ABSTRACT
Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.
Subject(s)
Gasdermins , Pyroptosis , Neoplasm Proteins/metabolism , Cardiolipins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Inflammasomes/metabolismABSTRACT
Chemogenetics refers to experimental systems that dynamically regulate the activity of a recombinant protein by providing or withholding the protein's specific biochemical stimulus. Chemogenetic tools permit precise dynamic control of specific signaling molecules to delineate the roles of those molecules in physiology and disease. Yeast d-amino acid oxidase (DAAO) enables chemogenetic manipulation of intracellular redox balance by generating hydrogen peroxide only in the presence of d-amino acids. Advances in biosensors have allowed the precise quantitation of these signaling molecules. The combination of chemogenetic approaches with biosensor methodologies has opened up new lines of investigation, allowing the analysis of intracellular redox pathways that modulate physiological and pathological cell responses. We anticipate that newly developed transgenic chemogenetic models will permit dynamic modulation of cellularredox balance in diverse cells and tissues and will facilitate the identification and validation of novel therapeutic targets involved in both physiological redox pathways and pathological oxidative stress.
Subject(s)
Hydrogen Peroxide , Oxidative Stress , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Oxidation-Reduction , Signal TransductionABSTRACT
INTRODUCTION: Centrifugal partition chromatography (CPC) is a liquid-liquid chromatography characterised by its solvent flexibility. The compounds undergoing separation are subjected to a continuous partition process between two immiscible phases in a column space free of solid support. In the context of green chemistry, it is important to substitute halogenated and petroleum-based solvents commonly used in purification processes. OBJECTIVES: The main goal of the current study was to replace classical solvents used in CPC (e.g., hexane and methanol) by green and renewable alternatives. METHODS: Solvents were first selected based on literature. Their commercial availability, price, recyclability, toxicity and ability to form two phases were particularly sought after. KEY FINDINGS: The new two-phase solvent systems were evaluated for the purification of two compounds of interest: piperine and cannabidiol. Using these alternative two-phase solvent systems allows us to isolate natural products with a high purity level (> 95%). CONCLUSION: Substituting petroleum-based solvents with bio-sourced, renewable alternatives reduces the environmental impact of CPC. Herein, new biphasic solvent systems were built using hexamethyldisiloxane, ethyl isobutyrate and 2-methyl tetrahydrofuran in combination with ethanol and water. Furthermore, this research provides a scientific basis for developing new and sustainable solvent systems in CPC.
Subject(s)
Biological Products , Petroleum , Solvents , Chromatography, Liquid , MethanolABSTRACT
BACKGROUND: Endothelial dysfunction is a critical component in the pathogenesis of cardiovascular diseases and is closely associated with nitric oxide (NO) levels and oxidative stress. Here, we report on novel findings linking endothelial expression of CD70 (also known as CD27 ligand) with alterations in NO and reactive oxygen species. METHODS: CD70 expression was genetically manipulated in human aortic and pulmonary artery endothelial cells. Intracellular NO and hydrogen peroxide (H2O2) were measured using genetically encoded biosensors, and cellular phenotypes were assessed. RESULTS: An unbiased phenome-wide association study demonstrated that polymorphisms in CD70 associate with vascular phenotypes. Endothelial cells treated with CD70-directed short-interfering RNA demonstrated impaired wound closure, decreased agonist-stimulated NO levels, and reduced eNOS (endothelial nitric oxide synthase) protein. These changes were accompanied by reduced NO bioactivity, increased 3-nitrotyrosine levels, and a decrease in the eNOS binding partner heat shock protein 90. Following treatment with the thioredoxin inhibitor auranofin or with agonist histamine, intracellular H2O2 levels increased up to 80% in the cytosol, plasmalemmal caveolae, and mitochondria. There was increased expression of NADPH oxidase 1 complex and gp91phox; expression of copper/zinc and manganese superoxide dismutases was also elevated. CD70 knockdown reduced levels of the H2O2 scavenger catalase; by contrast, glutathione peroxidase 1 expression and activity were increased. CD70 overexpression enhanced endothelial wound closure, increased NO levels, and attenuated the reduction in eNOS mRNA induced by TNFα. CONCLUSIONS: Taken together, these data establish CD70 as a novel regulatory protein in endothelial NO and reactive oxygen species homeostasis, with implications for human vascular disease.
Subject(s)
CD27 Ligand , Endothelial Cells , Nitric Oxide , CD27 Ligand/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Humans , Hydrogen Peroxide/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species/metabolismABSTRACT
Mitochondrial mRNAs encode key subunits of the oxidative phosphorylation complexes that produce energy for the cell. In Saccharomyces cerevisiae, mitochondrial translation is under the control of translational activators, specific to each mRNA. In Schizosaccharomyces pombe, which more closely resembles the human system by its mitochondrial DNA structure and physiology, most translational activators appear to be either lacking, or recruited for post-translational functions. By combining bioinformatics, genetic and biochemical approaches we identified two interacting factors, Cbp7 and Cbp8, controlling Cytb production in S. pombe. We show that their absence affects cytb mRNA stability and impairs the detection of the Cytb protein. We further identified two classes of Cbp7/Cbp8 partners and showed that they modulated Cytb or Cox1 synthesis. First, two isoforms of bS1m, a protein of the small mitoribosomal subunit, that appear mutually exclusive and confer translational specificity. Second, a complex of four proteins dedicated to Cox1 synthesis, which includes an RNA helicase that interacts with the mitochondrial ribosome. Our results suggest that S. pombe contains, in addition to complexes of translational activators, a heterogeneous population of mitochondrial ribosomes that could specifically modulate translation depending on the mRNA translated, in order to optimally balance the production of different respiratory complex subunits.
Subject(s)
Electron Transport Chain Complex Proteins/genetics , Mitochondria/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Mitochondrial/genetics , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Computational Biology/methods , Cytochromes b/genetics , Cytochromes b/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Electron Transport Chain Complex Proteins/metabolism , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Gene Expression Regulation, Fungal , Mitochondria/metabolism , Oxidative Phosphorylation , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Stability , RNA, Messenger/metabolism , RNA, Mitochondrial/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/metabolism , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
As an alternative to fossil volatile hydrocarbon solvents used nowadays in perfumery, investigation on essential oil of Commiphora wildii Merxm. oleo gum resin as a source of heptane is reported here. Heptane, representing up to 30 wt-% of this oleo gum resin, was successfully isolated from the C. wildii essential oil, using an innovative double distillation process. Isolated heptane was then used as a solvent in order to extract some noble plants of perfumery. It was found that extracts obtained with this solvent were more promising in terms of sensory analysis than those obtained from fossil-based heptane. In addition, in order to valorize the essential oil depleted from heptane, chemical composition of this oil was found to obtain, and potential biological activity properties were studied. A total of 172 different compounds were identified by GC-MS in the remaining oil. In vitro tests-including hyaluronidase, tyrosinase, antioxidant, elastase and lipoxygenase, as well as inhibitory tests against two yeasts and 21 bacterial strains commonly found on the skin-were carried out. Overall, bioassays results suggest this heptane-depleted essential oil is a promising active ingredient for cosmetic applications.
Subject(s)
Oils, Volatile , Oils, Volatile/chemistry , Plant Extracts/pharmacology , Plant Extracts/chemistry , Commiphora/chemistry , Skin , Resins, PlantABSTRACT
Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Qo site). We identified the G37V change within the cytochrome b Qi site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Qo site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.
Subject(s)
Ascomycota , Fungicides, Industrial , Ascomycota/genetics , Cytochromes b/genetics , Drug Resistance, Fungal/genetics , Fungicides, Industrial/pharmacology , Lactones , Plant Diseases/microbiology , Pyridines , Strobilurins/pharmacologyABSTRACT
The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H2O2) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H2O2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart.NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H2O2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.
Subject(s)
Cardiomyopathy, Dilated , Heart Failure , Alanine/pharmacology , Amino Acids/metabolism , Amino Acids/pharmacology , Amino Acids/therapeutic use , Animals , Cardiomyopathy, Dilated/metabolism , Dependovirus/metabolism , Disease Models, Animal , Heart Failure/genetics , Heart Failure/metabolism , Hydrogen Peroxide/metabolism , Myocytes, Cardiac/metabolism , Oxidative Stress , Rats , TranscriptomeABSTRACT
Nitric oxide (NO) synthesized by the endothelial isoform of nitric oxide synthase (eNOS) is a critical determinant of vascular homeostasis. However, the real-time detection of intracellular NO-a free radical gas-has been difficult, and surrogate markers for eNOS activation are widely utilized. eNOS phosphorylation can be easily measured in cells by probing immunoblots with phosphospecific antibodies. Here, we pursued multispectral imaging approaches using biosensors to visualize intracellular NO and Ca2+ and exploited chemogenetic approaches to define the relationships between NO synthesis and eNOS phosphorylation in cultured endothelial cells. We found that the G protein-coupled receptor agonists adenosine triphosphate (ATP) and histamine promoted rapid increases in eNOS phosphorylation, as did the receptor tyrosine kinase agonists insulin and Vascular Endothelial Growth Factor (VEGF). Histamine and ATP also promoted robust NO formation and increased intracellular Ca2+ By contrast, neither insulin nor VEGF caused any increase whatsoever in intracellular NO or Ca2+-despite eliciting strong eNOS phosphorylation responses. Our findings demonstrate an unexpected and striking discordance between receptor-modulated eNOS phosphorylation and NO formation in endothelial cells. Previous reports in which phosphorylation of eNOS has been studied as a surrogate for enzyme activation may need to be reassessed.
Subject(s)
Biosensing Techniques , Molecular Imaging , Nitric Oxide Synthase Type III/metabolism , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/metabolism , Calcium/metabolism , Cells, Cultured , Cytosol , Endothelial Cells/metabolism , Enzyme Activation , Molecular Imaging/methods , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Signal TransductionABSTRACT
Proguanil in combination with its synergistic partner atovaquone has been used for malaria treatment and prophylaxis for decades. However its mode of action is not fully understood. Here we used yeast to investigate its activity. Proguanil inhibits yeast growth, causes cell death and acts in synergy with atovaquone. It was previously proposed that the drug would target the system that maintains the mitochondrial membrane potential when the respiratory chain is inhibited. However our data did not seem to validate that hypothesis. We proposed that proguanil would not have a specific target but accumulate in the mitochondrial to concentrations that impair multiple mitochondrial functions leading to cell death. Selection and study of proguanil resistant mutants pointed towards an unexpected resistance mechanism: the decrease of CoQ level, which possibly alters the mitochondrial membrane properties and lowers proguanil intramitochondrial level.
Subject(s)
Antimalarials/pharmacology , Proguanil/pharmacology , Yeasts/drug effects , Atovaquone/pharmacology , Drug Resistance, Fungal/drug effects , Drug Resistance, Fungal/genetics , Drug Synergism , Drug Therapy, Combination , Membrane Potential, Mitochondrial/drug effects , Mutation , Oxygen/metabolism , Pyrimidines/pharmacology , Strobilurins/pharmacology , Ubiquinone/analogs & derivatives , Ubiquinone/metabolism , Ubiquinone/pharmacology , Vitamin K 3/analogs & derivatives , Vitamin K 3/pharmacology , Yeasts/genetics , Yeasts/growth & developmentABSTRACT
INTRODUCTION: The tomato plant, Solanum lycopersicum L. (Solanaceae), is one of the most widely consumed vegetables in the world and plays an important role in human diet. Tomato cultivars are hosts for diverse types of pests, implying diverse chemical defence strategies. Glycoalkaloids are the main specialised metabolites produced by tomato leaves and fruits to protect against pests. However, the roots have received little attention, leading to limited knowledge about their phytochemical content. OBJECTIVE: The main goal of the current study was the development of an untargeted ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) based metabolomic approach to study phytochemical variations in tomato roots at two different development stages (i.e. 34th and 62nd day after sowing). METHODS: UHPLC-HRMS was used to establish the fingerprint of 24 batches of tomato roots. Statistical analyses were performed to highlight the compounds that discriminated between young and mature tomato roots. A dereplication strategy using molecular networking and HRMS/MS data was set up to identify the metabolites regulated during early root development. KEY FINDINGS: The main biomarkers were guanidine and adenosine derivatives associated with tryptophan. Secondary metabolites such as glycoalkaloids and steroidal alkaloids were also characterised. Most of the metabolites were up-regulated in young tomato roots (34 days old) while tryptophan was up-regulated in the older roots (62 days old). CONCLUSION: The metabolic changes observed in this work contribute to a deeper understanding of early-stage root development and may help our understanding of the complex processes involved in the tomato root defence arsenal.
Subject(s)
Solanum lycopersicum , Chromatography, High Pressure Liquid , Solanum lycopersicum/genetics , Mass Spectrometry , Metabolomics , Plant LeavesABSTRACT
INTRODUCTION: Fine lavender and lavandin are perfume and medicinal plants originate from the South of France and are widely cultivated for their essential oils. Recently, cultivated plants suffered from a severe decline in France, due to the propagation of the yellow decline disease. This disease is caused by the stolbur phytoplasma, a bacterium transmitted by a sap-sucking insect, the planthopper. OBJECTIVES: In order to understand the complex relationships between host plant, pest, pathogen and environment responsible for the yellow decline of lavender, we use a metabolomic approach to highlight changes in chemical emissions from asymptomatic ("healthy") and symptomatic ("infected") plants. METHODS: Volatile compounds produced by fine lavender and lavandin were collected in the field using a dynamic headspace extraction approach. Afterwards, compounds trapped on Tenax adsorbent were thermodesorbed and analysed using an automated thermal desorption-gas chromatography-mass spectrometry (ATD-GC-MS). Multivariate statistical analyses was performed using principal component analysis and partial least square discriminant analyses. RESULTS: The untargeted screening of volatiles allowed the separation of asymptomatic and symptomatic plants according to their emissions. The approach was sufficiently accurate so as to separate the emissions according to the different stages of infection. Twelve compounds were found to be deregulated metabolites of yellow disease infection, common to fine lavender (variety 7713) and lavandin (variety abrial). CONCLUSION: The metabolomic approach allowed for the effective identification of chemical variations between infected and healthy plants in a complex field environment.
Subject(s)
Lavandula/chemistry , Metabolomics , Plant Diseases , Volatile Organic Compounds/metabolism , Discriminant Analysis , Gas Chromatography-Mass Spectrometry , Lavandula/growth & development , Lavandula/metabolism , Principal Component Analysis , Volatile Organic Compounds/analysisABSTRACT
In the course of phytochemical and chemotaxonomical investigations of Cornulaca monacantha (Amaranthaceae), two new isoflavones, 3-(2-hydroxyphenyl)-5,7-dimethoxy-6-(methoxymethyl)-4H-1-benzopyran-4-one (1) and 7-hydroxy-3-(4-hydroxyphenyl)-5-methoxy-6-(methoxymethyl)-4H-1-benzopyran-4-one (2) were isolated from the fresh aerial parts of C. monacantha among with three known compounds named vanillic acid (3), N-cis-feruloyltyramine (4) and N-trans-feruloyltyramine (5). Their structures were elucidated by means of spectroscopic methods including one- and two-dimensional NMR and HR-ESI-MS techniques. The isolated compounds exhibited interesting antioxidant activity determined by DPPH, ABTS and TAC tests.
Subject(s)
Amaranthaceae/chemistry , Antioxidants/isolation & purification , Antioxidants/pharmacology , Isoflavones/isolation & purification , Isoflavones/pharmacology , Magnetic Resonance Spectroscopy/methods , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
INTRODUCTION: In situ analysis of volatile organic compounds (VOCs) emitted by plants is an important challenge in chemical ecology. The traditional approach usually consists in trapping compounds using dynamic headspace extraction (DHS) in-field, followed by gas chromatography analysis coupled with mass spectrometry (GC-MS and/or GC-FID) in the laboratory. OBJECTIVES: In this study, we evaluated the use of the new portable Torion T-9 GC-MS system for rapid and in situ analysis of VOCs emitted by fine lavender and lavandin species. MATERIAL AND METHODS: All field analyses were performed using a person-portable low-thermal mass GC system coupled with a miniature toroidal ion trap mass analyser (ppGC-ITMS): Torion T-9 portable GC-MS. Subsequently, multivariate statistical analyses were performed to determine chemical differences between species. RESULTS: Thirty compounds were separated and detected in all lavender above-ground samples in only 3 min of analysis. CONCLUSIONS: The portable GC-MS device enabled a rapid in-field distinction of Lavandula species based on their detected volatile profiles.
Subject(s)
Lavandula , Volatile Organic Compounds , Gas Chromatography-Mass Spectrometry , Volatile Organic Compounds/analysisABSTRACT
We previously described a novel "chemogenetic" animal model of heart failure that recapitulates a characteristic feature commonly found in human heart failure: chronic oxidative stress. This heart failure model uses a chemogenetic approach to activate a recombinant yeast d-amino acid oxidase in rat hearts in vivo to generate oxidative stress, which then rapidly leads to the development of a dilated cardiomyopathy. Here we apply this new model to drug testing by studying its response to treatment with the angiotensin II (ANG II) receptor blocker valsartan, administered either alone or with the neprilysin inhibitor sacubitril. Echocardiographic and [18F]fluorodeoxyglucose positron emission tomographic imaging revealed that valsartan in the presence or absence of sacubitril reverses the anatomical and metabolic remodeling induced by chronic oxidative stress. Markers of oxidative stress, mitochondrial function, and apoptosis, as well as classical heart failure biomarkers, also normalized following drug treatments despite the persistence of cardiac fibrosis. These findings provide evidence that chemogenetic heart failure is rapidly reversible by drug treatment, setting the stage for the study of novel heart failure therapeutics in this model. The ability of ANG II blockade and neprilysin inhibition to reverse heart failure induced by chronic oxidative stress identifies a central role for cardiac myocyte angiotensin receptors in the pathobiology of cardiac dysfunction caused by oxidative stress.NEW & NOTEWORTHY The chemogenetic approach allows us to distinguish cardiac myocyte-specific pathology from the pleiotropic changes that are characteristic of other "interventional" animal models of heart failure. These features of the chemogenetic heart failure model facilitate the analysis of drug effects on the progression and regression of ventricular remodeling, fibrosis, and dysfunctional signal transduction. Chemogenetic approaches will be highly informative in the study of the roles of redox stress in heart failure providing an opportunity for the identification of novel therapeutic targets.
Subject(s)
Aminobutyrates/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Cardiomyopathy, Dilated/drug therapy , D-Amino-Acid Oxidase/metabolism , Fungal Proteins/metabolism , Hydrogen Peroxide/metabolism , Myocytes, Cardiac/drug effects , Oxidative Stress/drug effects , Protease Inhibitors/pharmacology , Tetrazoles/pharmacology , Animals , Apoptosis/drug effects , Biphenyl Compounds , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/physiopathology , D-Amino-Acid Oxidase/genetics , Dependovirus/genetics , Disease Models, Animal , Drug Combinations , Energy Metabolism/drug effects , Fungal Proteins/genetics , Genetic Vectors/administration & dosage , Injections, Intravenous , Male , Mitochondria, Heart/drug effects , Mitochondria, Heart/metabolism , Mitochondria, Heart/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Neprilysin/antagonists & inhibitors , Promoter Regions, Genetic , Rats, Wistar , Troponin T/genetics , Valsartan , Ventricular Function, Left/drug effects , Ventricular Remodeling/drug effectsABSTRACT
The present work deals with the assessment of the in vitro and in vivo estrogenic effects of the triterpenoids (lupenone, lupeol, and stigmastenone) isolated from Millettia macrophylla extract. The in vitro estrogenicity was performed by a reporter gene assay and estrogen receptor-α (ERα) target gene expression, whereas the in vivo estrogenicity was evaluated by a 3-day uterotrophic assay in ovariectomized rats. As results, lupenone and lupeol did not transactivate ERα as well as ERß of human embryonic kidney 293T (HEK293T) cells. However, lupeol seems to be antagonistic to estrogen (E2) only in HEK293T-ERα (10-9 and 10-8 µM). Furthermore, lupeol slightly upregulated GREB1 gene expression at the concentration of 1 µM, suggesting a weak activation of endogenous ERα. In vivo, only lupeol at a dose of 1 mg/kg significantly increased the uterine wet weight (p < 0.05), uterine (p < 0.05), and vaginal (p < 0.01) epithelial heights. The concomitant administration of lupeol (1 mg/kg) with a pure antiestrogen fulvestrant abrogated its effects only in the vagina, whereas in combination with E2, lupeol exhibited a significant antiestrogen-like effect in uterine wet weight and synergistic effects on endometrium. Lupeol has estrogenic effects that is partly through ERs transcriptional activity and does involve alternative mechanisms that are still to be uncovered.
Subject(s)
Millettia/chemistry , Pentacyclic Triterpenes/pharmacology , Phytoestrogens/pharmacology , Animals , Estrogen Receptor alpha/physiology , Female , HEK293 Cells , Humans , Methylene Chloride , Ovariectomy , Plant Extracts/pharmacology , Rats , Rats, WistarABSTRACT
A headspace solid-phase microextraction (HS-SPME) method combined with gas chromatography-mass spectrometry (GC/MS) was developed and optimized for the extraction and the analysis of volatile organic compounds (VOCs) from lavandin and fine lavender roots. Optimal parameters to extract volatile molecules from ground and intact roots were determined using a divinylbenzene-carboxen-polydimethylsiloxane (DVB/CAR/PDMS) coating fiber at 70 °C for 60â min. A total of 99 VOCs, including 40 monoterpenoids, 15 sesquiterpenoids, 1 diterpenoid and 2 coumarins were detected. The main compounds detected in lavandin roots were fenchol, borneol, and coumarin. Performances of the optimized SPME GC/MS method were evaluated via the comparison of VOC emissions between roots from different cultivars of fine lavender (7713 and maillette) and lavandin (abrial and grosso). Chemometric analysis, using partial least squares-discriminant analysis (PLS-DA), suggests fifteen significant features as potential discriminatory compounds. Among them, ß-phellandrene allows discrimination between lavender and lavandin varieties.
Subject(s)
Gas Chromatography-Mass Spectrometry , Lavandula/chemistry , Volatile Organic Compounds/analysis , Discriminant Analysis , Lavandula/metabolism , Least-Squares Analysis , Plant Extracts/chemistry , Plant Roots/chemistry , Plant Roots/metabolism , Solid Phase Microextraction , Volatile Organic Compounds/isolation & purificationSubject(s)
Angiotensin Receptor Antagonists/therapeutic use , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Hypertension/drug therapy , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/metabolism , Renin-Angiotensin System/drug effects , Angiotensin Receptor Antagonists/pharmacology , Angiotensin-Converting Enzyme 2 , Angiotensin-Converting Enzyme Inhibitors/pharmacology , COVID-19 , Coronavirus Infections/complications , Down-Regulation , Humans , Hypertension/complications , Pandemics , Pneumonia, Viral/complications , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/drug therapy , SARS-CoV-2ABSTRACT
Platelets are essential for hemostasis, and thrombocytopenia is a major clinical problem. Megakaryocytes (MKs) generate platelets by extending long processes, proplatelets, into sinusoidal blood vessels. However, very little is known about what regulates proplatelet formation. To uncover which proteins were dynamically changing during this process, we compared the proteome and transcriptome of round vs proplatelet-producing MKs by 2D difference gel electrophoresis (DIGE) and polysome profiling, respectively. Our data revealed a significant increase in a poorly-characterized MK protein, myristoylated alanine-rich C-kinase substrate (MARCKS), which was upregulated 3.4- and 5.7-fold in proplatelet-producing MKs in 2D DIGE and polysome profiling analyses, respectively. MARCKS is a protein kinase C (PKC) substrate that binds PIP2. In MKs, it localized to both the plasma and demarcation membranes. MARCKS inhibition by peptide significantly decreased proplatelet formation 53%. To examine the role of MARCKS in the PKC pathway, we treated MKs with polymethacrylate (PMA), which markedly increased MARCKS phosphorylation while significantly inhibiting proplatelet formation 84%, suggesting that MARCKS phosphorylation reduces proplatelet formation. We hypothesized that MARCKS phosphorylation promotes Arp2/3 phosphorylation, which subsequently downregulates proplatelet formation; both MARCKS and Arp2 were dephosphorylated in MKs making proplatelets, and Arp2 inhibition enhanced proplatelet formation. Finally, we used MARCKS knockout (KO) mice to probe the direct role of MARCKS in proplatelet formation; MARCKS KO MKs displayed significantly decreased proplatelet levels. MARCKS expression and signaling in primary MKs is a novel finding. We propose that MARCKS acts as a "molecular switch," binding to and regulating PIP2 signaling to regulate processes like proplatelet extension (microtubule-driven) vs proplatelet branching (Arp2/3 and actin polymerization-driven).
Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Megakaryocytes/metabolism , Membrane Proteins/physiology , Protein Processing, Post-Translational , Thrombopoiesis/physiology , Actin-Related Protein 2-3 Complex/metabolism , Actin-Related Protein 3/metabolism , Amino Acid Sequence , Angiopoietin-Like Protein 2 , Angiopoietin-like Proteins , Angiopoietins/metabolism , Animals , Apoptosis , Blood Platelets/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Liver/cytology , Liver/embryology , Membrane Proteins/deficiency , Membrane Proteins/metabolism , Mice , Mice, Knockout , Molecular Sequence Data , Myristoylated Alanine-Rich C Kinase Substrate , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphorylation , Protein Biosynthesis , Protein Kinase C/metabolism , Signal TransductionABSTRACT
Lichens are complex symbiotic organisms able to produce a vast array of compounds. The Algerian lichen diversity has only prompted little interest even given the 1085 species listed. Herein, the chemodiversity of four Algerian lichens including Cladonia rangiformis, Ramalina farinaceae, R. fastigiata, and Roccella phycopsis was investigated. A dereplication strategy, using ultra high performance liquid chromatography-high resolution-electrospray ionization-mass spectrometry (UHPLC-HRMS/MS), was carried out for a comprehensive characterization of their substances including phenolics, depsides, depsidones, depsones, dibenzofurans, and aliphatic acids. Some known compounds were identified for the first time in some species. Additionally, the lichenic extracts were evaluated for their antifungal and antimicrobial activities on human pathogenic strains (Candida albicans, C. glabrata, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli). Cyclohexane extracts were found particularly active against human pathogenic fungi with MIC80 values ranging from 8 to 62.5 µg/mL, without cytotoxicity. This study highlights the therapeutic and prophylactic potential of lichenic extracts as antibacterial and antifungal agents.