NADPH Oxidase 5 Is a Pro‐Contractile Nox Isoform and a Point of Cross‐Talk for Calcium and Redox Signaling‐Implications in Vascular Function

Background NADPH Oxidase 5 (Nox5) is a calcium‐sensitive superoxide‐generating Nox. It is present in lower forms and higher mammals, but not in rodents. Nox5 is expressed in vascular cells, but the functional significance remains elusive. Given that contraction is controlled by calcium and reactive oxygen species, both associated with Nox5, we questioned the role of Nox5 in pro‐contractile signaling and vascular function. Methods and Results Transgenic mice expressing human Nox5 in a vascular smooth muscle cell–specific manner (Nox5 mice) and Rhodnius prolixus, an arthropod model that expresses Nox5 endogenoulsy, were studied. Reactive oxygen species generation was increased systemically and in the vasculature and heart in Nox5 mice. In Nox5‐expressing mice, agonist‐induced vasoconstriction was exaggerated and endothelium‐dependent vasorelaxation was impaired. Vascular structural and mechanical properties were not influenced by Nox5. Vascular contractile responses in Nox5 mice were normalized by N‐acetylcysteine and inhibitors of calcium channels, calmodulin, and endoplasmic reticulum ryanodine receptors, but not by GKT137831 (Nox1/4 inhibitor). At the cellular level, vascular changes in Nox5 mice were associated with increased vascular smooth muscle cell [Ca2+]i, increased reactive oxygen species and nitrotyrosine levels, and hyperphosphorylation of pro‐contractile signaling molecules MLC20 (myosin light chain 20) and MYPT1 (myosin phosphatase target subunit 1). Blood pressure was similar in wild‐type and Nox5 mice. Nox5 did not amplify angiotensin II effects. In R. prolixus, gastrointestinal smooth muscle contraction was blunted by Nox5 silencing, but not by VAS2870 (Nox1/2/4 inhibitor). Conclusions Nox5 is a pro‐contractile Nox isoform important in redox‐sensitive contraction. This involves calcium‐calmodulin and endoplasmic reticulum–regulated mechanisms. Our findings define a novel function for vascular Nox5, linking calcium and reactive oxygen species to the pro‐contractile molecular machinery in vascular smooth muscle cells

NADPH oxidase 5 is a pro‐contractile Nox isoform and a point of cross‐talk for calcium and redox signaling‐implications in vascular function

Background: NADPH Oxidase 5 (Nox5) is a calcium‐sensitive superoxide‐generating Nox. It is present in lower forms and higher mammals, but not in rodents. Nox5 is expressed in vascular cells, but the functional significance remains elusive. Given that contraction is controlled by calcium and reactive oxygen species, both associated with Nox5, we questioned the role of Nox5 in pro‐contractile signaling and vascular function. Methods and Results: Transgenic mice expressing human Nox5 in a vascular smooth muscle cell–specific manner (Nox5 mice) and Rhodnius prolixus, an arthropod model that expresses Nox5 endogenoulsy, were studied. Reactive oxygen species generation was increased systemically and in the vasculature and heart in Nox5 mice. In Nox5‐expressing mice, agonist‐induced vasoconstriction was exaggerated and endothelium‐dependent vasorelaxation was impaired. Vascular structural and mechanical properties were not influenced by Nox5. Vascular contractile responses in Nox5 mice were normalized by N‐acetylcysteine and inhibitors of calcium channels, calmodulin, and endoplasmic reticulum ryanodine receptors, but not by GKT137831 (Nox1/4 inhibitor). At the cellular level, vascular changes in Nox5 mice were associated with increased vascular smooth muscle cell [Ca2+]i, increased reactive oxygen species and nitrotyrosine levels, and hyperphosphorylation of pro‐contractile signaling molecules MLC20 (myosin light chain 20) and MYPT1 (myosin phosphatase target subunit 1). Blood pressure was similar in wild‐type and Nox5 mice. Nox5 did not amplify angiotensin II effects. In R. prolixus, gastrointestinal smooth muscle contraction was blunted by Nox5 silencing, but not by VAS2870 (Nox1/2/4 inhibitor). Conclusions: Nox5 is a pro‐contractile Nox isoform important in redox‐sensitive contraction. This involves calcium‐calmodulin and endoplasmic reticulum–regulated mechanisms. Our findings define a novel function for vascular Nox5, linking calcium and reactive oxygen species to the pro‐contractile molecular machinery in vascular smooth muscle cells

Blood Meal-Derived Heme Decreases ROS Levels in the Midgut of Aedes aegypti and Allows Proliferation of Intestinal Microbiota

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme

Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

Contains fulltext : 172380.pdf (publisher's version ) (Open Access

Amino acids trigger down-regulation of superoxide via TORC pathway in the midgut of Rhodnius prolixus

Submitted by Sandra Infurna ([email protected]) on 2016-12-01T11:54:35Z No. of bitstreams: 1 fernando_genta_etal_IOC_2016.pdf: 833246 bytes, checksum: ffe49bf298fa139b61467e9b6dc81ed4 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-01T12:10:03Z (GMT) No. of bitstreams: 1 fernando_genta_etal_IOC_2016.pdf: 833246 bytes, checksum: ffe49bf298fa139b61467e9b6dc81ed4 (MD5)Made available in DSpace on 2016-12-01T12:10:03Z (GMT). No. of bitstreams: 1 fernando_genta_etal_IOC_2016.pdf: 833246 bytes, checksum: ffe49bf298fa139b61467e9b6dc81ed4 (MD5) Previous issue date: 2016Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Resposta ao Estresse. Rio de Janeiro, RJ, Brasil. / Universidade Federal do Rio de Janeiro. Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem. Laboratório de Inflamação e Metabolismo. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil.Universidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratório de Biologia Vascular. São Paulo, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Universidade de São Paulo. Escola de Medicina. Instituto do Coração. Laboratório de Biologia Vascular. São Paulo, SP, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Resposta ao Estresse. Rio de Janeiro, RJ, Brasil. / Universidade Federal do Rio de Janeiro. Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem. Laboratório de Inflamação e Metabolismo. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo De Meis. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica de Artrópodes Hematófagos. Rio de Janeiro, RJ, Brasil / Instituto Nacional e Tecnologia em Entomologia Molecular, Rio de Janeiro, RJ, Brasil.Sensing incoming nutrients is an important and critical event for intestinal cells to sustain life of the whole organism. The TORC is a major protein complex involved in monitoring the nutritional status and is activated by elevated amino acid concentrations. An important feature of haematophagy is that huge amounts of blood are ingested in a single meal, which results in the release of large quantities of amino acids, together with the haemoglobin prosthetic group, haem, which decomposes hydroperoxides and propagates oxygen-derived free radicals. Our previous studies demonstrated that reactive oxygen species (ROS) levels were diminished in the mitochondria and midgut of the Dengue fever mosquito, Aedes aegypti, immediately after a blood meal. We proposed that this mechanism serves to avoid oxidative damage that would otherwise be induced by haem following a blood meal. Studies also performed in mosquitoes have shown that blood or amino acids controls protein synthesis through TORC activation. It was already proposed, in different models, a link between ROS and TOR, however, little is known about TOR signalling in insect midgut nor about the involvement of ROS in this pathway. Here, we studied the effect of a blood meal on ROS production in the midgut of Rhodnius prolixus We observed that blood meal amino acids decreased ROS levels in the R. prolixus midgut immediately after feeding, via lowering mitochondrial superoxide production and involving the amino acid-sensing TORC pathway

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection

<div><p>Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite <i>Trypanosoma cruzi</i>, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and <i>T</i>. <i>cruzi</i>, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization <i>in vivo</i> by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total <i>T</i>. <i>cruzi</i> parasite counts in <i>R</i>. <i>prolixus</i> were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and <i>T</i>. <i>cruzi</i> life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and <i>T</i>. <i>cruzi</i> transmission.</p></div

Identification of a selenium-dependent glutathione peroxidase in the blood-sucking insect Rhodnius prolixus

The selenium-dependent glutathione peroxidase (SeGPx) is a well-studied enzyme that detoxifies organic and hydrogen peroxides and provides cells or extracellular fluids with a key antioxidant function. The presence of a SeGPx has not been unequivocally demonstrated in insects. In the present work, we identified the gene and studied the function of a Rhodnius prolixus SeGPx (RpSeGPx). The RpSeGPx mRNA presents the UGA codon that encodes the active site selenocysteine (Sec) and a corresponding Sec insertion sequence (SECIS) in the 3' UTR region. The encoded protein includes a signal peptide, which is consistent with the high levels of GPx enzymatic activity in the insect's hemolymph, and clusters phylogenetically with the extracellular mammalian GPx03. This result contrasts with all other known insect GPxs, which use a cysteine residue instead of Sec and cluster with the mammalian phospholipid hydroperoxide GPx04. RpSeGPx is widely expressed in insect organs, with higher expression levels in the fat body. RNA interference (RNAi) was used to reduce RpSeGPx gene expression and GPx activity in the hemolymph. Adult females were apparently unaffected by RpSeGPx RNAi, whereas first instar nymphs showed a three-day delay in ecdysis. Silencing of RpSeGPx did not alter the gene expression of the antioxidant enzymes catalase, xanthine dehydrognase and a cysteine-GPx, but it reduced the levels of the dual oxidase and NADPH oxidase 5 transcripts that encode for enzymes releasing extracellular hydrogen peroxide/superoxide. Collectively, our data suggest that RpSeGPx functions in the regulation of extracellular (hemolymph) redox homeostasis of R. prolixus. (C) 2015 Elsevier Ltd. All rights reserved

Schematic model of physiological consequences of blocked Hz formation in triatomine midgut.

<p>In the presence of QND, heme derived from blood meal forms stable complexes with this drug, impairing Hz formation in the midgut lumen. Non-crystallized heme levels build up in the midgut causing cytotoxic effects to <i>T</i>. <i>cruzi</i> trypomastigotes. Excessive heme is transported to hemolymph through the midgut cells by hemoxisomes/residual bodies, causing redox imbalance and autophagy in the midgut. Heme accumulates in the hemolymph, increasing RHBP production, as a compensatory defense against "free" heme. However, this mechanism is overwhelmed, as the levels of urate drop. Redox imbalance has a direct effect on oogenesis, reducing egg production.</p

Oogenesis and <i>Trypanosoma cruzi</i> infection depend on heme crystallization in the midgut.

<p>Insects were fed with blood (Ctrl) or blood supplemented with 100 μM QND or blood supplemented with 100 μM QND and 1 mM urate (QND+urate). <b>(A)</b> Stereoscope images of adult female abdomens from IBqM colony four days after feeding. Ovaries are depicted as dashed white lines. <b>(B)</b> The average number of eggs laid per females from Fiocruz colony insects fed with blood was determined along 24 days after blood meal. Ctrl: n≥3; 100 μM QND: n≥3; QND+urate: n≥3. Comparisons between groups were done by two-way ANOVA and a <i>posteriori</i> Bonferroni’s tests (^ap<0.0001 relative to QND and QND+urate, and ^bp<0.0001 relative to QND). <b>(C)</b> Total <i>T</i>. <i>cruzi</i> counts in the digestive tract of infected adult females from Fiocruz colony 15 days after blood meal. Ctrl: n = 18; 100 μM QND: n = 18; QND + urate: n = 8). Comparisons between groups were done by one-way ANOVA, (*<i>p</i> = 0.007 relative to Ctrl), with a <i>posteriori</i> Bonferroni’s tests. Data in Fig 4B were expressed as mean ± S.E.M., and in Fig 4C as scattered plot with gray lines representing medians.</p

Impaired heme crystallization affects posterior midgut organization.

<p><b>(A and B)</b> Light microscopy images of posterior midguts of adult insects fed with blood (Ctrl; <b>A</b>) and blood supplemented with 100 μM QND (<b>B</b>) from IBqM colony four days after blood meal (bars = 20 μm). Midgut lumen (ML), midgut cells (Mc), hemocoel (Hc), vacuoles (black arrows) and lipid droplets (white arrows) are indicated in the images. <b>(C-H)</b> Transmission electron microscopy images of posterior midguts from insects maintained at IBqM (<b>C-F</b>), and Fiocruz (<b>G,H</b>) colonies fed with blood (Ctrl, <b>C,F</b>), or blood + 100 μM QND (<b>D,E,G,H</b>) four days after blood meal. The general architecture of posterior midgut cells in control insects (<b>C,F</b>) includes mitochondria (white arrows), endoplasmic reticulum (dashed box) and microvilli (asterisks). In QND treated insects (<b>D,E,G,H</b>), extensive organelle disappearance contrasts with the presence of numerous electron-dense hemoxisomes/residual bodies (arrowheads), vacuoles (black arrow), and intracellular lipid droplets (LD). Electron-dense mitochondria found in the posterior midgut of control insects (<b>F</b>), contrast with swollen and washed out mitochondria from 100 μM QND treated insects (<b>G</b>). <b>(H)</b> Mitochondria inside an autophagosome. (Scale bars: C-E: 2 μm; F: 1.0 μm; G,H: 0.5 μm).</p