Nitric Oxide Synthase (NOS) Characterization in Leishmania amazonensis Axenic Amastigotes

Genestra, Marcelo; Guedes-Silva, Damiana; Souza, Wilson Jacinto Silva de; Cysne-Finkelstein, Léa; Soares‐Bezerra, Rômulo José; Monteiro, Fabiane Pereira; Leon, Leonor L.

doi:10.1016/j.arcmed.2005.07.011

Cited by 34 publications

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“…NOS enzyme in the native form exists as a dimer in Leishmania parasites (17) and has been characterized in both forms of the parasite (16,18,42). The presence of NOS activity prompted us to measure levels of intracellular NO in different Leishmania spp.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, sodium antimony gluconate, the main stay of treatment for leishmaniasis (14), generates free radicals within parasites (15). Studies revealed that orthologues of NOS (neuronal NOS or nNOS) are present in Leishmania (16,17) and its activity was measured by the NADPH consumption assay (18). In this study, we have established a flow cytometry based method to measure production of NO in parasites after treatment with conventional anti-leishmanial drugs.…”

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confidence: 99%

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Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

et al. 2010

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Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 lM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 lM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds. ' International Society for Advancement of Cytometry Key termsanti-leishmanial; DAF-2DA; flow cytometry; leishmaniasis; nitric oxide LEISHMANIA are intracellular protozoan parasites that infect host mononuclear phagocytes, the resultant complex of diseases being leishmaniasis affecting 12 million people world wide in 88 countries (1). This disease is characterized by development of dermal lesions that may be cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), or mucocutaneous leishmaniasis (MCL) as also there can be systemic involvement as in the case of visceral leishmaniasis (VL), which is potentially fatal if left untreated (2).This obligate intracellular parasite resides and multiplies within macrophages, and therefore it should be able to evade the cytotoxic mechanisms innately operative within the macrophages for its survival (3). One of the predominant cytotoxic mechanisms includes production of reactive nitrogen intermediates, which the Leishmania parasite effectively decreases to ensure its survival (4,5).

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Section: Resultsmentioning

confidence: 99%

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Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

et al. 2010

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“…The isolation and characterization of NOS from L. donovani promastigotes [44] and L. amazonensis axenic amastigotes and promastigotes [46] revealed that this parasite-derived enzyme closely resembles its mammalian constitutive NOS (cNOS), which requires Ca 2+ , calmoduline, and NADPH for its activity [46]. Importantly, culture media derived from amastigotes or highly infectious metacyclics appeared to contain relatively high NOS activities [46], implying a potential correlation between Leishmania NOS and parasite infectivity [47]. However, the biological relevance of these findings in parasitic infections remains unclear.…”

Section: L-arginine Metabolism In Leishmania Parasitesmentioning

confidence: 99%

L-arginine metabolism and its impact on host immunity against Leishmania infection

2007

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Leishmaniasis is a vector-borne disease found in many countries worldwide. The causative agent of the disease, Leishmania spp., lives as an obligate intracellular parasite within mammalian hosts. Since tissue macrophages are major target cells for parasite replication, the outcome of infection depends largely on the activation status of these cells. L-arginine is a crucial amino acid required for both nitric oxide (NO)-mediated parasite killing and polyamine-mediated parasite replication. This review highlights the significance of L-arginine as a factor determining the outcomes of Leishmania infection in vitro and its influences on host immune responses in vivo. Various therapeutic approaches targeting L-arginine metabolic pathways during infections with Leishmania are also discussed.

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“…In our researches of experimental chemotherapy to treat Leishmaniasis, we evaluated the effect of the more active 4-phenyl-5-cinnamoyl-1,3,4-thiadiazolium-2-phenylamine chlorides (3 0 -or 4 0 -methoxy) which showed a significative effect in the reduction of the NO production in promastigotes and axenic amastigotes cultures. The inhibition was evident through NADPH consumption by NOS purified from both evolutive forms of the parasite (that presented the cNOS form of the enzyme) [6,7]. No significant effect on the arginase activity was verified in IP, but the 3 0 -methoxy cinnamoyl compound was able to inhibit this enzyme activity.…”

Section: Resultsmentioning

confidence: 99%

“…Although the macrophages triggered their defense mechanism to neutralize the parasite, there are evidences that the NO pathway from L. amazonensis participates in the parasite-host interaction, and there is a correlation between NO production and the amount of metacyclic forms in the culture of infective forms [4 -6]. Additionally, recent results also demonstrated that L. amazonensis axenic amastigotes produce NO [7], as well as the fact that the NOS activity is evident in this evolutive form, suggesting that both the NOS pathway and the arginase are cross talking during the signaling involving host cell-parasite interaction [3][4][5][6][7][8].…”

Section: Introductionmentioning

confidence: 97%

Effect of mesoionic 4-phenyl-5-(cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivative salts on the activities of the nitric oxide synthase and arginase of Leishmania amazonensis

Soares‐Bezerra

Silva

Echevarrı́a

et al. 2008

Journal of Enzyme Inhibition and Medicinal Chemistry

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L-arginine is involved in the production of both nitric oxide (NO), mediated by nitric oxide synthase (NOS) and L-ornithine, by arginase activity. It is generally accepted that NO regulation occurs mainly at the transcriptional level of NOS. In a previous work we purported that there is evidence that Leishmania sp. can produce NO from L-arginine. An arginase activity in its gene sequence has also been reported in Leishmania parasites. In a search for intracellular targets as potential antileishmanicidal agents, such as the L-arginine metabolism, we used 1,3,4-thiadiazolium mesoionic compounds, that have been demonstrated to be cytotoxic to the Leishmania amazonensis, when compared to Pentamidine isethionate as a reference drug. Parasites were assayed in absence/presence of 4 0 -and 3 0 -methoxy mesoionic derivatives in order to verify the effect on NO production and arginase activity in L. amazonensis. The results indicated that the drugs reduce from 70 to 90% of the NO production by the parasite and act on a soluble nitric oxide synthase purified from L. amazonensis promastigotes and axenic amastigotes.

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Nitric Oxide Synthase (NOS) Characterization in Leishmania amazonensis Axenic Amastigotes

Cited by 34 publications

References 36 publications

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

L-arginine metabolism and its impact on host immunity against Leishmania infection

Effect of mesoionic 4-phenyl-5-(cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivative salts on the activities of the nitric oxide synthase and arginase of Leishmania amazonensis

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