Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca2+, phosphorylation, and protein-protein interactions on RBOH-dependent ROS production.
- Klíčová slova
- Human embryonic kidney 293T (HEK293T), Luminol, NADPH oxidase, Respiratory oxidase homolog (RBOH),
- MeSH
- HEK293 buňky MeSH
- ledviny metabolismus MeSH
- lidé MeSH
- NADPH-oxidasy * metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulace genové exprese u rostlin * MeSH
- rostliny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- NADPH-oxidasy * MeSH
- reaktivní formy kyslíku MeSH
Numerous pathological changes of subcellular structures are characteristic hallmarks of neurodegeneration. The main research has focused to mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomal networks as well as microtubular system of the cell. The sequence of specific organelle damage during pathogenesis has not been answered yet. Exposition to rotenone is used for simulation of neurodegenerative changes in SH-SY5Y cells, which are widely used for in vitro modelling of Parkinson´s disease pathogenesis. Intracellular effects were investigated in time points from 0 to 24 h by confocal microscopy and biochemical analyses. Analysis of fluorescent images identified the sensitivity of organelles towards rotenone in this order: microtubular cytoskeleton, mitochondrial network, endoplasmic reticulum, Golgi apparatus and lysosomal network. All observed morphological changes of intracellular compartments were identified before alphaS protein accumulation. Therefore, their potential as an early diagnostic marker is of interest. Understanding of subcellular sensitivity in initial stages of neurodegeneration is crucial for designing new approaches and a management of neurodegenerative disorders.
- MeSH
- apoptóza MeSH
- insekticidy toxicita MeSH
- lidé MeSH
- mikrotubuly účinky léků metabolismus patologie MeSH
- mitochondrie účinky léků metabolismus patologie MeSH
- nádorové buněčné linie MeSH
- NADPH-oxidasy metabolismus MeSH
- neuroblastom komplikace MeSH
- neurodegenerativní nemoci etiologie metabolismus patologie MeSH
- rotenon toxicita MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- insekticidy MeSH
- NADPH-oxidasy MeSH
- rotenon MeSH
For long time bilirubin was only considered as a potentially dangerous sign of liver diseases, but it now appears clear that it is also a powerful signaling molecule. Together with potent antioxidant activities that were only reported in the last few decades, many other biological effects have now been clearly described. These include especially profound inhibitory effects on almost all effectors of the immune system, with their clinical consequences in the bilirubin-mediated protection against autoimmune and inflammatory diseases. Separate from these, bilirubin activates various nuclear and cytoplasmic receptors, resembling the endocrine activities of actual hormonal substances. This is true for the "classical" hepatic nuclear receptors, including the aryl hydrocarbon receptor, or the constitutive androstane receptor; and also for some lesser-explored receptors such as peroxisome proliferator-activated receptors α and γ; Mas-related G protein-coupled receptor; or other signaling molecules including fatty acid binding protein 1, apolipoprotein D, or reactive oxygen species. All of these targets have broad metabolic effects, which in turn may offer protection against obesity, diabetes mellitus, and other metabolic diseases. The (mostly experimental) data are also supported by clinical evidence. In fact, data from the last three decades have convincingly demonstrated the protective effects of mildly elevated serum bilirubin concentrations against various "diseases of civilization." Additionally, even tiny, micromolar changes of serum bilirubin concentrations have been associated with substantial alteration in the risks of these diseases. It is highly likely that all of the biological activities of bilirubin have yet to be exhaustively explored, and thus we can expect further clinical discoveries about this evolutionarily old molecule into the future.
- Klíčová slova
- bilirubin, cell signalization, civilization diseases, endocrine effects, metabolism,
- MeSH
- bilirubin metabolismus MeSH
- cirkadiánní rytmus fyziologie MeSH
- lidé MeSH
- ligandy MeSH
- NADPH-oxidasy metabolismus MeSH
- receptory buněčného povrchu metabolismus MeSH
- signální transdukce * MeSH
- superoxidy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- bilirubin MeSH
- ligandy MeSH
- NADPH-oxidasy MeSH
- receptory buněčného povrchu MeSH
- superoxidy MeSH
S-nitrosoglutathione reductase (GSNOR) exerts crucial roles in the homeostasis of nitric oxide (NO) and reactive nitrogen species (RNS) in plant cells through indirect control of S-nitrosation, an important protein post-translational modification in signaling pathways of NO. Using cultivated and wild tomato species, we studied GSNOR function in interactions of key enzymes of reactive oxygen species (ROS) metabolism with RNS mediated by protein S-nitrosation during tomato root growth and responses to salinity and cadmium. Application of a GSNOR inhibitor N6022 increased both NO and S-nitrosothiol levels and stimulated root growth in both genotypes. Moreover, N6022 treatment, as well as S-nitrosoglutathione (GSNO) application, caused intensive S-nitrosation of important enzymes of ROS metabolism, NADPH oxidase (NADPHox) and ascorbate peroxidase (APX). Under abiotic stress, activities of APX and NADPHox were modulated by S-nitrosation. Increased production of H2O2 and subsequent oxidative stress were observed in wild Solanumhabrochaites, together with increased GSNOR activity and reduced S-nitrosothiols. An opposite effect occurred in cultivated S. lycopersicum, where reduced GSNOR activity and intensive S-nitrosation resulted in reduced ROS levels by abiotic stress. These data suggest stress-triggered disruption of ROS homeostasis, mediated by modulation of RNS and S-nitrosation of NADPHox and APX, underlies tomato root growth inhibition by salinity and cadmium stress.
- Klíčová slova
- S-nitrosation, S-nitrosoglutathione reductase, Solanum habrochaites, Solanum lycopersicum, abiotic stress, cadmium, nitric oxide, reactive oxygen species, root growth, salinity,
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- askorbátperoxidasa metabolismus MeSH
- benzamidy chemie metabolismus farmakologie MeSH
- chlorid sodný farmakologie MeSH
- fyziologický stres MeSH
- kadmium toxicita MeSH
- kořeny rostlin účinky léků růst a vývoj metabolismus MeSH
- NADPH-oxidasy metabolismus MeSH
- nitrosace MeSH
- oxid dusnatý metabolismus MeSH
- oxidační stres účinky léků MeSH
- peroxid vodíku metabolismus MeSH
- pyrroly chemie metabolismus farmakologie MeSH
- reaktivní formy dusíku chemie metabolismus MeSH
- reaktivní formy kyslíku chemie metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- rostlinné proteiny metabolismus MeSH
- S-nitrosoglutathion farmakologie MeSH
- S-nitrosothioly metabolismus MeSH
- Solanum lycopersicum účinky léků růst a vývoj metabolismus MeSH
- Solanum růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aldehydoxidoreduktasy MeSH
- askorbátperoxidasa MeSH
- benzamidy MeSH
- chlorid sodný MeSH
- formaldehyde dehydrogenase, glutathione-independent MeSH Prohlížeč
- kadmium MeSH
- N6022 MeSH Prohlížeč
- NADPH-oxidasy MeSH
- oxid dusnatý MeSH
- peroxid vodíku MeSH
- pyrroly MeSH
- reaktivní formy dusíku MeSH
- reaktivní formy kyslíku MeSH
- rostlinné proteiny MeSH
- S-nitrosoglutathion MeSH
- S-nitrosothioly MeSH
Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.
- MeSH
- acetofenony farmakologie MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosfatidylinositol-3-kinasy metabolismus MeSH
- fosforylace účinky léků MeSH
- myší embryonální kmenové buňky účinky léků metabolismus MeSH
- myši MeSH
- NADPH-oxidasy genetika metabolismus MeSH
- oniové sloučeniny farmakologie MeSH
- oxidační stres účinky léků MeSH
- proliferace buněk účinky léků MeSH
- proteiny Wnt metabolismus MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- synergismus léků MeSH
- transkripční faktor STAT3 metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetofenony MeSH
- acetovanillone MeSH Prohlížeč
- diphenyleneiodonium MeSH Prohlížeč
- extracelulárním signálem regulované MAP kinasy MeSH
- fosfatidylinositol-3-kinasy MeSH
- NADPH-oxidasy MeSH
- oniové sloučeniny MeSH
- proteiny Wnt MeSH
- protoonkogenní proteiny c-akt MeSH
- reaktivní formy kyslíku MeSH
- transkripční faktor STAT3 MeSH
INTRODUCTION: Chronic granulomatous disease (CGD) is an inherited mutational defect in any of the NADPH oxidase complex, CYBB (gp91-phox), NCF1 (p47-phox), CYBA (p22-phox), NCF2 (p67-phox), or NCF4 (p40-phox) leading to inability of phagocytes to perform effective respiratory burst and thus diminished killing of bacteria and fungi. The identification of defective proteins aids in establishing a diagnosis prior to genetic analysis, which is rather labor-intensive, expensive, and time-consuming. AIM: The present study aims at assessing the NADPH proteins by performing the intracellular staining with specific monoclonal antibodies and their assessment on flow cytometry. The use of flow cytometry is less laborious and faster to perform than western blot. It also confirms the diagnosis of CGD and detects the affected components allowing proper management of patients. MATERIALS AND METHODS: Twenty-eight patients from 25 different kindred, clinically suspected as CGD were recruited in Egypt. Dihydrorhodamine test was performed to confirm the diagnosis of the patients. Intracellular staining of NADPH components using specific monoclonal antibodies was performed followed by flow cytometric analysis. RESULTS: The present study revealed that the most common defective protein in our cohort is p22-phox, found in 13 patients (46.4 % of cases) followed by p47-phox in 8 patients (28.6 %), gp91-phox in 5 patients (17.9 %), and finally p67-phox in 2 patients (7.1 %). CONCLUSION: In countries with limited resources and yet large number of CGD patients, the analysis of the defective proteins by flow cytometry is an optimum solution for confirming the diagnosis and is a step for targeted sequencing in families seeking prenatal diagnosis.
- Klíčová slova
- CYBA, CYBB, Chronic granulomatous disease, Flow cytometry, NCF1, NCF2,
- MeSH
- biologické markery MeSH
- chronická granulomatózní nemoc diagnóza genetika imunologie metabolismus MeSH
- dítě MeSH
- genotyp MeSH
- imunofenotypizace MeSH
- kojenec MeSH
- lidé MeSH
- mutace MeSH
- NADP metabolismus MeSH
- NADPH-oxidasy metabolismus MeSH
- neutrofily imunologie metabolismus MeSH
- předškolní dítě MeSH
- průtoková cytometrie MeSH
- rizikové faktory MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Egypt MeSH
- Názvy látek
- biologické markery MeSH
- NADP MeSH
- NADPH-oxidasy MeSH
The c-Myb transcription factor is important for maintenance of immature cells of many tissues including colon epithelium. Overexpression of c-Myb occurring in colorectal carcinomas (CRC) as well as in other cancers often marks poor prognosis. However, the molecular mechanism explaining how c-Myb contributes to progression of CRC has not been fully elucidated. To address this point, we investigated the way how c-Myb affects sensitivity of CRC cells to anticancer drugs. Using CRC cell lines expressing exogenous c-myb we show that c-Myb protects CRC cells from the cisplatin-, oxaliplatin-, and doxorubicin-induced apoptosis, elevates reactive oxygen species via up-regulation of NOX1, and sustains the pro-survival p38 MAPK pathway. Using pharmacological inhibitors and gene silencing of p38 and NOX1 we found that these proteins are essential for the protective effect of c-Myb and that NOX1 acts upstream of p38 activation. In addition, our result suggests that transcription of NOX1 is directly controlled by c-Myb and these genes are strongly co-expressed in human tumor tissue of CRC patients. The novel c-Myb/NOX1/p38 signaling axis that protects CRC cells from chemotherapy described in this study could provide a new base for design of future therapies of CRC.
- Klíčová slova
- Cell survival, Colorectal carcinoma, NADPH oxidase, Reactive oxygen species, Signaling pathway, c-Myb,
- MeSH
- aktivace enzymů účinky léků MeSH
- apoptóza účinky léků MeSH
- cisplatina farmakologie MeSH
- doxorubicin farmakologie MeSH
- kolorektální nádory genetika patologie MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- mitogenem aktivované proteinkinasy p38 genetika metabolismus MeSH
- myši MeSH
- NADH, NADPH oxidoreduktasy genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- NADPH-oxidasa 1 MeSH
- NADPH-oxidasy genetika metabolismus MeSH
- organoplatinové sloučeniny farmakologie MeSH
- oxaliplatin MeSH
- oxidační stres účinky léků MeSH
- promotorové oblasti (genetika) genetika MeSH
- protoonkogenní proteiny c-myb metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulace genové exprese u nádorů * účinky léků MeSH
- sekvence nukleotidů MeSH
- upregulace účinky léků MeSH
- vazebná místa MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cisplatina MeSH
- doxorubicin MeSH
- messenger RNA MeSH
- mitogenem aktivované proteinkinasy p38 MeSH
- NADH, NADPH oxidoreduktasy MeSH
- NADPH-oxidasa 1 MeSH
- NADPH-oxidasy MeSH
- NOX1 protein, human MeSH Prohlížeč
- NOX1 protein, mouse MeSH Prohlížeč
- organoplatinové sloučeniny MeSH
- oxaliplatin MeSH
- protoonkogenní proteiny c-myb MeSH
- reaktivní formy kyslíku MeSH
Hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) play an important role in brain control of blood pressure (BP). One of the important mechanisms involved in the pathogenesis of hypertension is the elevation of reactive oxygen species (ROS) production by nicotine adenine dinucleotide phosphate (NADPH) oxidase. The aim of our present study was to investigate NADPH oxidase-mediated superoxide (O(2)(-)) production and to search for the signs of lipid peroxidation in hypothalamus and medulla oblongata as well as in renal medulla and cortex of hypertensive male rats transgenic for the murine Ren-2 renin gene (Ren-2 TGR) and their age-matched normotensive controls - Hannover Sprague Dawley rats (HanSD). We found no difference in the activity of NADPH oxidase measured as a lucigenin-mediated O(2)(-) production in the hypothalamus and medulla oblongata. However, we observed significantly elevated NADPH oxidase in both renal cortex and medulla of Ren-2 TGR compared with HanSD. Losartan (LOS) treatment (10 mg/kg body weight/day) for 2 months (Ren-2 TGR+LOS) did not change NADPH oxidase-dependent O(2)(-) production in the kidney. We detected significantly elevated indirect markers of lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS) in Ren-2 TGR, while they were significantly decreased in Ren-2 TGR+LOS. In conclusion, the present study shows increased NADPH oxidase activities in renal cortex and medulla with significantly increased TBARS in renal cortex. No significant changes of NADPH oxidase and markers of lipid peroxidation were detected in the studied brain regions.
- MeSH
- blokátory receptoru 1 pro angiotenzin II farmakologie terapeutické užití MeSH
- hypertenze farmakoterapie enzymologie MeSH
- ledviny účinky léků enzymologie MeSH
- losartan farmakologie terapeutické užití MeSH
- mozek účinky léků enzymologie MeSH
- NADPH-oxidasy metabolismus MeSH
- náhodné rozdělení MeSH
- peroxidace lipidů účinky léků MeSH
- potkani transgenní MeSH
- superoxidy metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- blokátory receptoru 1 pro angiotenzin II MeSH
- losartan MeSH
- NADPH-oxidasy MeSH
- superoxidy MeSH
Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), have been identified as a major source of reactive oxygen species (ROS) during plant-microbe interactions. The subcellular localization of the tobacco (Nicotiana tabacum) ROS-producing enzyme RBOHD was examined in Bright Yellow-2 cells before and after elicitation with the oomycete protein cryptogein using electron and confocal microscopy. The plasma membrane (PM) localization of RBOHD was confirmed and immuno-electron microscopy on purified PM vesicles revealed its distribution in clusters. The presence of the protein fused to GFP was also seen in intracellular compartments, mainly Golgi cisternae. Cryptogein induced, within 1h, a 1.5-fold increase in RBOHD abundance at the PM and a concomitant decrease in the internal compartments. Use of cycloheximide revealed that most of the proteins targeted to the PM upon elicitation were not newly synthesized but may originate from the Golgi pool. ROS accumulation preceded RBOHD transcript- and protein-upregulation, indicating that ROS resulted from the activation of a PM-resident pool of enzymes, and that enzymes newly addressed to the PM were inactive. Taken together, the results indicate that control of RBOH abundance and subcellular localization may play a fundamental role in the mechanism of ROS production.
- Klíčová slova
- BY-2 cells, Nicotiana tabacum, cryptogein, protein trafficking, protein trafficking., reactive oxygen species, respiratory burst oxidase homolog D (RBOHD),
- MeSH
- buněčná membrána metabolismus MeSH
- fungální proteiny metabolismus MeSH
- konfokální mikroskopie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- NADPH-oxidasy genetika metabolismus MeSH
- Phytophthora fyziologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- tabák genetika metabolismus mikrobiologie MeSH
- transmisní elektronová mikroskopie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fungální proteiny MeSH
- NADPH-oxidasy MeSH
- reaktivní formy kyslíku MeSH
- rostlinné proteiny MeSH
Spirulina platensis, a water blue-green alga, has been associated with potent biological effects, which might have important relevance in atheroprotection. We investigated whether S. platensis or phycocyanobilin (PCB), its tetrapyrrolic chromophore, can activate atheroprotective heme oxygenase-1 (Hmox1), a key enzyme in the heme catabolic pathway responsible for generation of a potent antioxidant bilirubin, in endothelial cells and in a mouse model of atherosclerosis. In vitro experiments were performed on EA.hy926 endothelial cells exposed to extracts of S. platensis or PCB. In vivo studies were performed on ApoE-deficient mice fed a cholesterol diet and S. platensis. The effect of these treatments on Hmox1, as well as other markers of oxidative stress and endothelial dysfunction, was then investigated. Both S. platensis and PCB markedly upregulated Hmox1 in vitro, and a substantial overexpression of Hmox1 was found in aortic atherosclerotic lesions of ApoE-deficient mice fed S. platensis. In addition, S. platensis treatment led to a significant increase in Hmox1 promoter activity in the spleens of Hmox-luc transgenic mice. Furthermore, both S. platensis and PCB were able to modulate important markers of oxidative stress and endothelial dysfunction, such as eNOS, p22 NADPH oxidase subunit, and/or VCAM-1. Both S. platensis and PCB activate atheroprotective HMOX1 in endothelial cells and S. platensis increased the expression of Hmox1 in aortic atherosclerotic lesions in ApoE-deficient mice, and also in Hmox-luc transgenic mice beyond the lipid lowering effect. Therefore, activation of HMOX1 and the heme catabolic pathway may represent an important mechanism of this food supplement for the reduction of atherosclerotic disease.
- MeSH
- aorta účinky léků enzymologie MeSH
- ateroskleróza farmakoterapie enzymologie prevence a kontrola MeSH
- cévní buněčněadhezivní molekula-1 genetika metabolismus MeSH
- fykobiliny aplikace a dávkování MeSH
- fykokyanin aplikace a dávkování MeSH
- hemoxygenasa-1 genetika metabolismus MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- myši MeSH
- NADPH-oxidasy genetika metabolismus MeSH
- oxidační stres účinky léků MeSH
- Spirulina chemie MeSH
- upregulace účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cévní buněčněadhezivní molekula-1 MeSH
- fykobiliny MeSH
- fykokyanin MeSH
- hemoxygenasa-1 MeSH
- NADPH-oxidasy MeSH
- phycocyanobilin MeSH Prohlížeč