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ABSTRACT: It is well known that chronic inflammation of the digestive tract is associated with an increased risk of malignant transformation. Because phagocytic leukocytes and cytokine-activated parenchymal cells produce large amounts of reactive metabolites of oxygen and nitrogen, there has been substantial interest in ascertaining whether these reactive intermediates may mediate mutagenesis and malignant transformation in vivo. However, very little information is available regarding the basic chemistry of how these oxygen and nitrogen-derived species may interact to yield potentially carcinogenic agents. This review will discuss our present understanding of the chemical and biochemical interactions between superoxide and nitric oxide and provide a model by which these reactive species may damage DNA and mediate mutagenesis.
Alimentary Pharmacology & Therapeutics 05/2000; 14 Suppl 1:3-9. · 3.77 Impact Factor
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ABSTRACT: Nitric oxide (NO) has been shown to be a key bioregulatory agent in a wide variety of biological processes, yet cytotoxic properties have been reported as well. This dichotomy has raised the question of how this potentially toxic species can be involved in so many fundamental physiological processes. We have investigated the effects of NO on a variety of toxic agents and correlated how its chemistry might pertain to the observed biology. The results generate a scheme termed the chemical biology of NO in which the pertinent reactions can be categorized into direct and indirect effects. The former involves the direct reaction of NO with its biological targets generally at low fluxes of NO. Indirect effects are reactions mediated by reactive nitrogen oxide species, such as those generated from the NO/O2 and NO/O2- reactions, which can lead to cellular damage via nitrosation or oxidation of biological components. This report discusses several examples of cytotoxicity involved with these stresses.
Journal of Inorganic Biochemistry 05/2000; 79(1-4):237-40. · 3.35 Impact Factor
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ABSTRACT: Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-gamma and either tumor necrosis factor-alpha or interleukin-1beta resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-gamma and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells 2-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O(2) reaction leading to N(2)O(3) formation. These data mimicked the pattern observed with a 5 microM concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1,2-diolate (PAPA/NO). The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N(2)O(3) and elicit nitrosative stress.
Journal of Biological Chemistry 05/2000; 275(15):11341-7. · 4.77 Impact Factor
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ABSTRACT: Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2)-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2)-Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N-oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO.
Free Radical Biology and Medicine 04/2000; 28(5):739-42. · 5.42 Impact Factor
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ABSTRACT: Nitric oxide is a key bioregulatory agent in a wide variety of biological processes, yet it also can have cytotoxic properties. This dichotomy raises the question of how this potentially toxic species can be involved in so many fundamental physiological processes. This articles discusses how the chemistry of nitric oxide might pertain to its observed biology as it relates to oxidative and nitrosative stress in different mechanisms of cytotoxicity.
Seminars in Perinatology 03/2000; 24(1):20-3. · 2.99 Impact Factor
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ABSTRACT: Macrophages respond to infection or injury by changing from a "resting" cellular phenotype to an "activated" state defined by the expression of various cytotoxic effector functions. Regulation of the transition from a resting to an activated state is effected by cytokine and/or pathogenic signals. Some signals do not directly induce activation, but instead "prime" the macrophage to respond more vigorously to a second signal. One example of this priming phenomenon involves induction of nitric oxide (NO) synthesis by the enzyme nitric oxide synthase (NOS2). Our experiments indicate that low doses (1-5 Gy) of ionizing radiation can enhance the induction of enzymatically active NOS2 by IFN-gamma or LPS in J774.1 and RAW264.7 murine macrophage cell lines. Radiation alone did not produce this induction, rather, it was effective as a priming signal; cells exposed to radiation produced more NO when a second signal, either IFN-gamma or LPS, was applied 24 h later.
Annals of the New York Academy of Sciences 02/2000; 899:61-8. · 3.15 Impact Factor
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ABSTRACT: Many cellular functions in physiology are regulated by the direct interaction of NO with target biomolecules. In many pathophysiologic and toxicologic mechanisms, NO first reacts with oxygen, superoxide or other nitrogen oxides to subsequently elicit indirect effects. The balance between nitrosative stress and oxidative stress within a specific biological compartment can determine whether the presence of NO will be ultimately deleterious or beneficial. Nitrosative stress can be defined primarily through reactions mediated by N2O3, a reactive nitrogen oxide species generated by high fluxes of NO in an aerobic environment. In contrast, oxidative stress is mediated primarily by superoxide and peroxides. In addition to reactive oxygen species, several reactive nitrogen oxide species such as peroxynitrite, nitroxyl, and nitrogen dioxide can also impose oxidative stress to a cell. We here describe how the mechanisms of cell death are interwoven in the balance between the different chemical intermediates involved in nitrosative and oxidative stress.
Annals of the New York Academy of Sciences 02/2000; 899:209-21. · 3.15 Impact Factor
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ABSTRACT: Recent experimental evidence suggests that reactive nitrogen oxide species can contribute significantly to postischemic myocardial injury. The aim of the present study was to evaluate the role of two reactive nitrogen oxide species, nitroxyl (NO(-)) and nitric oxide (NO(.)), in myocardial ischemia and reperfusion injury. Rabbits were subjected to 45 min of regional myocardial ischemia followed by 180 min of reperfusion. Vehicle (0.9% NaCl), 1 micromol/kg S-nitrosoglutathione (GSNO) (an NO(.) donor), or 3 micromol/kg Angeli's salt (AS) (a source of NO(-)) were given i.v. 5 min before reperfusion. Treatment with GSNO markedly attenuated reperfusion injury, as evidenced by improved cardiac function, decreased plasma creatine kinase activity, reduced necrotic size, and decreased myocardial myeloperoxidase activity. In contrast, the administration of AS at a hemodynamically equieffective dose not only failed to attenuate but, rather, aggravated reperfusion injury, indicated by an increased left ventricular end diastolic pressure, myocardial creatine kinase release and necrotic size. Decomposed AS was without effect. Co-administration of AS with ferricyanide, a one-electron oxidant that converts NO(-) to NO(.), completely blocked the injurious effects of AS and exerted significant cardioprotective effects similar to those of GSNO. These results demonstrate that, although NO(.) is protective, NO(-) increases the tissue damage that occurs during ischemia/reperfusion and suggest that formation of nitroxyl may contribute to postischemic myocardial injury.
Proceedings of the National Academy of Sciences 01/2000; 96(25):14617-22. · 9.68 Impact Factor
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ABSTRACT: Since the discovery of nitric oxide (NO) as an endogenous mediator of numerous physiological processes ranging from regulation
of cardiovascular function to memory formation, there has been some question as to the effect its chemistry has on biology
(IGNARRO 1989; MONCADA et al. 1991; DAWSON et al. 1992; FELDMAN et al. 1992). In the immune system, this diatomic radical is involved in numerous anti-pathogenic and tumoricidal processes (Hibbs 1991; MAC
MICKING et al. 1997). Yet, despite these properties critical to maintaining homeostasis, NO has been implicated as a participant
in different pathophysiological conditions (GROSS and WOLIN 1995; Wink et al. 1998c). To further complicate definition of the exact roles of NO in vivo, both protective and deleterious effects have been attributed
to NO, even in the same biological event. Mechanistic explanations to account for these differences are still being pursued.
12/1999: pages 7-29;
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ABSTRACT: Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-D-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.
The American journal of physiology 11/1999; 277(4 Pt 1):C673-83.
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ABSTRACT: The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity. We examined the effects of H(4)Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H(4)Bip in a competitive manner. Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min). NOS-associated H(4)Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control). A similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.
Journal of Biological Chemistry 09/1999; 274(35):24921-9. · 4.77 Impact Factor
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ABSTRACT: We evaluated the effects of the xanthine oxidase (XO)-derived reactive oxygen metabolites on the permeability of bovine pulmonary artery-endothelial monolayers and examined how iron and nitric oxide (NO) participate in these changes in permeability.
Permeability was measured using a cell-column chromatographic method in which monolayers were exposed to combinations of agents.
Exposure of monolayers to a superoxide/peroxide generator, xanthine (X, 0.1 mM)/XO (25 mU/mL), increased solute permeability after 10 minutes, but the same dose of either X or XO alone did not. Exposure of monolayers to peroxide (0.1 mM) also increased permeability, but only after 70 minutes. This X/XO permeability was attenuated by either catalase, superoxide dismutase, methionine (1 mM), an oxy-radical scavenger, or desferrioxamine (0.1 mM), an iron chelator. Spermine NONOate (SNO), an NO donor, attenuated X/XO permeability at 0.1 mM, but this protection was not significant at 0.01 or 1 mM. Spermine NONOate (0.1 mM) did not alter the permeability produced by 0.1 mM peroxide. L-N5-(1-iminoethyl)-ornithine (10 microM), an NO synthase inhibitor, completely blocked peroxide-, and partially attenuated X/XO-mediated permeability. However, 3-morphosynodiomine (SIN-1, 1 mM) plus catalase (1,000 U/mL), a peroxynitrite generator, did not alter permeability.
Xanthine/Xanthine Oxidase permeability involves peroxide, superoxide, oxy-radicals, and iron. Endogenous NO may regulate peroxide-, but not superoxide-mediated permeability. The protective effects of exogenous NO on the X/XO permeability may represent interactions between superoxide, peroxide, and cell surface-bound iron.
Microcirculation 07/1999; 6(2):107-16. · 2.57 Impact Factor
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ABSTRACT: Nitric oxide (NO) synthases (NOSs), which catalyse the oxidation of L-arginine to L-citrulline and an oxide of nitrogen, possibly NO or nitroxyl (NO-), are subject to autoinhibition by a mechanism that has yet to be fully elucidated. In the present study we investigated the actions of NO and other NOS-derived products as possible autoregulators of enzyme activity. With the use of purified NOS-I, L-arginine turnover was found to operate initially at Vmax (0-15 min, phase I) although, despite the presence of excess substrate and cofactors, prolonged catalysis (15-90 min, phase II) was associated with a rapid decline in L-arginine turnover. Taken together, these observations suggested that one or more NOS products inactivate NOS. Indeed, exogenously applied reactive nitrogen oxide species (RNSs) decreased Vmax during phase I, although with different potencies (NO->NO> ONOO-) and efficacies (NO>NO-=ONOO-). The NO scavengers oxyhaemoglobin (HbO2; 100 microM) and 1H-imidazol-1 - yloxy - 2 - (4-carboxyphenyl) - 4,5 - dihydro - 4,4,5,5 - tetramethyl - 3 -oxide (CPTIO; 10 microM) and the ONOO- scavenger GSH (7 mM) had no effect on NOS activity during phase I, except for an endogenous autoinhibitory influence of NO and ONOO-. However, superoxide dismutase (SOD; 300 units/ml), which is thought either to increase the half-life of NO or to convert NO- to NO, lowered Vmax in an NO-dependent manner because this effect was selectively antagonized by HbO2 (100 microM). This latter observation demonstrated the requirement of SOD to reveal endogenous NO-mediated autoinhibition. Importantly, during phase II of catalysis, NOS became uncoupled and began to form H2O2 because catalase, which metabolizes H2O2, increased enzyme activity. Consistent with this, exogenous H2O2 also inhibited NOS activity during phase I. Thus during catalysis NOS is subject to complex autoinhibition by both enzyme-derived RNS and H2O2, differentially affecting enzyme activity.
Biochemical Journal 06/1999; 340 ( Pt 3):745-52. · 4.90 Impact Factor
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ABSTRACT: The primary product of the interaction between nitric oxide (NO) and superoxide () is peroxynitrite (ONOO-), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or can further react with ONOO- to form species which mediate nitrosation. Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and. Since ONOO- reacts not only with NO and but also with CO2, the effects of bicarbonate () on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3-diaminonaphthalene and reduced glutathione were examined. Nitric oxide and were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2 does not affect the scavenging of ONOO- by either NO or. However, an increase in the rate of DHR oxidation by ONOO- in the presence of suggests that a CO2-ONOO- adduct does play a role in the interaction of NO or with a product derived from ONOO-. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO- nor cis-HOONO. It was concluded that NO reacts with both trans-HOONO and a CO2 adduct of ONOO- to form nitrosating species which have similar oxidation chemistry and reactivity with and NO.
Archives of Biochemistry and Biophysics 06/1999; 365(1):92-100. · 2.93 Impact Factor
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ABSTRACT: Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.
Cancer Research 06/1999; 59(9):2142-9. · 7.86 Impact Factor
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ABSTRACT: Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO+ equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2+, whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 microM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.
Journal of Neurochemistry 06/1999; 72(5):1843-52. · 4.06 Impact Factor
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ABSTRACT: The role of nitric oxide (NO) in inflammation represents one of the most studied yet controversial subjects in physiology. A number of reports have demonstrated that NO possesses potent anti-inflammatory properties, whereas an equally impressive number of studies suggest that NO may promote inflammation-induced cell and tissue dysfunction. The reasons for these apparent paradoxical observations are not entirely clear; however, we propose that understanding the physiological chemistry of NO and its metabolites will provide a blueprint by which one may distinguish the regulatory/anti-inflammatory properties of NO from its deleterious/proinflammatory effects. The physiological chemistry of NO is complex and encompasses numerous potential reactions. In an attempt to simplify the understanding of this chemistry, the physiological aspects of NO chemistry may be categorized into direct and indirect effects. This type of classification allows for consideration of timing, location, and rate of production of NO and the relevant targets likely to be affected. Direct effects are those reactions in which NO interacts directly with a biological molecule or target and are thought to occur under normal physiological conditions when the rates of NO production are low. Generally, these types of reactions may serve regulatory and/or anti-inflammatory functions. Indirect effects, on the other hand, are those reactions mediated by NO-derived intermediates such as reactive nitrogen oxide species derived from the reaction of NO with oxygen or superoxide and are produced when fluxes of NO are enhanced. We postulate that these types of reactions may predominate during times of active inflammation. Consideration of the physiological chemistry of NO and its metabolites will hopefully allow one to identify which of the many NO-dependent reactions are important in modulating the inflammatory response and may help in the design of new therapeutic strategies for the treatment of inflammatory tissue injury.
The American journal of physiology 03/1999; 276(2 Pt 1):G315-21.
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Methods in Enzymology 02/1999; 301:413-24. · 2.04 Impact Factor
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Methods in Enzymology 02/1999; 301:220-7. · 2.04 Impact Factor
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Methods in Enzymology 02/1999; 301:201-11. · 2.04 Impact Factor
