Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy

Komarov, Andrei M.; Lai, Ching‐San

doi:10.1016/0925-4439(95)00061-8

Cited by 108 publications

(42 citation statements)

References 19 publications

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“…An NO˙-specific electron paramagnetic resonance (EPR) spin trapping technique was used (19). Ten millimolar Fe(II) (N-methyl-D-glucamine dithiocarbamate) 2 complex was prepared by anoxic mixing of FeSO 4 and N-methyl-D-glucamine dithiocarbamate and was incubated with 0.471 mg of purified M. tuberculosis H37Rv KatG ml Ϫ1 (37) with 10 mM INH and 10 mM H 2 O 2 in 10 mM phosphate buffer, pH 7, at 37°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo

et al. 2004

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Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species. Here we investigated the requirements and the pathway of nitric oxide (NO˙) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro.We also provide in vivo evidence that INH-derived NO˙can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH.Mycobacterium tuberculosis infections are of serious concern; they cause 2 million deaths every year and latently persist in over a billion individuals worldwide (41). Isoniazid (isonicotinic acid hydrazide [INH]) remains a front-line antituberculosis agent some 50 years after its development, and millions of doses are prescribed worldwide. M. tuberculosis is exceptionally sensitive to INH (12, 44), a prodrug, which is peroxidatively activated intracellularly by the M. tuberculosis catalase-peroxidase KatG to produce a range of reactive radicals that act as damaging species within the bacteria. Although the mechanism(s) of action and cellular targets of KatG-activated INH continue to be uncovered (22,27), its full range of effects on mycobacterial cells still remains to be resolved (20,33).The critical role of INH activation via KatG is clearly apparent based on the findings that the katG gene represents the main site for mutations causing INH resistance in M. tuberculosis (24, 45). Several INH-derived intermediates generated from isoniazid activation, such as isonicotinic acyl NADH (27), and mycobacterial targets including enzymes from the mycobacterial type II fatty acid synthase system (1, 22) have been identified. Other work on INH activation has centered upon INH-derived free radicals as important antimycobacterial intermediates (18,31,38). Despite these advances, the exact mechanism(s) of INH action that results in its exceptional and specific potency against M. tuberculosis are not yet fully delineated, as multiple targets and pathways have been considered (12,20,23,32).A range of reactive nitrogen species, such as nitric oxide (NO˙) and peroxynitrite (ONOO Ϫ ) are known to have various levels of activity against M. tuberculosis (8, 21, 43). In addition to the action of exogenously added NO˙immune-derived NOḟ rom the action of inducible nitric oxide synthase is considered to contribute to defenses against mycobacterial infection (29). Previous reports of tyrosine nitration during oxidation of INH (34) and NO˙formation from peroxidative activation of hydroxyurea (16, 17) led us to hypothesize that NO˙might be generated as a result of INH activation by KatG. We further hypothesized that the exceptional sensitivity of M. tuberculosis to NO˙could potentially account for at least some of the potency of INH (8,21,43). Here we used spin trapping techniques to document KatG generation of nitric oxide during activation of INH and to probe potential pathways for its production from INH. We also present in vivo analysis indica...

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Section: Methodsmentioning

confidence: 99%

Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo

et al. 2004

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“…2, experiments done by incubating A. thaliana leaf homogenates of wild-type and mutant plants (control or Psm-inoculated) with the spin trap in 100 mM phosphate buffer for 1 h showed no (MGD) 2 Fe(II)NO production. However, the incubation of wild-type leaf homogenates with all mammalian NOS cofactors, including 1 mM NADPH, and the spin trap for 1 h resulted in the characteristic three-line spectrum of the (MGD) 2 Fe(II)NO complex [26] providing unequivocal evidence for the production of Å NO. In contrast, the complex was barely detectable in homogenates of mutant plants.…”

Section: 2mentioning

confidence: 96%

“…To further verify the participation of NOS activity in the production of the Å NO detected by EPR, incubations were done using arginine isotopically labeled with 15 N (L L-[ 15 N 2 -guanidineimino]arginine) [23,26]. This substrate would produce labeled Å15 NO which, when complexed with (MGD) 2 Fe(II), should yield a two-line EPR spectrum.…”

Section: 2mentioning

confidence: 99%

Nitrite as the major source of nitric oxide production by Arabidopsis thaliana in response to Pseudomonas syringae

et al. 2005

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Å NO production in Arabidopsis thaliana in response to Pseudomonas syringae pv maculicola. NOS activity increased substantially in leaves inoculated with P. syringae. However, electron paramagnetic resonance experiments showed a much higher Å NO formation that was dependent on nitrite and mitochondrial electron transport rather than on arginine or nitrate. Overall, these results indicate that NOS, NR and a mitochondrial-dependent nitrite-reducing activity cooperate to produce Å NO during A. thaliana-P. syringae interaction.

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“…Isolated chloroplasts (0.8 mg protein mL 21 ) were supplemented with equal volumes of 100 mM phosphate buffer containing the spin trap (10 mM MGD, 1 mM FeSO4; Komarov and Lai, 1995) and the appropriate cofactors as described in each experiment. For Arg-dependent NO generation, chloroplasts were incubated up to 10 min in the presence or absence of 1 mM Arg, 0.1 mM NADPH, 1 mM Ca 21 , 10 mg mL 21 calmodulin, and 5 mM L-NAME and L-NNA as Arg analogs.…”

Section: Epr Detection Of Nomentioning

confidence: 99%

Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins

Jasid

Simontacchi²,

Bártoli³

et al. 2006

Plant Physiology

281

139

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Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and L-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 6 0.2 nmol min 21 mg 21 protein in the presence of 1 mM NaNO 2 . Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 6 0.04 nmol min 21 mg 21 protein) of NO generation. Chloroplasts incubated with 1 mM Arg showed NO production of 0.76 6 0.04 nmol min 21 mg 21 protein that was not affected either by omission of Ca 21 or by supplementation with Ca 21 and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N v -nitro-L-Arg methyl ester hydrochloride and N v -nitro-L-Arg. In vitro exposure of chloroplasts to an NO donor (250 mM S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mM peroxynitrite (ONOO 2 ) led to an increase in lipid-derived radicals (34%). The effect of ONOO 2 on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO 2 treatment significantly affected both O 2 evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

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Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy

Cited by 108 publications

References 19 publications

Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo

Requirements for Nitric Oxide Generation from Isoniazid Activation In Vitro and Inhibition of Mycobacterial Respiration In Vivo

Nitrite as the major source of nitric oxide production by Arabidopsis thaliana in response to Pseudomonas syringae

Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins

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