Intracellular Nitrite Research Articles

Health-Promoting Properties of Pectin-Polyphenol Complex Extracted from Olive Oil By-Product Alperujo: Antioxidant, Antiproliferative, and Anti-Inflammatory Activities.

This research explores the health-promoting properties of the pectin-polyphenol complex extracted from alperujo, a by-product of olive oil production. This study investigates the chemical composition and antioxidant activity of the extracts, revealing their high antioxidant activity in vitro. Cell viability assays conducted on colon carcinoma cells (Caco-2) demonstrate the inhibitory effect of the extracts on cell proliferation. However, the extracts do not affect the viability of differentiated Caco-2 cells, suggesting a selective antiproliferative action. Additionally, the extracts reduce intracellular reactive oxygen species (ROS) and nitrite (NO) production in LPS-stimulated murine peritoneal macrophages. Furthermore, the extracts exhibit anti-inflammatory effects by downregulating the secretion of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in these macrophages. These findings highlight the potential of pectin-polyphenol complexes as functional ingredients with significant health benefits, demonstrating antioxidant, antiproliferative, and anti-inflammatory properties.

Effect of Carbon Source on Endogenous Partial Denitrification Process: Characteristics of Intracellular Carbon Transformation and Nitrite Accumulation

This study focused on the start-up and operating characteristics of the endogenous partial denitrification (EPD) process with different carbon sources. Two sequencing batch reactors (SBRs) with sodium acetate (SBR1#) and glucose (SBR2#) as carbon sources were operated under anaerobic/oxic (A/O) and anaerobic/anoxic/oxic (A/A/O) modes successively for 240 d. The results showed that COD removal efficiency reached 85% and effluent COD concentrations were below 35 mg/L in both SBRs. The difference was that faster absorption and transformation of sodium acetate was achieved compared to glucose (COD removal rate (CRR) was 7.54 > 2.22 mgCOD/(L·min) in SBR1# compared to SBR2#). EPD could be started up with sodium acetate and glucose as carbon sources, respectively, and desirable high nitrite accumulations were both obtained at influent NO3−−N (NO3−-Ninf) increased from 20 to 40 mg/L with nitrate-to-nitrite transformation ratio (NTR) and specific NO3−-N deduction rate (rNa) of 88.4~90% and 2.41~2.38 mgN/(gVSS·h), respectively. However, at NO3−-N of 50~60 mg/L, both the NTR and rNa in SBR1# were higher compared to SBR2# (86.5% > 83.9% and 1.58 > 1.20 mgN/(gVSS·h), respectively). Hereafter, when NO3−-N was increased by 70~90 mg/L, lower NTR and rNa were observed in SBR1# than in SBR2# (72% and 78%, 1.16 and 1.32 mgN/(gVSS·h), respectively). Additionally, similar internal carbon transformations were observed to drive EPD for NO2−−N accumulation, especially for higher and faster carbon transformation with sodium acetate as carbon source compared to glucose. However, precise control of anoxic time as the peak point of nitrite (TNi,max) was still the key to achieve high NO2−−N accumulation.

Nitric oxide augments signaling for contraction in hypoxic pulmonary arterial smooth muscle-Implications for hypoxic pulmonary hypertension.

Introduction: Hypoxic persistent pulmonary hypertension in the newborn (PPHN) is usually treated with oxygen and inhaled nitric oxide (NO), both pulmonary arterial relaxants. But treatment failure with NO occurs in 25% of cases. We previously demonstrated that 72h exposure to hypoxia, modeling PPHN, sensitized pulmonary artery smooth muscle cells (PASMC) to the contractile agonist thromboxane and inhibited relaxant adenylyl cyclase (AC) activity. Methods: In this study, we examined the effects of sodium nitroprusside (SNP), as NO donor, on the thromboxane-mediated contraction and NO-independent relaxation pathways and on reactive oxygen species (ROS) accumulation in PASMC. In addition, we examined the effect of the peroxynitrite scavenger 5,10,15,20-Tetrakis (4-sulfonatophenyl)porphyrinato Iron (III) (FeTPPS) on these processes. Results: Exposure of PASMC to 72h hypoxia increased total intracellular ROS compared to normoxic control cells and this was mitigated by treatment of cells with either SNP or FeTPPS. Total protein nitrosylation was increased in hypoxic PASMC compared to controls. Both normoxic and hypoxic cells treated with SNP exhibited increased total protein nitrosylation and intracellular nitrite; this was reduced by treatment with FeTPPS. While cell viability and mitochondrial number were unchanged by hypoxia, mitochondrial activity was decreased compared to controls; addition of FeTPPS did not alter this. Basal and maximal mitochondrial metabolism and ATP turnover were reduced in hypoxic PASMC compared to controls. Hypoxic PASMC had higher basal Ca2+, and a heightened peak Ca2+ response to thromboxane challenge compared to controls. Addition of SNP further elevated the peak Ca2+ response, while addition of FeTPPS brought peak Ca2+ response down to control levels. AC mediated relaxation was impaired in hypoxic PASMC compared to controls but was normalized following treatment with FeTPPS. Addition of SNP inhibited adenylyl cyclase activity in both normoxic and hypoxic PASMC. Moreover, addition of the Ca2+ chelator BAPTA improved AC activity, but the effect was minimal. Discussion: We conclude that NO independently augments contraction and inhibits relaxation pathways in hypoxic PASMC, in part by a mechanism involving nitrogen radical formation and protein nitrosylation. These observations may partially explain impaired effectiveness of NO when treating hypoxic pulmonary hypertension.

Endothelial alpha globin is a nitrite reductase

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin’s inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36–39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36–39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36–39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.

(-)-Methyl-Oleocanthal, a New Oleocanthal Metabolite Reduces LPS-Induced Inflammatory and Oxidative Response: Molecular Signaling Pathways and Histones Epigenetic Modulation.

The antioxidant and anti-inflammatory responses of (−)-methyl-oleocanthal (met-OLE), a new metabolite of the extra virgin olive oil (EVOO) phenolic oleocanthal (OLE), were explored in lipopolysaccharide (LPS)-induced murine peritoneal macrophages. Possible signaling pathways and epigenetic modulation of histones were studied. Met-OLE inhibited LPS-induced intracellular reactive oxygen species (ROS) and nitrite (NO) production and decreased the overexpression of the pro-inflammatory enzymes COX-2, mPGES-1 and iNOS in murine macrophages. In addition, met-OLE was able to significantly decrease the activation of p38, JNK, and ERK mitogen-activated protein kinases (MAPKs) and blocked canonical and non-canonical inflammasome signaling pathways. On the contrary, met-OLE upregulated haem oxigenase 1 (HO-1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) expression in treated cells. Finally, met-OLE pretreated spleen cells counteracted LPS induction, preventing H3K18 acetylation or H3K9 and H3K27 demethylation. Overall, these results provide novel mechanistic insights into the beneficial effects of met-OLE regarding the regulation of the immune–inflammatory response through epigenetic changes in histone markers. This revealing evidence suggests that the methylated metabolite of OLE may contribute significantly to the beneficial effects that are associated with the secoiridoid-related compound and the usual consumption of EVOO.

Characteristics of nitrogen transformation and intracellular nitrite accumulation by the hypothermia bacterium Arthrobacter arilaitensis

A new nitrite accumulation pathway was discovered in the nitrogen conversion process of Arthrobacter arilaitensis. The extracellular nitrite reached 0.65 and 43.66 mg/L with hydroxylamine and nitrate as the sole nitrogen source, respectively. The enzyme activities of ammonia monooxygenase, hydroxylamine oxidoreductase and nitrate reductase were 0.42, 0.0014 and 0.0049 U/mg protein, respectively. The activity of nitrite reductase was completely inhibited by diethyldithiocarbamate. Intriguingly, the intracellular nitrite accumulated as high as 43.0, 42.26, 39.94 and 35.01 mg/L, when the Arthrobacter arilaitensis was incubated with Luria-Bertani medium, ammonium, nitrate and nitrite as the nitrogen source, respectively. These results confirmed that the highest concentration of intracellular nitrite was accumulated when LB was selected as the nitrogen source, followed by ammonium and nitrate, then nitrite was the least. To date, biochemical mechanism responsible for the accumulation of a high concentration of intracellular nitrite is unknown.

Nitrate uptake and metabolism in human skeletal muscle cell cultures

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear. We hypothesized that exogenous nitrate can be taken up and metabolized to nitrite/NO inside the skeletal muscle. We found rapid uptake of exogeneous nitrate in both myoblasts and myotubes, increasing nitrite levels in myotubes, but not myoblasts. During differentiation we found increased expression of molybdenum containing proteins, such as xanthine oxidoreductase (XOR) and the mitochondrial amidoxime-reducing component (MARC); nitrate and nitrite reductases. Sialin, a known nitrate transporter, was detected in myoblasts; nitrate uptake decreased after sialin knockdown. Inhibition of chloride channel 1 (CLC1) also led to significantly decreased uptake of nitrate. Addition of exogenous nitrite, which resulted in higher intracellular nitrite levels, increased intracellular cGMP levels in myotubes. In summary, our results demonstrate for the first time the presence of the nitrate/nitrite/NO pathway in skeletal muscle cells, namely the existence of strong uptake of exogenous nitrate into cells and conversion of intracellular nitrate to nitrite and NO. Our results further support our previously formulated hypothesis about the importance of the nitrate to nitrite to NO intrinsic reduction pathways in skeletal muscles, which likely contributes to improved exercise tolerance after nitrate ingestion.

The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells.

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future.

Syntheses, toxicities and anti-inflammation of H2S-donors based on non-steroidal anti-inflammatory drugs

Three series of H2S donors based on NSAIDs were synthesized and characterized by 1H-NMR, IR and ESI-HRMS. The H2S-release abilities of all compounds were evaluated in the presence of TECP or cysteine. The results show all compounds were fast H2S-releasers, and their half-lives were in range of 0–20 min. Under the same condition, H2S released from compound 9 was more than any other compounds. In cytotoxicity aspect, all compounds but 1 and 2 displayed much lower toxicities to both LO2 and HepG2 cell lines, and the IC50 values of most compounds were over 800 μM. Compounds 1 and 2 had a stronger anti-proliferative activity to both cell lines, but they displayed lower toxicities to LO2 than to HepG2. Based on the cytotoxicity, the developmental toxicities of the compounds were assessed using zebrafish embryos. The results show all tested compounds 2, 9 and 15 had effects on the mortality, hatching rate and spontaneous movements of zebrafish embryos, and caused embryos teratogenesis; and the compounds had dose-dependent toxicities to both embryonic and larval zebrafish. In addition, all compounds had a better anti-inflammatory activity. In the test of anti-inflammatory activities, the tested compounds all reduced the levels of intracellular nitrite and pro-inflammatory cytokines (TNF-α, COX-2), increased the levels of anti-inflammatory cytokines (IL-10, HO-1). All these suggest these H2S donors based on NSAIDs have a potential to be a candidate medicine.

Syntheses, properties and bio-activities of water-soluble CO-releasing molecule based on manganese.

A series of water-soluble CO-releasing molecules, [Mn(CO)3NH2CHRCO2]2 (1-3), [M(CO)3Br[(Py-C=N)(Gly) n CO2] (M=Mn, Re, 4-7), Mn(CO)4[S2CNC m H n CO2] (8-12), were synthesized and characterized by (1)H NMR, IR and ESI-HRMS. The stability of all the complexes in solution was evaluated by means of UV, IR and (1)H NMR. Among all the complexes, complex 4 and complex 8 were stable in H2O, acidic aqueous solution and basic media; complex 1 was stable in acidic aqueous solution and weak basic media (pH < 9.4). The assays showed that each complex has CO-release ability; excess sodium dithionite can enhance CO release. Among them, complexes 8-12 were fast CO-releasers. In the test of the cell proliferation, all the complexes showed anti-proliferative activities for HeLa and HepG2. In particular, complex 8 displayed a 3.5-fold anti-proliferative activity on HeLa cells (IC50 23.13μM) and fivefold on HepG2 cells (34.00μM) compared with 5-FU. What is more, the complexes distinctly influenced cell cycle and promoted cell apoptosis; complex 1 arrested HeLa cells in S phase, whereas complex 4 and complex 8 arrested in G2/M phase; all the complexes induced HeLa cells "Early apoptosis". In addition, all complexes 1, 4 and 8 decreased intracellular nitrite levels, and complex 8 was stronger than both of the others. All these data demonstrate that complex 8 has potential to be a drug candidate. Three different categories of water-soluble CORMs 1-12 were synthesized, and their stability were evaluated. The biological activities were preliminarily evaluated. This includes anti-proliferation and anti-inflammatory properties.

Intracellular nitrite accumulation: The cause of growth inhibition of Microcystis aeruginosa exposure to high nitrite level

SummaryThe aim of the present study is to test the role of intracellular nitrite in external nitrite suppressing algal growth. We examined the growth of Microcystis aeruginosa at different nitrite levels under high nitrate conditions and without nitrate conditions. There were higher intracellular nitrite and lower Pmchla, Rd chla, αchl, maximum cell density and specific growth rate in high nitrate group than nitrate absence group at 5 mg NO2−‐N L−1. At 10 and 15 mg NO2−‐N L−1, Pmchla, Rd chla, αchl, maximum cell densities and specific growth rates in the high nitrate group became higher than those of the nitrate absence group, while a lower intracellular nitrite in the high nitrate group than nitrate absence group was observed. In addition, the intracellular nitrite and the growth of M. aeruginosa in the high nitrate group did not change from 5 to 10 mg NO2−‐N L−1. In the nitrite uptake experiment, with nitrite concentration increasing from 5 to 15 mg NO2−‐N L−1, maximum nitrite uptake rate of alga increased, and half‐saturation constant of alga decreased. These results indicate that external nitrite inhibited algal growth through stimulating intracellular nitrite rise, which resulted from overexpression of nitrite transporter.

Nitric oxide-triggered remodeling of chloroplast bioenergetics and thylakoid proteins upon nitrogen starvation in Chlamydomonas reinhardtii.

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b6f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b6f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b6f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b6f degradation pathway is activated.

Nitrite Reductase Activity and Inhibition of H2S Biogenesis by Human Cystathionine ß-Synthase

Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS) as a new player in nitrite reduction with implications for the nitrite-dependent control of H2S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (FeII-NO) CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR) and nitrite. Formation of FeII-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H2S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H2S biology.

Nitrous Oxide (N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O) production in axenic &amp;lt;i&amp;gt;Chlorella vulgaris&amp;lt;/i&amp;gt; microalgae cultures: evidence, putative pathways, and potential environmental impacts

Abstract. Using antibiotic assays and genomic analysis, this study demonstrates nitrous oxide (N2O) is generated from axenic Chlorella vulgaris cultures. In batch assays, this production is magnified under conditions favouring intracellular nitrite accumulation, but repressed when nitrate reductase (NR) activity is inhibited. These observations suggest N2O formation in C. vulgaris might proceed via NR-mediated nitrite reduction into nitric oxide (NO) acting as N2O precursor via a pathway similar to N2O formation in bacterial denitrifiers, although NO reduction to N2O under oxia remains unproven in plant cells. Alternatively, NR may reduce nitrite to nitroxyl (HNO), the latter being known to dimerize to N2O under oxia. Regardless of the precursor considered, an NR-mediated nitrite reduction pathway provides a unifying explanation for correlations reported between N2O emissions from algae-based ecosystems and NR activity, nitrate concentration, nitrite concentration, and photosynthesis repression. Moreover, these results indicate microalgae-mediated N2O formation might significantly contribute to N2O emissions in algae-based ecosystems (e.g. 1.38–10.1 kg N2O-N ha−1 yr−1 in a 0.25 m deep raceway pond operated under Mediterranean climatic conditions). These findings have profound implications for the life cycle analysis of algae biotechnologies and our understanding of the global biogeochemical nitrogen cycle.

Unsaponifiable fraction from extra virgin olive oil inhibits the inflammatory response in LPS-activated murine macrophages

Extra virgin olive oil (EVOO) has demonstrated great anti-inflammatory properties. Nowadays, it is clear that its minor components have a key role in these beneficial effects. However, the contribution of the unsaponifiable fraction (UF) to these healthy effects remains unknown. The present study was designed to evaluate UF in LPS stimulated peritoneal macrophages isolated from mice. NO production was analysed by the Griess method and intracellular ROS by fluorescence analysis. In addition, MAPK family activation, IKBα degradation, NFκB-p65, iNOS and COX-2 expression were determined by Western blot. UF exerted anti-inflammatory and anti-oxidant effects inhibiting LPS-induced intracellular ROS and nitrite production. Additionally, UF decreased COX-2 and iNOS protein expression. These effects were related with a down-regulation in NFκB signal signalling pathways and in MAPK phosphorylation. UF of EVOO compounds could play an important role in the anti-inflammatory effect of virgin olive oils and probably provide an attractive complement in management of inflammatory diseases.

Impact of mitochondria on nitrite metabolism in HL-1 cardiomyocytes

Apart from ATP synthesis mitochondria have many other functions, one being nitrite reductase activity. Nitric oxide (NO) released from nitrite has been shown to protect the heart from ischemia/reperfusion (I/R) injury in a cGMP-dependent manner. However, the exact impact of mitochondria on the release of NO from nitrite in cardiomyocytes is not completely understood. Besides mitochondria, a number of non-mitochondrial metalloproteins have been suggested to facilitate this process. The aim of this study was to investigate the impact of mitochondria on the bioactivation of nitrite in HL-1 cardiomyocytes. The levels of nitrosyl complexes of hemoglobin (NO-Hb) and cGMP levels were measured by electron spin resonance spectroscopy and enzyme immunoassay. In addition the formation of free NO was determined by confocal microscopy as well as intracellular nitrite and S-nitrosothiols by chemoluminescence analysis. NO was released from nitrite in cell culture in an oxygen-dependent manner. Application of specific inhibitors of the respiratory chain, p450, NO synthases (NOS) and xanthine oxidoreductase (XOR) showed that all four enzymatic systems are involved in the release of NO, but more than 50% of NO is released via the mitochondrial pathway. Only NO released by mitochondria activated cGMP synthesis. Cardiomyocytes co-cultured with red blood cells (RBC) competed with RBC for nitrite, but free NO was detected only in HL-1 cells suggesting that RBC are not a source of NO in this model. Apart from activation of cGMP synthesis, NO formed in HL-1 cells diffused out of the cells and formed NO-Hb complexes. In addition nitrite was converted by HL-1 cells to S-nitrosyl complexes. In HL-1 cardiomyocytes, several enzymatic systems are involved in nitrite reduction to NO but only the mitochondrial pathway of NO release activates cGMP synthesis. Our data suggest that this pathway may be a key regulator of myocardial contractility especially under hypoxic conditions.

Sensing Hypoxia by Mitochondria: A Unifying Hypothesis Involving S-Nitrosation

Sudden hypoxia requires a rapid response in tissues with high energy demand. Mitochondria are rapid sensors for a lack of oxygen, but no consistent mechanism for the sensing process and the subsequent counter-regulation has been described. In the present hypothesis review, we suggest an oxygen-sensing mechanism by mitochondria that is initiated at low oxygen tension by electrons from the respiratory chain, leading to the reduction of intracellular nitrite to nitric oxide ((•)NO) that would subsequently compete with oxygen for binding to cytochrome c oxidase. This allows superoxide ((•)O2(-)) formation in hypoxic areas, leading to S-nitrosation and the inhibition of mitochondrial Krebs cycle enzymes. With more formation of (•)O2(-), peroxynitrite is generated and known to damage the connection between the mitochondrial matrix and the outer membrane. A fundamental question on a regulatory mechanism is its reversibility. Readmission of oxygen and opening of the mitochondrial KATP-channel would allow electrons from glycerol-3-phosphate to selectively reduce the ubiquinone pool to generate (•)O2(-) at both sides of the inner mitochondrial membrane. On the cytosolic side, superoxide is dismutated and will support H2O2/Fe(2+)-dependent transcription processes and on the mitochondrial matrix side, it could lead to the one-electron reduction and reactivation of S-nitrosated proteins. It remains to be elucidated up to which stage the herein proposed silencing of mitochondria remains reversible and when irreversible changes that ultimately lead to classical reperfusion injury are initiated.

Curcumin induces differentiation of embryonic stem cells through possible modulation of nitric oxide-cyclic GMP pathway

Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 μmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.

Effects of nitrate on nitrite toxicity to Microcystis aeruginosa

The aim of this study was to investigate the effect of nitrate on nitrite toxicity to Microcystis aeruginosa. Short-term uptake experiment revealed nitrate could depress the nitrite active uptake. The long-term experiment of nitrate effects on nitrite toxicity showed a negative relationship between the growth and intracellular nitrite levels of M. aeruginosa. The net nitrite uptake displayed a decline in the range of 0–150mg NO3--NL-1 and an elevation at 200mg NO3--NL-1, whereas the activities of nitrate reductase (NR) were more sensitive to nitrate than nitrite reductase (NiR). As a result, the total intracellular nitrite levels tended to decrease up to 100mg NO3--NL-1 then increased at 150–200mg NO3--NL-1. These results suggested the existence of external nitrate could affect the toxicity of nitrite to alga through changing intracellular nitrite content.

Expression, and Molecular and Enzymatic Characterization of Cu-Containing Nitrite Reductase from a Marine Ammonia-Oxidizing Gammaproteobacterium, &lt;i&gt;Nitrosococcus oceani&lt;/i&gt;

Ammonia-oxidizing bacteria (AOB) remove intracellular nitrite to prevent its toxicity by a nitrifier denitrification pathway involving two denitrifying enzymes, nitrite reductase and nitric oxide reductase. Here, a Cu-containing nitrite reductase from Nitrosococcus oceani strain NS58, a gammaproteobacterial marine AOB, was expressed in Escherichia coli and purified to homogeneity. Sequence homology analysis indicated that the nitrite reductase from N. oceani was phylogenetically closer to its counterparts from denitrifying bacteria than that of the betaproteobacterium Nitrosomonas europaea. The recombinant enzyme was a homotrimer of a 32 kDa subunit molecule. The enzyme was green in the oxidized state with absorption peaks at 455 nm and 575 nm. EPR spectroscopy indicated the presence of type 2 Cu. Molecular activities and the affinity constant for the nitrite were determined to be 1.6×103 s−1 and 52 μM, respectively.