The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.     

Nitric oxide, a biological double-faced janus--is this good or bad?

Nitric oxide (NO) is a biological messenger molecule produced by one of the essential amino acids L-arginine by the catalytic action of the enzyme NO synthase (NOS). The dual role of NO as a protective or toxic molecule is due to several factors, such as; the isoform of NOS involved, concentration of NO and the type of cells in which it is synthesised, the availability of the substrate L-arginine, generation of guanosine 3,5'-cyclic monophosphate (cGMP) from soluble guanylate cyclase and the overall extra and intracellular environment in which NO is produced. NOS activation as a result of trauma (calcium influx) or infection leads to NO production, which activates its downstream receptor sGC to synthesise cGMP and/or leads to protein nitrosylation. This may lead to one or more systemic effects including altered neurotransmission which can be protective or toxic, vaso/bronchodilatation in the cardiovascular and respiratory systems and enhanced immune activity against invading pathogens. In addition to these major functions, NO plays important role in thermoregulation, renal function, gastrointestinal motility, endocrine function, and various functions of the urogenital system ranging from renin secretion to micturation; spermatogenesis to penile erection; and ovulation to implantation and parturition. A schematic summary of the functions of NO and the various isoforms of NOS expressed in body systems is shown in figure 1. In this review, the historical background, biochemistry and biosynthesis of NO and its enzymes together with the mechanism of NO actions in physiology and pathophysiology are discussed.     

Cytokinin- and auxin-induced stomatal opening is related to the change of nitric oxide levels in guard cells in broad bean

The relationship between cytokinin- and auxin-induced stomatal opening and nitric oxide (NO) levels in guard cells in broad bean was studied. Results indicate that cytokinins and auxins reduced the levels of NO in guard cells and induced stomatal opening in darkness. In addition, cytokinins not only reduced NO levels in guard cells caused by sodium nitroprusside (SNP) in light but also abolished NO that had been generated by dark, and then promoted the closed stomata reopening, as did NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. However, unlike cytokinins, auxins not only had incapability to reduce NO levels by SNP but also could not abolish NO having been generated by dark, so auxins could not promote the closed stomata to reopen. The above-mentioned effects of auxins were similar to that of nitric oxide synthase (enzyme commission 1.14.13.39) inhibitor N ^G-nitro- l -Arg-methyl ester. Hence, it is concluded that cytokinins reduced probably the levels of NO in guard cells via scavenging, and auxins reduced NO levels through restraining NO generation in all probability, and then induced stomatal opening in darkness.     

Nitrification and denitrification in lake and estuarine sediments measured by the N dilution technique and isotope pairing

The transformation of nitrogen compounds in lake and estuarine sediments incubated in the dark was analyzed in a continuous-flowthrough system. The inflowing water contained NO(3), and by determination of the isotopic composition of the N(2), NO(3), and NH(4) pools in the outflowing water, it was possible to quantify the following reactions: total NO(3) uptake, denitrification based on NO(3) from the overlying water, nitrification, coupled nitrification-denitrification, and N mineralization. In sediment cores from both lake and estuarine environments, benthic microphytes assimilated NO(3) and NH(4) for a period of 25 to 60 h after darkening. Under steady-state conditions in the dark, denitrification of NO(3) originating from the overlying water accounted for 91 to 171 mumol m h in the lake sediments and for 131 to 182 mumol m h in the estuarine sediments, corresponding to approximately 100% of the total NO(3) uptake for both sediments. It seems that high NO(3) uptake by benthic microphytes in the initial dark period may have been misinterpreted in earlier investigations as dissimilatory reduction to ammonium. The rates of coupled nitrification-denitrification within the sediments contributed to 10% of the total denitrification at steady state in the dark, and total nitrification was only twice as high as the coupled process.     

The nitric oxide-iron interplay in mammalian cells: transport and storage of dinitrosyl iron complexes.

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl-dithiolato-Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl-diglutathionyl-Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.     

Effect of exogenous and endogenous nitric oxide on biofilm formation by Lactobacillus plantarum

Biofilms are a widespread form of occurrence of microorganisms in nature, and understanding the mechanism of regulation of their formation is of unquestionable practical significance for medicine and biotechnology. In the present work, the effect of nitric oxide (NO) on biofilm formation by Lactobacillus plantarum was investigated and the micromolar concentrations of exogenous NO were shown to have a negative effect on this process due to its toxic effect on the cells. However, the decrease in the level of endogenous NO in bacteria in the presence of a nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) impaired the characteristics of the forming biofilms, as was evident from the decrease in their size.     

Biological role of glutathione in nitric oxide-induced toxicity in cell culture and animal models

Glutathione (GSH) plays an important role in cellular defense response in many in vitro and in vivo models. Here we investigated its role in NO()-induced toxicity in cell culture and mouse models. Wild-type (TK6) and p53-null (NH32) human lymphoblastoid cells were treated with NO(.) at a steady-state concentration of 0.6 muM, similar to the level estimated to occur in inflamed tissues. In both cell types, GSH was depleted by this exposure in a dose- and time-dependent manner. Contrary to expectations, prior depletion of GSH by treatment with l-buthionine-SR-sulfoximine did not potentiate NO(.)-induced cell killing or DNA deamination in TK6 cells. In activated RAW264.7 murine macrophages producing NO(.), intracellular GSH content did not change, although gamma-glutamate-cysteine ligase was upregulated. NO(.) overproduction in RcsX lymphoma-bearing SJL mice resulted in significantly elevated GSH levels in various organs. Administration of the NO(.) synthase inhibitor N-methylarginine abolished the increase in GSH in these animals. Collectively, these data indicate a multifaceted and complex involvement of GSH in responses of cells and tissues to toxic levels of NO(.). NO(.) treatment effectively depleted GSH levels in human lymphoblastoid cells, but this alteration was not a critical initiating factor for NO(.)-mediated toxicity. Murine macrophages maintained GSH homeostasis when exposed to endogenously produced NO(.). In RcsX lymphoma-bearing mice, upregulation of de novo synthesis of GSH appeared to be a response to the toxic effects of NO(.).     

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25-50 microM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.     

The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.     

Expression of peptidergic and nitrergic structures in dorsal root ganglia of the rabbit

In this study we have demonstrated the presence of neuropeptide substance P (SP)and nonpeptide neurotransmiter NO (nitric oxide) in the dorsal root ganglia (DRG) of rabbits. NADPH-diaphorase histochemical staining was used for detection of NO and an immunohistochemical method for detection of substance P. A number of DRG cells were stained by SP- and NADPH-d reactions. The presence of SP and NADPH-diaphorase positive cells varied depending upon the spinal level of the DRG. Positively stained neurons were only small and intermediate in size. Cells of large diameter profiles showed no staining. Substance P immunoreactive cells were of brown and dark brown colour, the intensity of NADPH-d staining varied from light to very dark blue. In some DRG cells, there was very significant neuronal co-localization of immunoreactivity for SP and reactivity for NADPH-d. In summary, DRG cells appear to express diaphorase and substance P activity, and some of them show the presence of both neurotransmitters. Recent studies on the participation of NO in the regulation of SP release in the spinal cord suggest, that also in the DRG neurons there may be a close interaction between NO and SP.     

Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.     

Inhibition of mitochondrial aldehyde dehydrogenase by nitric oxide-mediated S-nitrosylation

Mitochondrial aldehyde dehydrogenase (ALDH2) is responsible for the metabolism of acetaldehyde and other toxic lipid aldehydes. Despite many reports about the inhibition of ALDH2 by toxic chemicals, it is unknown whether nitric oxide (NO) can alter the ALDH2 activity in intact cells or in vivo animals. The aim of this study was to investigate the effects of NO on ALDH2 activity in H4IIE-C3 rat hepatoma cells. NO donors such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, and 3-morpholinosydnonimine significantly increased the nitrite concentration while they inhibited the ALDH2 activity. Addition of GSH-ethylester (GSH-EE) completely blocked the GSNO-mediated ALDH2 inhibition and increased nitrite concentration. To directly demonstrate the NO-mediated S-nitrosylation and inactivation, ALDH2 was immunopurified from control or GSNO-treated cells and subjected to immunoblot analysis. The anti-nitrosocysteine antibody recognized the immunopurified ALDH2 only from the GSNO-treated samples. All these results indicate that S-nitrosylation of ALDH2 in intact cells leads to reversible inhibition of ALDH2 activity.     

Identification of the receptor binding domain of the mouse mammary tumor virus envelope protein

Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe(40) to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe(40) residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation.     

Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNA-damaging anti-tumour agents

Nitric-oxide synthase (NOS) activity has been detected in many human tumours, although its function is unclear. Here we show that exposure of cells to nitric oxide (NO) results in a 4-5-fold increase in expression of the DNA-dependent protein-kinase catalytic subunit (DNA-PKcs), one of the key enzymes involved in repairing double-stranded DNA breaks. This NO-mediated increase in enzymatically active DNA-PK not only protects cells from the toxic effects of NO, but also provides crossprotection against clinically important DNA-damaging agents, such as X-ray radiation, adriamycin, bleomycin and cisplatin. The NO-mediated increase in DNA-PKcs described here demonstrates the presence of a new and highly effective NO-mediated mechanism for DNA repair.     

The role of nitric oxide in inflammatory reactions

Nitric oxide (NO) was initially described as a physiological mediator of endothelial cell relaxation, an important role in hypotension. NO is an intercellular messenger that has been recognized as one of the most versatile players in the immune system. Cells of the innate immune system--macrophages, neutrophils and natural killer cells--use pattern recognition receptors to recognize the molecular patterns associated with pathogens. Activated macrophages then inhibit pathogen replication by releasing a variety of effector molecules, including NO. In addition to macrophages, a large number of other immune-system cells produce and respond to NO. Thus, NO is important as a toxic defense molecule against infectious organisms. It also regulates the functional activity, growth and death of many immune and inflammatory cell types including macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils and natural killer cells. However, the role of NO in nonspecific and specific immunity in vivo and in immunologically mediated diseases and inflammation is poorly understood. This Minireview will discuss the role of NO in immune response and inflammation, and its mechanisms of action in these processes.     

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport

Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.     

The Cellular Phenotype of Roberts Syndrome Fibroblasts as Revealed by Ectopic Expression of ESCO2

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.     

星型孢菌素(STS)对蚕豆气孔运动的调控效应

用激光扫描共聚焦显微技术,初步研究广谱性蛋白激酶抑制剂星型孢菌素(STS)对蚕豆气孔运动的调控效应.结果表明:(1)光下STS对气孔开度无影响但暗中显著促进气孔开放,表明蛋白激酶参与光/暗对气孔运动的调控,光下蛋白激酶活性低而暗中高;(2)与H2O2清除剂抗坏血酸(ASA)和NO清除剂羧基-2-苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)一样,STS既降低暗处理和光下外源H2O2、硝普钠(SNP)处理保卫细胞H2O2、NO水平,也促进气孔开放,表明暗中蛋白激酶通过抑制H2O2、NO清除机制提高保卫细胞内源H2O2、NO水平并促进气孔关闭.     

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-α, interleukin-1 β and interferon-γ, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25–50 μM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.