Quantitative determination of nitric oxide production in haemocytes: Nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae

Semenova, Alexandra; Glazachev, Yuriy I.; Slepneva, Irina A.; Глупов, В. В.

doi:10.1016/j.niox.2013.12.011

Cited by 12 publications

(8 citation statements)

References 50 publications

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“…This fluorescent probe is widely used to register nitric oxide; however, it does not react directly with nitric oxide but with its oxidized forms, NO 2 27 or N 2 O 3 20 , resulting in the formation of a highly fluorescent product, DAF-FM triazole. In our previous study 21 , we have observed that the rate of DAF-FM triazole formation is proportional to generation rate of NO 2 . In the present study, we added PO to the system PAPA/NO and DAF-FM.…”

Section: Nitric Oxide Oxidation In the Presence Of Pomentioning

confidence: 86%

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Dual effect of nitric oxide on phenoloxidase-mediated melanization

Sanzhaeva

Vorontsova

Glazachev

et al. 2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO 2 at the active site of PO followed by oxidation of DOPA by NO 2 . On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect's haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.

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Section: Nitric Oxide Oxidation In the Presence Of Pomentioning

confidence: 86%

“…Fluorescence kinetics were recorded using a microplate fluorometer. Experimental conditions are described in 21 .…”

Section: Detection Of Daf-fm Triazole Formationmentioning

confidence: 99%

Dual effect of nitric oxide on phenoloxidase-mediated melanization

Sanzhaeva

Vorontsova

Glazachev

et al. 2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…The groups in which heat-killed lactobacilli triggered hemolymph melanization, initially, had no maintenance of this response and when the larvae were infected with S. aureus the insect defense system recognized the threat and induced greater NO production, which is an important molecule in the immune response to microorganisms and parasites in insects. In addition, Semenova et al (2014) reported that a quantitative analysis of NO production in insects is more complicated than in mammals due to a deceleration of metabolic activities and Whitten et al (2007) observed that the NO-mediated immune response in insects is specific for each pathogen and that through inoculation of LPS, it was more remarkable in the body fat than in haemocytes.…”

Section: Discussionmentioning

confidence: 99%

Effect of Lactobacillus rhamnosus on the response of Galleria mellonella against Staphylococcus aureus and Escherichia coli infections

et al. 2017

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This study evaluated the prophylactic effects of the live or heat-killed probiotic strain Lactobacillus rhamnosus ATCC 7469 in Galleria mellonella, inoculated with Staphylococcus aureus or Escherichia coli. L. rhamnosus suspension was prepared and a part of it was autoclaved to obtain heat-killed lactobacilli. The larvae were inoculated of these suspensions and pathogenic. The survival of the larvae was observed during 7 days and after 24 h of inoculation haemocytes counted, melanization and nitric oxide production were analyzed. Larvae survival rate increased in the group inoculated with heat-killed L. rhamnosus, however, with no statistical difference. There was a significant increase in total haemocyte counts in all test groups. Haemolymph melanization and nitric oxide production were higher in the group inoculated with L. rhamnosus and infected with S. aureus. It was concluded that, in this model of infection, heat-killed L. rhamnosus ATCC 7469 promoted greater protection in Galleria mellonella infected with S. aureus or E. coli.

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“…Therefore, in addition the G. mellonella infection model was employed. Haemocytes from G. mellonella are able to produce NO when they are confronted with pathogens or LPS (Krishnan et al, 2006; Semenova et al, 2014). Also in this invertebrate model that does not require immunosuppression, virulence of the mutants was not affected.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence

et al. 2014

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Aspergillus fumigatus is a saprophytic mold that can cause life-threatening infections in immunocompromised patients. In the lung, inhaled conidia are confronted with immune effector cells that attack the fungus by various mechanisms such as phagocytosis, production of antimicrobial proteins or generation of reactive oxygen intermediates. Macrophages and neutrophils can also form nitric oxide (NO) and other reactive nitrogen intermediates (RNI) that potentially also contribute to killing of the fungus. However, fungi can produce several enzymes involved in RNI detoxification. Based on genome analysis of A. fumigatus, we identified two genes encoding flavohemoglobins, FhpA, and FhpB, which have been shown to convert NO to nitrate in other fungi, and a gene encoding S-nitrosoglutathione reductase GnoA reducing S-nitrosoglutathione to ammonium and glutathione disulphide. To elucidate the role of these enzymes in detoxification of RNI, single and double deletion mutants of FhpA, FhpB, and GnoA encoding genes were generated. The analysis of mutant strains using the NO donor DETA-NO indicated that FhpA and GnoA play the major role in defense against RNI. By generating fusions with the green fluorescence protein, we showed that both FhpA-eGFP and GnoA-eGFP were located in the cytoplasm of all A. fumigatus morphotypes, from conidia to hyphae, whereas FhpB-eGFP was localized in mitochondria. Because fhpA and gnoA mRNA was also detected in the lungs of infected mice, we investigated the role of these genes in fungal pathogenicity by using a murine infection model for invasive pulmonary aspergillosis. Remarkably, all mutant strains tested displayed wild-type pathogenicity, indicating that the ability to detoxify host-derived RNI is not essential for virulence of A. fumigatus in the applied mouse infection model. Consistently, no significant differences in killing of ΔfhpA, ΔfhpB, or ΔgnoA conidia by cells of the macrophage cell line MH-S were observed when compared to the wild type.

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Quantitative determination of nitric oxide production in haemocytes: Nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae

Cited by 12 publications

References 50 publications

Dual effect of nitric oxide on phenoloxidase-mediated melanization

Dual effect of nitric oxide on phenoloxidase-mediated melanization

Effect of Lactobacillus rhamnosus on the response of Galleria mellonella against Staphylococcus aureus and Escherichia coli infections

Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence

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