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

Chronic exposure to warm temperature causes low sperm abundance and quality in Drosophila melanogaster

Abstract Temperature influences male fertility across organisms; however, how suboptimal temperatures affect adult spermatogenesis remains understudied. In a recent study on Drosophila melanogaster oogenesis, we observed a drastic reduction in the fertility of adult males exposed to warm temperature (29 °C). Here, we show that males become infertile at 29 °C because of low sperm abundance and quality. The low sperm abundance at 29 °C does not stem from reduced germline stem cell or spermatid numbers, as those numbers remain comparable between 29 °C and control 25 °C. Notably, males at cold 18 °C and 29 °C had similarly increased frequencies of spermatid elongation and individualization defects which, considering the high sperm abundance and male fertility measured at 18 °C, indicate that spermatogenesis has a high tolerance for elongation and individualization defects. Interestingly, the abundance of sperm at 29 °C decreases abruptly and with no evidence of apoptosis as they transition into the seminal vesicle near the end of spermatogenesis, pointing to sperm elimination through an unknown mechanism. Finally, sperm from males at 29 °C fertilize eggs less efficiently and do not support embryos past the first stage of embryogenesis, indicating that poor sperm quality is an additional cause of male infertility at 29 °C

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

Abstract Background NADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not been examined. In this study, we analyzed the evolution of this gene family in arthropods. A thorough search of genomes and transcriptomes was performed enabling us to browse most branches of arthropod phylogeny. Results We have found that the subfamilies NOX5 and DUOX are present in all arthropod groups. We also show that a NOX gene, closely related to NOX4 and previously found only in mosquitoes (NOXm), can also be found in other taxonomic groups, leading us to rename it as NOX4-art. Although the accessory protein p22-phox, essential for NOX1-4 activation, was not found in any of the arthropods studied, NOX4-art of Aedes aegypti encodes an active protein that produces H2O2. Although NOX4-art has been lost in a number of arthropod lineages, it has all the domains and many signature residues and motifs necessary for ROS production and, when silenced, H2O2 production is considerably diminished in A. aegypti cells. Conclusions Combining all bioinformatic analyses and laboratory work we have reached interesting conclusions regarding arthropod NOX gene family evolution. NOX5 and DUOX are present in all arthropod lineages but it seems that a NOX2-like gene was lost in the ancestral lineage leading to Ecdysozoa. The NOX4-art gene originated from a NOX4-like ancestor and is functional. Although no p22-phox was observed in arthropods, there was no evidence of neo-functionalization and this gene probably produces H2O2 as in other metazoan NOX4 genes. Although functional and present in the genomes of many species, NOX4-art was lost in a number of arthropod lineages

Heme crystallization in the midgut of triatomine insects

Submitted by Martha Martínez Silveira ([email protected]) on 2015-05-20T17:50:01Z No. of bitstreams: 1 Oliveira MF Heme crystalization in the midgut......pdf: 470893 bytes, checksum: 07f8324e561a2a3503de46cea2de522c (MD5)Approved for entry into archive by Martha Martínez Silveira ([email protected]) on 2015-05-20T18:02:53Z (GMT) No. of bitstreams: 1 Oliveira MF Heme crystalization in the midgut......pdf: 470893 bytes, checksum: 07f8324e561a2a3503de46cea2de522c (MD5)Made available in DSpace on 2015-05-20T18:02:53Z (GMT). No. of bitstreams: 1 Oliveira MF Heme crystalization in the midgut......pdf: 470893 bytes, checksum: 07f8324e561a2a3503de46cea2de522c (MD5) Previous issue date: 2007Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilPetrobrás. CENPES. Setor de Química. Rio de Janeiro, RJ, BrasilUniversidade Estadual do Norte Fluminense. Centro de Biociências e Biotecnologia. Campos de Goytacazes, RJ, BrasilUniversidade Estadual do Norte Fluminense. Centro de Biociências e Biotecnologia. Campos de Goytacazes, RJ, BrasilUniversidade Estadual do Norte Fluminense. Centro de Biociências e Biotecnologia. Campos de Goytacazes, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Rio de Janeiro, RJ, BrasilHemozoin (Hz) is a heme crystal produced by several blood-feeding organisms in order to detoxify free heme released upon hemoglobin (Hb) digestion. Here we show that heme crystallization also occurs in three species of triatomine insects. Ultraviolet-visible and infrared light absorption spectra of insoluble pigments isolated from the midgut of three triatomine species Triatoma infestans, Dipetalogaster maximus and Panstrongylus megistus indicated that all produce Hz. Morphological analysis of T. infestans and D. maximus midguts revealed the close association of Hz crystals to perimicrovillar membranes and also as multicrystalline assemblies, forming nearly spherical structures. Heme crystallization was promoted by isolated perimicrovillar membranes from all three species of triatomine bugs in vitro in heat-sensitive reactions. In conclusion, the data presented here indicate that Hz formation is an ancestral adaptation of Triatominae to a blood-sucking habit and that the presence of perimicrovillar membranes plays a central role in this process. © 2007 Elsevier Inc. All rights reserve

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

ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells

Submitted by sandra infurna ([email protected]) on 2016-03-03T12:54:57Z No. of bitstreams: 1 flavio_lara_etal_IOC_2015.PDF: 6748083 bytes, checksum: 0febe6bad842ab7fb38d1aad3c3aad7e (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-03-03T13:21:47Z (GMT) No. of bitstreams: 1 flavio_lara_etal_IOC_2015.PDF: 6748083 bytes, checksum: 0febe6bad842ab7fb38d1aad3c3aad7e (MD5)Made available in DSpace on 2016-03-03T13:21:47Z (GMT). No. of bitstreams: 1 flavio_lara_etal_IOC_2015.PDF: 6748083 bytes, checksum: 0febe6bad842ab7fb38d1aad3c3aad7e (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio Grande do Sul. Faculdade de Veterinária. Centro de Biotecnologia. Porto Alegre, RS, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.Universidade Estadual Paulista. Faculdade de Ciências Agrárias e Veterinárias. Departamento de Patologia Veterinária. Jaboticabal, SP, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular–INCTEM. Rio de Janeiro, RJ, Brasil.University of Texas at El Paso. The Border Biomedical Research Center. El Paso, Texas, USA.Universidade Federal do Rio Grande do Sul. Faculdade de Veterinária. Centro de Biotecnologia. Porto Alegre, RS, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular–INCTEM. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular–INCTEM. Rio de Janeiro, RJ, Brasil.In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a wellknown inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides