Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress

The nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) molecules play important roles in the redox homeostasis of plant cells. Using tomato (Solanum lycopersicum) plants grown with 120 mM NaCl, we studied the redox state of NADPH and GSH as well as ascorbate, nitric oxide (NO) and S-nitrosoglutathione (GSNO) content and the activity of the principal enzymes involved in the metabolism of these molecules in roots. Salinity caused a significant reduction in growth parameters and an increase in oxidative parameters such as lipid peroxidation and protein oxidation. Salinity also led to an overall decrease in the content of these redox molecules and in the enzymatic activities of the main NADPH-generating dehydrogenases, S-nitrosoglutathione reductase and catalase. However, NO content as well as gluthahione reductase and glutathione peroxidase activity increased under salinity stress. These findings indicate that salinity drastically affects redox and NO homeostasis in tomato roots. In our view, these molecules, which show the interaction between ROS and RNS metabolisms, could be excellent parameters for evaluating the physiological conditions of plants under adverse stress conditions.     

O(2)-dependent stimulation of the pentose phosphate pathway by S-nitrosocysteine in human erythrocytes

In the present study we analysed the effects of S-nitrosocysteine (CysNO) on adult human red blood cell metabolism and observed that metabolic response depended on the degree of cell oxygenation. In particular, glucose metabolised through the pentose phosphate pathway (PPP) was higher in treated erythrocytes than in untreated cells only at high O(2) pressure. Since, following the treatment of intact cells with CysNO, glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase (PFK) activities did not evidence any significant alteration, the possibility that the stimulation of PPP was triggered by a CysNO mediated modification of these enzymes was excluded. Intracellular S-nitrosoglutathione (GSNO), detected only in treated red blood cells, may be linked solely to the exposition to the NO donor. A possible rationalisation of the different metabolic behaviour shown by erythrocytes as a function of their oxygenation state is proposed. It takes into account the different route of catabolic degradation observed in vitro for GSNO under aerobic and anaerobic condition.     

Nitric oxide and S-nitrosylation: excitotoxic and cell signaling mechanism

This review focuses on the important physiological messenger, nitric oxide (NO), and its role in N-methyl-D-aspartate (NMDA) excitotoxicity. NO has been shown to be a key mediator of voltage-gated Ca(+2) transmembrane proteins. It remains unclear whether NO is implicated during hypoxia, or ischemic/reperfusion injuries as a neuroprotective or neurodegenerative factor. Excitotoxicity results from the excessive stimulation of excitatory glutamate receptors within the CNS. This review maintains that the feed-forward pathway precipitated by oxidative stress is the discriminating factor in the neuroprotective or neurodegenerative actions of NO.     

S-Nitroso compounds interfere with zinc probing by Zinquin

The intracellular homeostasis of zinc is postulated to be controlled by signaling through nitric oxide (NO). Administration of the NO donor S-nitrosocysteine (SNOC) caused a rapid drop in the fluorescence of the zinc-specific fluorescence of the zinc probe zinquin in C6 glioma cells. Tentatively, a strong effect of NO on the level of mobile intracellular zinc ions was concluded. However, zinc analysis with atomic absorption spectrometry demonstrated that the total cellular zinc level was not changed under these conditions. Sodium nitrite or an NO donor devoid of sulfhydryl groups (diethylamine NONOate) exerted no degrading effect on the Zn/zinquin fluorescence, but cysteine alone evoked a similar decline as SNOC. Hence, the sulfhydryl groups of cysteine seem to compete for zinc from the Zn/zinquin complex. Analysis of the reaction products by mass spectrometry demonstrated that cysteine caused a depletion of zinc from the Zn/zinquin complex, whereas an NO donor without sulfhydryl groups (diethylamine NONOate) did not. It is concluded that great caution should be employed when using S-nitroso compounds together with zinquin in investigations of intracellular zinc homeostasis.     

Protein S-nitrosylation: Role for nitric oxide signaling in neuronal death

Background

One of the signaling mechanisms mediated by nitric oxide (NO) is through S-nitrosylation, the reversible redox-based modification of cysteine residues, on target proteins that regulate a myriad of physiological and pathophysiological processes. In particular, an increasing number of studies have identified important roles for S-nitrosylation in regulating cell death.

Scope of review

The present review focuses on different targets and functional consequences associated with nitric oxide and protein S-nitrosylation during neuronal cell death.

Major conclusions

S-Nitrosylation exhibits double-edged effects dependent on the levels, spatiotemporal distribution, and origins of NO in the brain: in general Snitrosylation resulting from the basal low level of NO in cells exerts anti-cell death effects, whereas S-nitrosylation elicited by induced NO upon stressed conditions is implicated in pro-cell death effects.

General Significance

Dysregulated protein S-nitrosylation is implicated in the pathogenesis of several diseases including degenerative diseases of the central nervous system (CNS). Elucidating specific targets of S-nitrosylation as well as their regulatory mechanisms may aid in the development of therapeutic intervention in a wide range of brain diseases.     

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.     

S-nitrosothiol and nitric oxide reactivity at β-diketiminato zinc thiolates

The β-diketiminato zinc halide [Me₂NN]ZnCl₂Li(THF)₃ (1) is prepared in 51% isolated yield by addition of the lithium β-diketiminate Li[Me₂NN] to ZnCl₂ in THF. Reaction of 1 with 2 equiv. of the thallium thiolate TlSCy provides {[Me₂NN]Zn(μ-SCy)₂Tl}₂ (2), a TlSCy adduct of [Me₂NN]ZnSCy, as colorless crystals in 51% yield. Reaction of 1 with 1 equiv. TlSR provides the dinuclear {[Me₂NN]Zn(μ-SR)}₂ (R = Cy (3), ^tBu (4)) which possess unsymmetrically bridging thiolate ligands with pairs of dissimilar Zn-S distances in the solid state (2.350(3) and 2.417(3) Å for 3; 2.312(1) and 2.415(1) Å for 4). Reaction of 1 with LiSCPh₃ results in the mononuclear zinc thiolates [Me₂NN]ZnSCPh₃(THF) (5) and [Me₂NN]ZnSCPh₃ (6) with shorter, but similar Zn-SR distances of 2.225(2) and 2.214(1) Å. Variable temperature ¹H NMR studies of 3 and 4 in CDCl₃ suggest that the aliphatic thiolates exist predominately as monomeric species in solution near room temperature, though at −50 °C two different β-diketiminato species are observed for 3. Thiolate exchange among 3, 4, and 6 also takes place on the NMR timescale near room temperature. Both 4 and 6 undergo transnitrosylation with CySNO in CDCl₃ to give {[Me₂NN]ZnSCy}₂ (3) and the corresponding S-nitrosothiol ^tBuSNO or Ph₃CSNO. Nitric oxide does not react with 4 or 6 under anaerobic conditions, but in the presence of O₂, NO cleaves the zinc-thiolate bond of 4 to rapidly give ^tBuSNO. Similarly, anaerobic NO₂ reacts with 4 to give ^tBuSNO providing insight into the active nitrogen oxide species capable of cleaving Zn-SR bonds.     

Nitric oxide modulation of voltage-gated calcium current by S-nitrosylation and cGMP pathway in cultured rat hippocampal neurons

Nitric oxide (NO) plays an important role in many physiological and pathophysiological processes in the brain. In this study, we examined the mechanistic effects of an NO donor, diethylenetriamine/nitric oxide adduct (DETA/NO) on the voltage-gated calcium currents in cultured rat hippocampal neurons. DETA/NO stimulated the calcium currents and slightly increased the channel sensitivity to depolarizing voltages. The effect of DETA/NO on the calcium current was blocked by either depleting the NO in DETA/NO or by pretreating the neurons with NEM, a thiol-specific alkylating agent, suggesting an involvement of S-nitrosylation in the current response to NO. In addition, activation of the cGMP pathway by 8-Br-cGMP inhibited the calcium current in the neurons. Also, inhibition of guanylyl cyclase by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) increased the current response to DETA/NO. Taken together, our results demonstrate that both S-nitrosylation and cGMP pathway are involved in the NO modulation of the hippocampal calcium current.     

The reaction mechanism of nitrosothiols with copper(I)

Copper and other transition metal ions and their complexes are catalysts for the decomposition of nitrosothiols. In this way they catalyze the biological functions of nitrosothiols. The kinetics and mechanism of the reaction of two nitrosothiols, S-nitrosothiolactic acid and S-nitrosoglutathione (GSNO), with copper(I) are reported. The kinetics of the reaction of Cu(MeCN) _n ⁺ (n=0–3) with the nitrosothiols were studied. The results indicate that Cu⁺ _aq is the active species in the GSNO system, with k(Cu⁺ _aq+GSNO)=(9.4 ±2.0)×10⁷ dm³ mol⁻¹ s⁻¹. The results also indicate that the Cu(MeCN) _n ⁺ (n=0–3) complexes react with S-nitrosothiolactic acid. Transient species are formed in these processes. The results suggest that these species contain copper(I) and thiol. The results shed light on the catalytic role of copper complexes in the decomposition of S-nitrosothiols. Received 10 April 1999 / Accepted 17 December 1999     

Acute hypoxia enhances proteins' S-nitrosylation in endothelial cells

Hypoxia-induced responses are frequently encountered during cardiovascular injuries. Hypoxia triggers intracellular reactive oxygen species/nitric oxide (NO) imbalance. Recent studies indicate that NO-mediated S-nitrosylation (S-NO) of cysteine residue is a key posttranslational modification of proteins. We demonstrated that acute hypoxia to endothelial cells (ECs) transiently increased the NO levels via endothelial NO synthase (eNOS) activation. A modified biotin-switch method coupled with Western blot on 2-dimentional electrophoresis (2-DE) demonstrated that at least 11 major proteins have significant increase in S-NO after acute hypoxia. Mass analysis by CapLC/Q-TOF identified those as Ras-GTPase-activating protein, protein disulfide-isomerase, human elongation factor-1-delta, tyrosine 3/tryptophan 5-monooxygenase activating protein, and several cytoskeleton proteins. The S-nitrosylated cysteine residue on tropomyosin (Cys 170) and β-actin (Cys 285) was further verified with the trypsic peptides analyzed by MASCOT search program. Further understanding of the functional relevance of these S-nitrosylated proteins may provide a molecular basis for treating ischemia-induced vascular disorders.     

The role of thioredoxin in the regulation of cellular processes by S-nitrosylation

Background

S-nitrosylation (or S-nitrosation) by Nitric Oxide (NO), i.e., the covalent attachment of a NO group to a cysteine thiol and formation of S-nitrosothiols (R-S-N=O or RSNO), has emerged as an important feature of NO biology and pathobiology. Many NO-related biological functions have been directly associated with the S-nitrosothiols and a considerable number of S-nitrosylated proteins have been identified which can positively or negatively regulate various cellular processes including signaling and metabolic pathways.

Scope of the review

Taking account of the recent progress in the field of research, this review focuses on the regulation of cellular processes by S-nitrosylation and Trx-mediated cellular homeostasis of S-nitrosothiols.

Major conclusions

Thioredoxin (Trx) system in mammalian cells utilizes thiol and selenol groups to maintain a reducing intracellular environment to combat oxidative/nitrosative stress. Reduced glutathione (GSH) and Trx system perform the major role in denitrosylation of S-nitrosylated proteins. However, under certain conditions, oxidized form of mammalian Trx can be S-nitrosylated and then it can trans-S-nitrosylate target proteins, such as caspase 3.

General significance

Investigations on the role of thioredoxin system in relation to biologically relevant RSNOs, their functions, and the mechanisms of S-denitrosylation facilitate the development of drugs and therapies. This article is part of a Special Issue entitled Regulation of Cellular Processes.     

Mechanistic studies of S-nitrosothiol formation by NO*/O2 and by NO*/methemoglobin

The mechanisms of formation of S-nitrosothiols under physiological conditions and, in particular, of generation of SNO-Hb (the hemoglobin form in which the cysteine residues beta93 are S-nitrosated) are still not completely understood. In this paper, we investigated whether, in the presence of O2, NO* is more efficient to nitrosate protein-bound thiols such as Cysbeta93 or low molecular weight thiols such as glutathione. Our results show that when substoichiometric amounts of NO* are mixed slowly with the protein solution, NO*, O2, and possibly NO2* and/or N2O3 accumulate in hydrophobic pockets of hemoglobin. Since the environment of the cysteine residue beta93 is rather hydrophobic, these conditions facilitate SNO-Hb production. Moreover, we show that S-nitrosation mediated by reaction of NO* with the iron(III) forms of Hb or Mb is significantly more effective when it can take place intramolecularly, as in metHb. Intermolecular reactions lead to lower S-nitrosothiol yields because of the concurring hydrolysis to nitrite.     

Further evidence that rat liver microsomal glutathione transferase 1 is not a cellular protein target for S-nitrosylation

By adopting biotin switch method, we recently reported that liver microsomal glutathione transferase 1 (MGST1) might not be a protein target for S-nitrosylation in rat microsomes or in vivo. However, alternative analytic methods are needed to confirm this observation, as a single biotin switch method in judging specific protein S-nitrosylation in biological samples is increasingly recognized as insufficient, or even unreliable. Besides, only MGST1 localized on endoplasmic reticulum (ER), but not mitochondria which favors protein S-nitrosylation was examined in the previous report. Present study was therefore carried out to address these issues. Primary cultured hepatocytes were used. A physiological existing nitric oxide (NO) donor S-nitrosoglutathione (GSNO) was adopted to trigger protein S-nitrosylation. MGST1 was immunoprecipitated and its S-nitrosothiol content was measured by the NO probe 2,3-diaminonaphthalene. In parallel, S-nitrosylated proteins were immunoprecipitated by a monoclonal anti-S-nitrosocysteine antibody and probed with an anti-MGST1 antibody. In hepatocytes, neither ER nor mitochondria were found to contain S-nitrosylated MGST1 after GSNO treatment, showing that differently distributed MGST1 was consistently un-nitrosylable in the cellular environment. But under broken cell conditions, when samples were incubated directly with GSNO, MGST1 S-nitrosylation was indeed detectable in both the microsomal and mitochondrial proteins, indicating that previous failure in detecting MGST1 S-nitrosylation in microsomes is due to the limitations of biotin switch method. These results clearly, if not definitely, demonstrate that MGST1 is not a ready candidate for S-nitrosylation in the cellular content, despite its susceptibility to S-nitrosylation under broken cell conditions.     

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.     

Sulfur K-edge X-ray absorption spectroscopy as an experimental probe for S-nitroso proteins

X-ray absorption spectroscopy at the sulfur K-edge (2.4-2.6keV) provides a sensitive and specific technique to identify S-nitroso compounds, which have significance in nitric oxide-based cell signaling. Unique spectral features clearly distinguish the S-nitroso-form of a cysteine residue from the sulfhydryl-form or from a methionine thioether. Comparison of the sulfur K-edge spectra of thiolate, thiol, thioether, and S-nitroso thiolate compounds indicates high sensitivity of energy positions and intensities of XAS pre-edge features as determined by the electronic environment of the sulfur absorber. A new experimental setup is being developed for reaching the in vivo concentration range of S-nitroso thiol levels in biological samples.     

Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with l-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of l-arginine, N(omega)-hydroxy-l-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein from a pathogenic bacterium opens up avenues for further studies in understanding the importance of this protein in pathogenicity.     

Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin

The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b₅ is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b₅ reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b₅ and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b₅ to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b₅ is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.     

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Regulatory roles of nitric oxide during larval development and metamorphosis in Ciona intestinalis

Metamorphosis in the ascidian Ciona intestinalis is a very complex process which converts a swimming tadpole to an adult. The process involves reorganisation of the body plan and a remarkable regression of the tail, which is controlled by caspase-dependent apoptosis. However, the endogenous signals triggering apoptosis and metamorphosis are little explored. Herein, we report evidence that nitric oxide (NO) regulates tail regression in a dose-dependent manner, acting on caspase-dependent apoptosis. An increase or decrease of NO levels resulted in a delay or acceleration of tail resorption, without affecting subsequent juvenile development. A similar hastening effect was induced by suppression of cGMP-dependent NO signalling. Inhibition of NO production resulted in an increase in caspase-3-like activity with respect to untreated larvae. Detection of endogenously activated caspase-3 and NO revealed the existence of a spatial correlation between the diminution of the NO signal and caspase-3 activation during the last phases of tail regression. Real-time PCR during development, from early larva to early juveniles, showed that during all stages examined, NO synthase (NOS) is always more expressed than arginase and it reaches the maximum value at late larva, the stage immediately preceding tail resorption. The spatial expression pattern of NOS is very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system (CNS), tail and new forming juvenile digestive organs. NO detection revealed free diffusion from the production sites to other cellular districts. Overall, the results of this study provide a new important link between NO signalling and apoptosis during metamorphosis in C. intestinalis and hint at novel roles for the NO signalling system in other developmental and metamorphosis-related events preceding and following tail resorption.     

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.