitric oxide adducts that participate in many cell signaling pathways in living organisms. SNOs receive widespread attention in cell biology, biochemistry and chemistry because they can donate nitric oxide and/or nitrosonium ions in S-nitrosylation reactions, which are comparable to phosphorylation, acetylation, glutathionylation, and palmitoylation reactions. SNOs have advantageous effects in respiratory diseases and other systems in the body. S-nitrosylation signaling is a metabolically regulated physiological process that leads to specific post-translational protein modifications. S-nitrosylation signaling is faulty in cystic fibrosis (CF) and many other lung diseases. CF is an inherited, lethal autosomal recessive multisystem disease resulting from mutations in the gene encoding the CF transmembrane conductance regulatory (CFTR) protein. F508del CFTR is the most common mutation associated with CF, which results in CFTR misfolding because a phenylalanine is deleted from the primary structure of CFTR. The majority of wild-type CFTR and almost all F508del is degraded before reaching the cell surface. Ultimately, CF researchers have been looking to correct the mutated CFTR protein in the CF patients. Remarkably, researchers have found that SNOs levels are low in the CF lower airway compared to non-CF patients. We have been interested in determining whether SNOs increase CFTR maturation through S-nitrosylation. Maturation of both wild type and mutant F508del CFTR increases SNOs, which up-regulate CFTR maturation. In this review, we summarized our current knowledge of S-nitrosothiols signaling in cystic fibrosis airways.

4′-Fluoropyrrolidinononanophenone elicits neuronal cell apoptosis through elevating production of reactive oxygen and nitrogen species

α-Pyrrolidinononanophenone (α-PNP) is a highly lipophilic synthetic cathinone (SC) that possesses a long hydrocarbon chain. It is considered that abuse of α-PNP and its derivatives causes serious health hazards, but there has been little evidence to date on the toxicological, pharmacological and pharmacokinetic properties. The purpose of this study was to elucidate the structure–toxicity relationship and the mechanism of neurocytotoxicity of α-PNP derivatives. We synthesized three α-PNP derivatives [4′-fluoro, 4′-methoxy and 3′,4′-methylenedioxy substituents on the aromatic ring] and examined their toxicities against four human cells. The mechanism of 4′-fluoro-α-PNP (F-α-PNP)-induced neurocytotoxicity was investigated in terms of productions of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Cytotoxicites of α-PNP derivatives were augmented by the presence of 4′-fluoro or 3′,4′-methylenedioxy group on α-PNP, and F-α-PNP exhibited the most potent cytotoxicity. The F-α-PNP treatment resulted in ROS production, cytochrome-c release into cytosol, caspase-9 and caspase-3 activation and DNA fragmentation in neuronal SK-N-SH cells. In addition, subcellular fractionation analysis demonstrated that F-α-PNP is localized in the mitochondria as early as 12 h after the incubation. α-PNP derivatives increased level of nitric oxide (NO) and 3-nitrotyrosine adducts, indicative of peroxynitrite formation, in SK-N-SH cells. Pretreatment with a NO or peroxynitrite donor also exacerbated the cellular toxicity of F-α-PNP. The presence of 4′-fluoro or 3′,4′-methylenedioxy group in the aromatic ring on SCs likely elevates the risk of occurrence of health hazards. Additionally, enhanced production of ROS and RNS plays central roles in neuronal cell apoptotic mechanism of F-α-PNP.

Nitric oxide: To be or not to be an endocrine hormone?

Nitric oxide (NO), a highly reactive gasotransmitter, is critical for a number of cellular processes and has multiple biological functions. Due to its limited lifetime and diffusion distance, NO has been mainly believed to act in autocrine/paracrine fashion. The increasingly recognized effects of pharmacologically delivered and endogenous NO at a distant site have changed the conventional wisdom and introduced NO as an endocrine signalling molecule. The notion is greatly supported by the detection of a number of NO adducts and their circulatory cycles, which in turn contribute to the transport and delivery of NO bioactivity, remotefrom the sites ofits synthesis. The existence of endocrine sites of synthesis, negative feedback regulation of biosynthesis, integrated storage and transport systems, having an exclusive receptor, that is, soluble guanylyl cyclase (sGC), and organized circadian rhythmicity make NO something beyond a simple autocrine/paracrine signalling molecule that could qualify for being an endocrine signalling molecule. Here, we discuss hormonal features of NO from the classical endocrine point of view and review available knowledge supporting NO as a true endocrine hormone. This new insight can provide a new framework within which to reinterpret NO biology and its clinical applications.

Nitric oxide loading reduces sickle red cell adhesion and vaso-occlusion in vivo.

Sickle red blood cells (SSRBCs) are adherent to the endothelium, activate leukocyte adhesion, and are deficient in bioactive nitric oxide (NO) adducts such as S-nitrosothiols (SNOs), with reduced ability to induce vasodilation in response to hypoxia. All these pathophysiologic characteristics promote vascular occlusion, the hallmark of sickle cell disease (SCD). Loading hypoxic SSRBCs in vitro with NO followed by reoxygenation significantly decreased epinephrine-activated SSRBC adhesion to the endothelium, the ability of activated SSRBCs to mediate leukocyte adhesion in vitro, and vessel obstruction in vivo. Because transfusion is frequently used in SCD, we also determined the effects of banked (SNO-depleted) red blood cells (RBCs) on vaso-occlusion in vivo. Fresh or 14-day-old normal RBCs (AARBCs) reduced epinephrine-activated SSRBC adhesion to the vascular endothelium and prevented vaso-occlusion. In contrast, AARBCs stored for 30 days failed to decrease activated SSRBC adhesivity or vaso-occlusion, unless these RBCs were loaded with NO. Furthermore, NO loading of SSRBCs increased S-nitrosohemoglobin and modulated epinephrine's effect by upregulating phosphorylation of membrane proteins, including pyruvate kinase, E3 ubiquitin ligase, and the cytoskeletal protein 4.1. Thus, abnormal SSRBC NO/SNO content both contributes to the vaso-occlusive pathophysiology of SCD, potentially by affecting at least protein phosphorylation, and is potentially amenable to correction by (S)NO repletion or by RBC transfusion.

Upregulation of the nitrosylome in bipolar disorder type 1 (BP1) and major depression, but not BP2: Increased IgM antibodies to nitrosylated conjugates are associated with indicants of leaky gut

ObjectiveMajor depression (MDD) and a lifetime history of MDD are characterized by increased nitrosylation, while bipolar disorder type 1 (BP1), but not BP2, is accompanied by highly increased levels of oxidative stress and nitric oxide (NO) production. Nevertheless, it is unknown whether nitrosylation is involved in BP and whether there are differences in nitrosylation between BP1 and BP2. MethodsSerum IgM antibodies directed against nitroso (NO)-adducts were examined in MDD, BP1, BP2 and healthy controls, namely IgM responses to NO-cysteine, NO-tryptophan (NOW), NO-arginine and NO-albumin (SBA) in association with IgA/IgM responses to LPS of Gram-negative bacteria, IgG responses to oxidized low-density lipoprotein (ox-LDL) and serum peroxides. ResultsSerum IgM levels against NO adducts were significantly higher in BP1 and MDD as compared with healthy controls, whereas BP2 patients occupied an intermediate position. IgM responses to NO-albumin were significantly higher in BP1 and MDD than in BP2 patients. There were highly significant associations between the IgM responses to NO-adducts and IgG responses to ox-LDL and IgA/IgM responses to Gram-negative bacteria. ConclusionsBP1 and MDD are characterized by an upregulation of the nitrosylome (the proteome of nitrosylated proteins) and increased IgM responses to nitrosylated conjugates. Increased nitrosylation may be driven by increased bacterial translocation and is associated with lipid peroxidation processes. Innate-like (B1 and marginal zone) B cells and increased nitrosylation may play a key role in the major affective disorders through activation of immune-inflammatory and oxidative pathways, cardiovascular comorbidity and impairments in antioxidant defenses, neuro-glial interactions, synaptic plasticity, neuroprotection, neurogenesis.

The Thiolate Trans Effect in Heme {FeNO}6 Complexes and Beyond: Insight into the Nature of the Push Effect.

Cyt P450 nitric oxide (NO) reductase (P450nor) is an important enzyme in fungal denitrification, responsible for the large-scale production of the greenhouse gas N2O. In the first step of catalysis, the ferric heme-thiolate active site of P450nor binds NO to produce a ferric heme-nitrosyl or {FeNO}6 intermediate (in the Enemark-Feltham notation). In this paper, we present the low-temperature preparation of six new heme-thiolate {FeNO}6 model complexes, [Fe(TPP)(SPh*)(NO)], using a unique series of electron-poor thiophenolates (SPh*-), and their detailed spectroscopic characterization. Our data show experimentally, for the first time, that a direct correlation exists between the thiolate donor strength and the Fe-NO and N-O bond strengths, evident from the corresponding stretching frequencies. This is due to a σ-trans effect of the thiolate ligand, which manifests itself in the population of an Fe-N-O σ-antibonding (σ*) orbital. Via control of the thiolate donor strength (using hydrogen bonds), nature is therefore able to exactly control the degree of activation of the FeNO unit in P450nor. Vice versa, NO can be used as a sensitive probe to quantify the donor strength of a thiolate ligand in a model system or protein, by simply measuring the Fe-NO and N-O frequencies of the ferric NO adduct and then projecting those data onto the correlation plot established here. Finally, we are able to show that the σ-trans effect of the thiolate is the electronic origin of the "push" effect, which is proposed to mediate O-O bond cleavage and Compound I formation in Cyt P450 monooxygenase catalysis.

Synthetic Iron Porphyrins for Probing the Differences in the Electronic Structures of Heme a3, Heme d, and Heme d1.

A variety of heme derivatives are pervasive in nature, having different architectures that are complementary to their function. Herein, we report the synthesis of a series of iron porphyrinoids, which bear electron-withdrawing groups and/or are saturated at the β-pyrrolic position, mimicking the structural variation of naturally occurring hemes. The effects of the aforementioned factors were systematically studied using a combination of electrochemistry, spectroscopy, and theoretical calculations with the carbon monoxide (CO) and nitric oxide (NO) adducts of these iron porphyinoids. The reduction potentials of iron porphyrinoids vary over several hundreds of millivolts, and the X-O (X = C, N) vibrations of the adducts vary over 10-15 cm-1. Density functional theory calculations indicate that the presence of electron-withdrawing groups and saturation of the pyrrole ring lowers the π*-acceptor orbital energies of the macrocycle, which, in turn, attenuates the bonding of iron to CO and NO. A hypothesis has been presented as to why cytochrome c containing nitrite reductases and cytochrome cd1 containing nitrite reductases follow different mechanistic pathways of nitrite reduction. This study also helps to rationalize the choice of heme a3 and not the most abundant heme b cofactor in cytochrome c oxidase.

Spectral Characterization of a Novel NO Sensing Protein in Bacteria: NosP.

A novel family of bacterial hemoproteins named NosP has been discovered recently; its members are proposed to function as nitric oxide (NO) responsive proteins involved in bacterial group behaviors such as quorum sensing and biofilm growth and dispersal. Currently, little is known about molecular activation mechanisms in NosP. Here, functional studies were performed utilizing the distinct spectroscopic characteristics associated with the NosP heme cofactor. NosPs from Pseudomonas aeruginosa ( Pa), Vibrio cholerae ( Vc), and Legionella pneumophila ( Lpg) were studied in their ferrous unligated forms as well as their ferrous CO, ferrous NO, and ferric CN adducts. The resonance Raman (rR) data collected on the ferric forms strongly support the existence of a distorted heme cofactor, which is a common feature in NO sensors. The ferrous spectra exhibit a 213 cm-1 feature, which is assigned to the Fe-Nhis stretching mode. The Fe-C and C-O frequencies in the spectra of ferrous CO NosP complexes are inversely correlated with relatively similar frequencies, consistent with a proximal histidine ligand and a relatively hydrophobic environment. The rR spectra obtained for isotopically labeled ferrous NO adducts provide evidence of formation of a 5-coordinate NO complex, resulting from proximal Fe-Nhis cleavage, which is believed to play a role in biological heme-NO signal transduction. Additionally, we found that of the three NosPs studied, Lpg NosP contains the most electropositive ligand binding pocket, while Pa NosP has the most electronegative ligand binding pocket. This pattern is also observed in the measured heme reduction potentials for these three proteins, which may indicate distinct functions for each.

Effect of Hyperglycemia on Purinergic and Nitrergic Inhibitory Neuromuscular Transmission in the Antrum of the Stomach: Implications for Fast Gastric Emptying.

Hyperglycemia has been reported to enhance vagovagal reflex that causes the release of inhibitory neurotransmitter, nitric oxide (NO), at the neuromuscular junction in the antrum to relax the antrum and slow gastric emptying by stimulating glucose-sensitive afferent neurons. However, hyperglycemia has also been reported to cause fast gastric emptying that may be due to suppression of the inhibitory motor neurons. The purpose of the present study was to investigate changes in inhibitory neuromuscular transmission in the gastric antrum due to hyperglycemia. Inhibitory electrical junction potentials were recorded from gastric antral muscle strips, using intracellular electrodes under non-adrenergic, non-cholinergic conditions. Studies were performed in non-hyperglycemic NOD (NH-NOD), NOD mice as they develop hyperglycemia (H-NOD) and their age-matched controls. The purinergic inhibitory junction potential (pIJP) and nitrergic IJP (nIJP) were isolated pharmacologically. The control pIJP was large, around -18 mV and nIJP was small, around -9 mV. In NH-NOD the IJPs were not affected, but in H-NOD pIJP was nearly abolished and nIJP was significantly reduced. In H-NOD mice, membrane hyperpolarization caused by exogenous α,β-MeATP or diethylenetriamine NO adduct was similar to that in wild-type controls (P > 0.05). H-NOD smooth muscles were significantly depolarized as compared to NH-NOD smooth muscles. These observations show that hyperglycemia causes suppression of purinergic and nitrergic transmission by acting on the motor neurons that form the last neuron in the vagovagal circuit. Moreover, the loss the neurotransmission is due to a defect in neurotransmitter release rather than a defect in signal transduction. Hyperglycemia also causes depolarization of smooth muscles that may increase their excitability.

Exogenous nitric oxide enhances the prophylactic effect of aminoguanidine, a preferred iNOS inhibitor, on bleomycin-induced fibrosis in the lung: Implications for the direct roles of the NO molecule in vivo

ObjectiveInducible nitric oxide synthase (iNOS) aggravates and endothelial nitric oxide synthase (eNOS) ameliorates fibrosis in the lung. Our previous study demonstrated that aminoguanidine (AG), a preferred iNOS inhibitor, prevents bleomycin-induced injury and fibrosis in the lung. The diethylenetriamine nitric oxide adduct (DETA/NO) is a slow-release NO donor. Here, to clarify the exact role of the nitric oxide (NO) molecule in the pathogenesis of pulmonary fibrosis in vivo, we observed the effects of inhalation of aerosolized DETA/NO on fibrosis in the lungs of bleomycin-exposed rats with AG treatment, including the effects on the myofibroblast number, collagen deposition, peroxynitrite anion (ONOO−) formation, and injury in the lung. Design and methodsRats received a single intratracheal instillation of bleomycin or normal saline (NS) on day 0, followed by a daily intraperitoneal injection of AG or NS from day 1 to day 13. Each group was additionally given a daily inhalation of DETA/NO or placebo from day 1 to day 13. On day 14, half of the rats in each group was euthanized, and plasma nitrite and nitrate (NOx), myofibroblasts, type I collagen, ONOO− and injury in the lung were estimated by the Griess reaction, western blotting, immunohistochemical staining, sirius red staining, and hematoxylin and eosin (HE) staining, respectively. On day 28, the other half of the rats in each group was euthanized, and the total collagen of the lung was evaluated by hydroxyproline assay. Results① At the day 14 time point, AG reduced the plasma NOx level in bleomycin rats, while this drug had no significant effect on sham rats. Inhalation of aerosolized DETA/NO increased the plasma NOx level of bleomycin + AG rats, sham rats and sham + AG rats. However, due to large areas of airspace obliteration in the lungs of bleomycin rats, DETA/NO inhalation had no significant effect on the plasma NOx level in these rats. ② At the day 14 time point, AG reduced ONOO− formation (marked by nitrotyrosine, NT), injury, myofibroblast number, and type I collagen deposition in the lungs of bleomycin rats, while this drug had no significant impact on the above parameters in the lungs of sham rats. Interestingly, DETA/NO inhalation enhanced the preventive effects afforded by AG on myofibroblast number and type I collagen deposition, but had no significant impact on ONOO− and injury in lung. ③ At the day 28 time point, because rats were not exposed to DETA/NO after day 13, there was no significant difference of the plasma NOx level in sham rats, sham + AG rats, bleomycin rats, and bleomycin + AG rats between DETA/NO inhalation and placebo inhalation. Interestingly, rats administered both DETA/NO and AG still showed a reduction in total collagen of the entire lung compared to rats administered AG alone at this time point. ConclusionsExogenous NO enhances the prophylactic effect afforded by AG on the myofibroblast number and collagen deposition in the lungs of bleomycin-treated rats in vivo. These results suggest that NO has a direct antifibrotic effect in lungs, except for the formation of ONOO− in the development of pulmonary fibrosis in vivo.

The Medicinal Chemistry of Nitrite as a Source of Nitric Oxide Signaling.

Conventional understanding of nitric oxide (NO) signaling in biology is commonly based on the premise that it simply diffuses randomly from its site of production by NO synthases to its site of action or inactivation. This notion has been challenged on a systemic cardiovascular scale with the realization that NO has endocrine effects despite being unable to exist in blood for more than a few milliseconds. Investigation of this phenomenon has led to the understanding that many of the chemical pathways that consume NO may not render it inactive as once thought. Instead, many of NO's metabolic products are still capable of carrying out NO signaling, or participate in NO-independent signaling in their own right. Nitrite and nitrate are two such products of NO metabolism that were once thought to be inert at physiological concentrations but are now known to contribute to NO bioactivity. The activity of nitrate is dependent upon its reduction to nitrite by bacterial nitrate reductase activity in the mouth. Nitrite can be reduced to NO by several metal-containing proteins under hypoxic conditions, or by nonenzymatic reactions under acidic conditions. Reduction and oxidation products of nitrite metabolism may also result in the production of NO adducts with a wide array of biological functions. The following review provides a general overview of the basic pathways underlying the physiological activity of nitrate and nitrite, as well as insight into the therapeutic potential of these pathways.

Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function

Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (although smaller values of nH are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role, safeguarding DNA from DNDGIC, display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs. Beside the well known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity.

Differentiation of Mouse Embryonic Stem Cells toward Functional Pancreatic β-Cell Surrogates through Epigenetic Regulation of Pdx1 by Nitric Oxide.

Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation toward β cells. Previously, we have shown that exposure of mouse embryonic stem cells (mESCs) to high concentrations of diethylenetriamine nitric oxide adduct (DETA-NO) triggers differentiation events and promotes the expression of Pdx1. Here we report evidence that Pdx1 expression is associated with release of polycomb repressive complex 2 (PRC2) and P300 from its promoter region. These events are accompanied by epigenetic changes in bivalent markers of histones trimethylated histone H3 lysine 27 (H3K27me3) and H3K4me3, site-specific changes in DNA methylation, and no change in H3 acetylation. On the basis of these findings, we developed a protocol to differentiate mESCs toward insulin-producing cells consisting of sequential exposure to DETA-NO, valproic acid, and P300 inhibitor (C646) to enhance Pdx1 expression and a final maturation step of culture in suspension to form cell aggregates. This small molecule-based protocol succeeds in obtaining cells that express pancreatic β-cell markers such as PDX1, INS1, GCK, and GLUT2 and respond in vitro to high glucose and KCl.

Preconditioning c-Kit-positive Human Cardiac Stem Cells with a Nitric Oxide Donor Enhances Cell Survival through Activation of Survival Signaling Pathways

Cardiac stem cell therapy has shown very promising potential to repair the infarcted heart but is severely limited by the poor survival of donor cells. Nitric oxide (NO) has demonstrated cytoprotective properties in various cells, but its benefits are unknown specifically for human cardiac stem cells (hCSCs). Therefore, we investigated whether pretreatment of hCSCs with a widely used NO donor, diethylenetriamine nitric oxide adduct (DETA-NO), promotes cell survival. Results from lactate dehydrogenase release assays showed a dose- and time-dependent attenuation of cell death induced by oxidative stress after DETA-NO preconditioning; this cytoprotective effect was abolished by the NO scavenger. Concomitant up-regulation of several cell signaling molecules after DETA-NO preconditioning was observed by Western blotting, including elevated phosphorylation of NRF2, NFκB, STAT3, ERK, and AKT, as well as increased protein expression of HO-1 and COX2. Furthermore, pharmaceutical inhibition of ERK, STAT3, and NFκB activities significantly diminished NO-induced cytoprotection against oxidative stress, whereas inhibition of AKT or knockdown of NRF2 only produced a minor effect. Blocking PI3K activity or knocking down COX2 expression did not alter the protective effect of DETA-NO on cell survival. The crucial roles of STAT3 and NFκB in NO-mediated signaling pathways were further confirmed by stable expression of gene-specific shRNAs in hCSCs. Thus, preconditioning hCSCs with DETA-NO promotes cell survival and resistance to oxidative stress by activating multiple cell survival signaling pathways. These results will potentially provide a simple and effective strategy to enhance survival of hCSCs after transplantation and increase their efficacy in repairing infarcted myocardium.

Supplementation of host response by targeting nitric oxide to the macrophage cytosol is efficacious in the hamster model of visceral leishmaniasis and adds to efficacy of amphotericin B

We investigated efficacy of nitric oxide (NO) against Leishmania donovani. NO is a mediator of host response to infection, with direct parasiticidal activity in addition to its role in signalling to evoke innate macrophage responses. However, it is short-lived and volatile, and is therefore difficult to introduce into infected cells and maintain inracellular concentrations for meaningful periods of time. We incorporated diethylenetriamine NO adduct (DETA/NO), a prodrug, into poly(lactide-co-glycolide) particles of ∼200 nm, with or without amphotericin B (AMB). These particles sustained NO levels in mouse macrophage culture supernatants, generating an area under curve (AUC0.08-24h) of 591.2 ± 95.1 mM × h. Free DETA/NO resulted in NO peaking at 3 h and declining rapidly to yield an AUC of 462.5 ± 193.4. Particles containing AMB and DETA/NO were able to kill ∼98% of promastigotes and ∼76% of amastigotes in 12 h when tested in vitro. Promastigotes and amastigotes were killed less efficiently by particles containing a single drug– either DETA/NO (∼42%, 35%) or AMB (∼90%, 50%) alone, or by equivalent concentrations of drugs in solution. In a pre-clinical efficacy study of power >0.95 in the hamster model, DETA/NO particles were non-inferior to Fungizone® but not Ambisome®, resulting in significant (∼73%) reduction in spleen parasites in 7 days. Particles containing both DETA/NO and AMB were superior (∼93% reduction) to Ambisome®. We conclude that NO delivered to the cytosol of macrophages infected with Leishmania possesses intrinsic activity and adds significantly to the efficacy of AMB.

INTRALUMINAL NITRIC OXIDE CONCENTRATION FOLLOWING PERIVASCULAR NITRIC OXIDE DONOR: IN VITRO HUMAN VEIN MODEL

Nitric oxide (NO) has vasculoprotective effects against atherosclerosis. Intraluminal delivery of NO to saphenous vein grafts in coronary artery bypass grafting (CABG) may be effective in reducing the restenosis rate following surgery, which complicates a considerable percent of the patients within a year. In this study, we studied transmural diffusion of NO delivered by two NO-releasing agents, trinitroglycerin (TNG) and diethylenetriamine NO adduct (DETA/NO). They were applied perivascularly to saphenous vein segments to investigate whether perivascular application of such agents is capable of increasing the intraluminal concentration of NO. Saphenous vein segments considered of no use during Coronary Artery Bypass Grafting were chosen by the surgeon. Being filled with normal saline, they were suspended in solutions of 250μg/ml TNG in normal saline, 250μg/ml DETA/NO in normal saline, or normal saline alone, and intraluminal concentration of peroxynitrite, a stable metabolite of NO, was measured after 1 and 6 hours of suspension. Analysis revealed that after 1 hour, the intraluminal concentration of this metabolite was significantly higher in the DETA/NO than the TNG groups. After 6 hours, the intraluminal concentrations of peroxynitrite in the TNG group and in the DETA/NO group were significantly higher than that of NaCl alone, and this concentration in the DETA/NO groups was significantly higher than the TNG group. Perivascular application of TNG and DETA/NO are probably capable of delivering NO to the lumen and may lead to the vasculoprotective effects of NO on the venous graft wall.