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1.
Clin Hemorheol Microcirc ; 79(1): 231-243, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34487034

RESUMO

Cell-based in vitro liver models are an important tool in the development and evaluation of new drugs in pharmacological and toxicological drug assessment. Hepatic microsomal enzyme complexes, consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs), play a decisive role in catalysing phase-1 biotransformation of pharmaceuticals and xenobiotics. For a comprehensive understanding of the phase-1 biotransformation of drugs, the availability of well-characterized substances for the targeted modulation of in vitro liver models is essential. In this study, we investigated diphenyleneiodonium (DPI) for its ability to inhibit phase-1 enzyme activity and further its toxicological profile in an in vitro HepG2 cell model with and without recombinant expression of the most important drug metabolization enzyme CYP3A4.Aim of the study was to identify effective DPI concentrations for CPR/CYP activity modulation and potentially associated dose and time dependent hepatotoxic effects. The cells were treated with DPI doses up to 5,000nM (versus vehicle control) for a maximum of 48 h and subsequently examined for CYP3A4 activity as well as various toxicological relevant parameters such as cell morphology, integrity and viability, intracellular ATP level, and proliferation. Concluding, the experiments revealed a time- and concentration-dependent DPI mediated partial and complete inhibition of CYP3A4 activity in CYP3A4 overexpressing HepG2-cells (HepG2-CYP3A4). Other cell functions, including ATP synthesis and consequently the proliferation were negatively affected in both in vitro cell models. Since neither cell integrity nor cell viability were reduced, the effect of DPI in HepG2 can be assessed as cytostatic rather than cytotoxic.


Assuntos
Citostáticos , Hepatoblastoma , Neoplasias Hepáticas , Biotransformação , Linhagem Celular , Células Clonais , Citocromo P-450 CYP3A/genética , Células Hep G2 , Hepatoblastoma/tratamento farmacológico , Humanos , Oniocompostos
2.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502052

RESUMO

The role of reactive oxygen species (ROS) in ABA-induced increase in hydraulic conductivity was hypothesized to be dependent on an increase in aquaporin water channel (AQP) abundance. Single ABA application or its combination with ROS manipulators (ROS scavenger ascorbic acid and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI)) were studied on detached roots of barley plants. We measured the osmotically driven flow rate of xylem sap and calculated root hydraulic conductivity. In parallel, immunolocalization of ABA and HvPIP2;2 AQPs was performed with corresponding specific antibodies. ABA treatment increased the flow rate of xylem, root hydraulic conductivity and immunostaining for ABA and HvPIP2;2, while the addition of antioxidants prevented the effects of this hormone. The obtained results confirmed the involvement of ROS in ABA effect on hydraulic conductivity, in particular, the importance of H2O2 production by ABA-treated plants for the effect of this hormone on AQP abundance.


Assuntos
Ácido Abscísico/farmacologia , Aquaporinas/metabolismo , Osmose , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Inibidores Enzimáticos/farmacologia , Hordeum/efeitos dos fármacos , Hordeum/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Raízes de Plantas/efeitos dos fármacos , Xilema/efeitos dos fármacos , Xilema/metabolismo
3.
Chem Commun (Camb) ; 57(76): 9656-9671, 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34472551

RESUMO

N-(Acyloxy)phthalimide and oxime derivatives containing N-O bonds are important chemicals and synthetic intermediates, and visible light photoredox reductions of the N-O bonds provide carbon- or nitrogen-centered radicals for N-(acyloxy)phthalimide derivatives and iminyl radicals for oxime derivatives. This feature article summarises the recent progress in the visible light photoredox organic reactions, including decarboxylative addition reactions, alkylation, allylation, alkenylation, alkynylation, arylation, heteroarylation and cascade annulation of N-(acyloxy)phthalimide derivatives through the formation of carbon-carbon bonds, decarboxylative borylation, amination, oxygenation, sulfuration, selenylation, fluorination and iodination of N-(acyloxy)phthalimide derivatives through the formation of carbon-heteroatom bonds, and additions to arenes and alkenes, hydrogen atom transfer and the cleavage of α-carbon-carbon bonds via the iminyl radical intermediates for oxime derivatives.


Assuntos
Nitrogênio/química , Oniocompostos/química , Oxigênio/química , Ftalimidas/química , Catálise , Estrutura Molecular , Processos Fotoquímicos
4.
Exp Cell Res ; 406(1): 112719, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34273405

RESUMO

Hepatic ischemia/reperfusion injury (IRI) is an adverse effect for liver transplantation which is characterized by immune response mediated inflammation. Recent studies report that neutrophil extracellular traps (NETs) are implicated in hepatic IRI. The aim of this study was to explore the mechanism of action of tetramethylpyrazine (TMP), the main chemical composition of Ligusticum chuanxiong in treatment of ischemic related diseases. Data showed that hepatic IRI increases the leak of alanine aminotransferase (ALT) and aspartate transaminase (AST), and stimulates formation of NETs. Extracellular DNA/NETs assay, hematoxylin-eosin (HE) staining, immunofluorescence assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and Western blot assay, showed that TMP significantly reduces formation of NETs and alleviates hepatic IRI. Moreover, TMP and Diphenyleneiodonium (DPI) suppressed ROS production in neutrophils. In addition, analysis showed that activation of NADPH oxidase plays a role in formation of NETs triggered by hepatic IRI. Notably, TMP inhibited formation of NETs though inhibition of NADPH oxidase. Additionally, Combination treatment using TMP and DPI was more effective compared with monotherapy of either of the two drugs. These findings show that combination therapy using TMP and DPI is a promising method for treatment hepatic IRI.


Assuntos
Antioxidantes/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Transplante de Fígado/reabilitação , Oniocompostos/farmacologia , Pirazinas/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Ácidos Nucleicos Livres/antagonistas & inibidores , Ácidos Nucleicos Livres/sangue , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Armadilhas Extracelulares/metabolismo , Marcação In Situ das Extremidades Cortadas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/cirurgia , Transplante de Fígado/métodos , Masculino , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/cirurgia , Resultado do Tratamento
5.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205065

RESUMO

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-ß-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. The results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measurements of Et+ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably decreases in the presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.


Assuntos
DNA/química , Etídio/análise , Salmonella typhimurium/crescimento & desenvolvimento , Espermatozoides/química , Animais , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Etídio/química , Masculino , Oniocompostos/química , Compostos Organofosforados/química , Salmão , Salmonella typhimurium/metabolismo , Espectrometria de Fluorescência , Espermatozoides/metabolismo
6.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065225

RESUMO

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.


Assuntos
Íons/metabolismo , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismo , Oniocompostos/metabolismo , Linhagem Celular Tumoral , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Células HCT116 , Células HT29 , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
7.
Org Lett ; 23(12): 4813-4817, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34032454

RESUMO

Arenes are broadly found motifs in societally important molecules. Access to diverse arene chemical space is critically important, and the ability to do so from common reagents is highly desirable. Aryl(TMP)iodonium tosylates provide one such access point to arene chemical space via diverse aryl intermediates. Here we demonstrate that controlling reaction pathways selectively leads to arynes with a broad scope of arenes and arynophiles (24 examples, 70% average yield) and efficient access to biologically active compounds.


Assuntos
Indicadores e Reagentes/química , Oniocompostos/química , Compostos de Tosil/química , Estrutura Molecular , Paládio/química
8.
Neurochem Res ; 46(8): 2089-2096, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34008119

RESUMO

Repeated morphine administration results in analgesic tolerance. However, the underlying mechanism of morphine analgesic tolerance remains unclear. NADPH-oxidase 2 (NOX2) is the first discovered NADPH oxidase, which mainly functions to produce reactive oxygen species. Its specific role in morphine tolerance has not been fully investigated. In this work, we found that chronic morphine administration significantly increased the expression of NOX2 in spinal cord. Pretreatment of NOX2 inhibitor blocked the upregulation of NOX2 and autophagy markers, including LC3B and P62, and consequently the development of morphine tolerance. NOX2 and LC3B were both colocalized with NeuN in spinal dorsal horn in morphine-tolerant rats. Our results suggest that the increased autophagy activity in spinal neurons promoted by NOX2 activation contributes to the development of morphine tolerance. NOX2 may be considered as a new therapeutic target for morphine tolerance.


Assuntos
Analgésicos Opioides/farmacologia , Autofagia/efeitos dos fármacos , Tolerância a Medicamentos/fisiologia , Morfina/farmacologia , NADPH Oxidase 2/metabolismo , Neurônios/efeitos dos fármacos , Animais , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Oxidase 2/antagonistas & inibidores , Oniocompostos/farmacologia , Ratos Sprague-Dawley , Corno Dorsal da Medula Espinal/citologia
9.
J Pharmacol Sci ; 146(1): 29-32, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33858652

RESUMO

Hydroxyl radical (•OH) production in the rat striatum during carbon monoxide (CO) poisoning, which inhibits complex IV, was enhanced synergistically by malonate, a mitochondrial complex II inhibitor, but not N-methyl-4-phenylpyridinium or NaCN, complex I and IV inhibitors, respectively. No such enhancement appeared in the case of NaCN combined with malonate. Intrastriatal dopamine, which is involved in •OH production by malonate, did not synergistically enhance CO-induced •OH production. Diphenyleneiodonium, a nonselective NADPH oxidase inhibitor, partly suppressed the potentiation of CO-induced •OH production by malonate. Impairment of mitochondrial functions might potentiate oxidative stress and intensify CO toxicity in the brain.


Assuntos
Intoxicação por Monóxido de Carbono/metabolismo , Corpo Estriado/metabolismo , Radical Hidroxila/metabolismo , Animais , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Masculino , Malonatos/farmacologia , Mitocôndrias/metabolismo , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley
10.
Sci Rep ; 11(1): 6556, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33753859

RESUMO

Hyperactivation of ABC transporter ABCB1 and induction of epithelial-mesenchymal transition (EMT) are the most common mechanism of acquired cancer chemoresistance. This study describes possible mechanisms, that might contribute to upregulation of ABCB1 and synergistically boost the acquisition of doxorubicin (DOX) resistance in breast cancer MX-1 cell line. DOX resistance in MX-1 cell line was induced by a stepwise increase of drug concentration or by pretreatment of cells with an ABCB1 transporter activator tetraphenylphosphonium (TPP+) followed by DOX exposure. Transcriptome analysis of derived cells was performed by human gene expression microarrays and by quantitative PCR. Genetic and epigenetic mechanisms of ABCB1 regulation were evaluated by pyrosequencing and gene copy number variation analysis. Gradual activation of canonical EMT transcription factors with later activation of ABCB1 at the transcript level was observed in DOX-only treated cells, while TPP+ exposure induced considerable activation of ABCB1 at both, mRNA and protein level. The changes in ABCB1 mRNA and protein level were related to the promoter DNA hypomethylation and the increase in gene copy number. ABCB1-active cells were highly resistant to DOX and showed morphological and molecular features of EMT. The study suggests that nongenotoxic ABCB1 inducer can possibly accelerate development of DOX resistance.


Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Oniocompostos/farmacologia , Compostos Organofosforados/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/agonistas , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Biologia Computacional/métodos , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Transcriptoma
11.
Plant Signal Behav ; 16(5): 1889251, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33632064

RESUMO

Heterotrimeric G-protein α and ß-subunits regulate H2O2-mediated aerenchyma formation. The rice G-protein γ-subunit, dense and erect panicle 1 (DEP1), is known to interact with the α-subunit and regulate nitrogen utilization and yield. However, it is unclear whether DEP1 regulates cell death for aerenchyma formation in rice roots. Using wild-type WYJ8 and its transgenic line WYJ8(DEP1), we confirmed that DEP1 is involved in H2O2-mediated aerenchyma formation. The rates of aerenchyma formation varied in different parts of the roots in both varieties, with the highest rate in the 4-7 cm segments, reaching a plateau in the 7-8 cm segments. Compared with WYJ8, the aerenchyma area and H2O2 content in WYJ8(DEP1) were increased by 55.98% and 53.37%, respectively; however, the responses of aerenchyma formation to exogenous H2O2 were basically the same in the two varieties. Diphenylene iodonium (DPI) treatment had no effect on H2O2 production and elimination processes in WYJ8, but significantly reduced the activity of the key enzyme that catalyzes H2O2 biosynthesis in WYJ8(DEP1). Importantly, exogenous H2O2 treatment did not offset the effect of the decrease in endogenous H2O2 level caused by DPI on aerenchyma formation. These results indicated that DEP1 enhanced H2O2 biosynthesis and promoted the cell death of the root cortex, thus contributing to aerenchyma development in WYJ8(DEP1).


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Peróxido de Hidrogênio/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais , Amitrol (Herbicida)/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oniocompostos/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética
12.
Cell Death Dis ; 12(2): 189, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594044

RESUMO

Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.


Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Genes ras , Glutationa/biossíntese , Metionina/análogos & derivados , Mutação , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Sulfóxidos/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Morte Celular/efeitos dos fármacos , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Genes p53 , Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutamato-Cisteína Ligase/metabolismo , Células HCT116 , Humanos , Metionina/farmacologia , Camundongos Nus , Camundongos Transgênicos , NADPH Oxidases/metabolismo , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Estresse Oxidativo , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Food Chem Toxicol ; 150: 112055, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33577942

RESUMO

Patulin (PAT) is a kind of mycotoxins that commonly found in decayed fruits and their products. Our previous studies have shown that PAT induced cell apoptosis and the overproduction of reactive oxygen species (ROS) in human embryonic kidney (HEK293) cells. The present study aimed to further investigate the functional role of NADPH oxidase, one of the main cellular sources of ROS, in PAT-induced apoptosis and oxidative damage in HEK293 cells. We demonstrated that the protein and mRNA expression levels of NADPH oxidase catalytic subunit NOX2 and regulatory subunit p47phox were up-regulated under PAT stress. Inhibiting of NADPH oxidase with the specific antagonist diphenyleneiodonium (DPI) suppressed cytotoxicity and apoptosis induced by PAT as evidenced by the increase of cell viability, the decrease of LDH release and the inhibition of caspase activities. Furthermore, DPI re-established mitochondrial membrane potential (MMP) and enhanced cellular ATP content. Importantly, DPI supplementation elevated endogenous GSH contents as well as the ratio of GSH/GSSG. Meanwhile, the antioxidant-enzyme activities of GPx, GR, CAT and SOD were significantly promoted. Collectively, our results suggested that NADPH oxidase played a critical role in PAT-induced nephrotoxicity, and inhibition of NADPH oxidase by DPI attenuated cell injury and apoptosis via regulation of oxidative damage.


Assuntos
Compostos de Bifenilo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Patulina/toxicidade , Trifosfato de Adenosina/metabolismo , Caspases/genética , Caspases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Células HEK293 , Humanos , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mutagênicos/toxicidade , NADPH Oxidases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Int J Phytoremediation ; 23(9): 945-957, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33472408

RESUMO

The present work was conducted to assess the effects of arsenic (As, 1000 µM), diphenyleneiodonium (DPI, 10 µM) and reduced glutathione (GSH, 500 µM) on Isatis cappadocica. As treatment decreased plant growth and fresh and dry weight of shoot and root and also enhanced the accumulation of As. As stress also enhanced the oxidative stress biomarkers, hydrogen peroxide (H2O2) and malondialdehyde (MDA) content. However, the application of GSH decreased the content of H2O2 and MDA by 43% and 55%, respectively, as compared to As treatment. The antioxidants like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione S-transferase (GST) also enhanced with As stress. NADPH oxidase inhibitor, the DPI, enhances the effect of As toxicity by increasing the accumulation of As, H2O2, MDA. DPI also enhances the activity of antioxidant enzymes except GR and GST, However, the application GSH increased the plant growth and biomass yield, decreases accumulation of As, H2O2 and MDA content in As as well as As + DPI treated plants. The thiols content [total thiol (TT), non-protein thiol (NPT) protein thiols (PT), and glutathione (GSH)] were decreased in the As + DPI treatment but supplementation of GSH enhanced them. Novelty statement: The study reveals the beneficial role of GSH in mitigating the deleterious effects of Arsenic toxicity through its active involvement in the antioxidant metabolism, thiol synthesis and osmolyte accumulation. Apart from As, We provided the plants NADPH oxidase inhibitor, the diphenyleneiodonium (DPI), which boosts the As toxicity. At present, there is dearth of information pertaining to the effects of DPI on plants growth and their responses under heavy metal stress.GSH application reversed the effect of diphenyleneiodonium (DPI) under As stress preventing the oxidative damage to biomolecules through the modulation of different antioxidant enzymes. The application of GSH for As stressed soil could be a sustainable approach for crop production.


Assuntos
Arsênio , Isatis , Antioxidantes , Arsênio/toxicidade , Ascorbato Peroxidases/metabolismo , Biodegradação Ambiental , Catalase/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio , Isatis/metabolismo , NADPH Oxidases , Oniocompostos , Estresse Oxidativo
15.
J Integr Plant Biol ; 63(3): 583-596, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33017089

RESUMO

Salicylic acid (SA) plays a crucial role in plant immunity. However, its function in plant development is poorly understood. The quiescent center (QC), which maintains columella stem cells (CSCs) in the root apical meristem and typically exhibits low levels of cell division, is critical for root growth and development. Here, we show that the Arabidopsis thaliana SA overaccumulation mutant constitutively activated cell death 1 (cad1), which exhibits increased cell division in the QC, is rescued by additional mutations in genes encoding the SA biosynthetic enzyme SALICYLIC ACID INDUCTION DEFFICIENT2 (SID2) or the SA receptor NONEXPRESSER OF PR GENES1 (NPR1), indicating that QC cell division in the cad1 mutant is promoted by the NPR1-dependent SA signaling pathway. The application of exogenous SA also promoted QC cell division in wild-type plants in a dose-dependent manner and largely suppressed the expression of genes involved in QC maintenance, including those encoding the APETALA2 (AP2) transcription factors PLETHORA1 (PLT1) and PLT2, as well as the homeodomain transcription factor WUSCHEL-RELATED HOMEOBOX5 (WOX5). Moreover, we showed that SA promotes reactive oxygen species (ROS) production, which is necessary for the QC cell division phenotype in the cad1 mutant. These results provide insight into the function of SA in QC maintenance.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Divisão Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/farmacologia , Fatores de Transcrição/genética , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Ácidos Indolacéticos/metabolismo , Mutação/genética , Oniocompostos/farmacologia , Fenótipo , Plantas Geneticamente Modificadas , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo
16.
Plant Signal Behav ; 16(2): 1848086, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33210579

RESUMO

Cutting is a frequently used model to study the process of adventitious root formation, and excision of cuttings leads to rapid wound response signaling. We recently showed that as a wound signal, reactive oxygen species (ROS, mainly hydrogen peroxide) participate in adventitious root induction of hypocotyl cuttings through regulation of auxin biosynthesis and transport. Here, superoxide anion (O2 -•), an early type of ROS, exhibited rapid burst at the cutting site immediately in response to wounding in Arabidopsis hypocotyl cuttings. Diphenylene iodonium chloride (DPI, inhibitor of NADPH oxidase) overwhelmingly suppressed O2 -• propagation through the hypocotyl. Compared to wild type, O2 -• burst only occur in cut base, and upward transduction were inhibited completely in NADPH oxidase mutant AtRbohD. These results indicate O2 -• generation and propagation in response to wound and via NADPH oxidase in adventitious root induction of hypocotyl cuttings.


Assuntos
Arabidopsis/metabolismo , Superóxidos/metabolismo , Arabidopsis/efeitos dos fármacos , Oniocompostos/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
Plant Signal Behav ; 16(3): 1856546, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33315520

RESUMO

The plasma membrane NADPH Oxidase-derived ROS as signaling molecules play crucial roles in salt stress response. As the motor organelle of cells, mitochondria are also important for salt tolerance. However, the possible interaction between NADPH Oxidase-derived ROS and mitochondria is not well studied. Here, a transgenic Arabidopsis expressing mitochondrial matrix-targeted pH-sensitive indicator cpYFP was used to monitor the pH dynamics in root cells under salt stress. A significant alkalization in mitochondria was observed when the root was exposed to NaCl or KCl, but not osmotic stress such as isotonic mannitol. Interestingly, when pretreated with the NADPH Oxidase inhibitor DPI, the mitochondrial alkalization in root cells was largely abolished. Genetic evidence further showed that salt-induced mitochondrial alkalization was significantly reduced in the loss of function mutant atrbohF . Pretreatment with endocytosis-related inhibitor PAO or TyrA23, which inhibited the ROS accumulation under salt treatment, almost abolished this effect. Furthermore, [Ca2+]cyt increase might also play important roles by affecting ROS generation to mediate salt-induced mitochondrial alkalization as indicated by treatment with plasma membrane Ca2+ channel inhibitor LaCl3 and mitochondrial Ca2+ uniporter inhibitor Ruthenium Red. Together, these results suggest that the plasma membrane NADPH Oxidase-derived ROS promote the mitochondrial alkalization under salt treatment, providing a possible link between different cellular compartments under salt stress.


Assuntos
Álcalis/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Raízes de Plantas/citologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Salino/fisiologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/antagonistas & inibidores , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Mitocôndrias/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Oniocompostos/farmacologia , Estresse Salino/efeitos dos fármacos , Cloreto de Sódio/farmacologia
18.
Biochim Biophys Acta Biomembr ; 1863(1): 183483, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002452

RESUMO

To clarify the contribution of charge delocalization in a lipophilic ion to the efficacy of its permeation through a lipid membrane, we compared the behavior of alkyl derivatives of triphenylphosphonium, tricyclohexylphosphonium and trihexylphosphonium both in natural and artificial membranes. Exploring accumulation of the lipophilic cations in response to inside-negative membrane potential generation in mitochondria by using an ion-selective electrode revealed similar mitochondrial uptake of butyltricyclohexylphosphonium (C4TCHP) and butyltriphenylphosphonium (C4TPP). Fluorescence correlation spectroscopy also demonstrated similar membrane potential-dependent accumulation of fluorescein derivatives of tricyclohexyldecylphosphonium and decyltriphenylphosphonium in mitochondria. The rate constant of lipophilic cation translocation across the bilayer lipid membrane (BLM), measured by the current relaxation method, moderately increased in the following sequence: trihexyltetradecylphosphonium ([P6,6,6,14]) < triphenyltetradecylphosphonium (C14TPP) < tricyclohexyldodecylphosphonium (C12TCHP). In line with these results, measurements of the BLM stationary conductance indicated that membrane permeability for C4TCHP is 2.5 times higher than that for C4TPP. Values of the difference in the free energy of ion solvation in water and octane calculated using the density functional theory and the polarizable continuum solvent model were similar for methyltriphenylphosphonium, tricyclohexylmethylphosphonium and trihexylmethylphosphonium. Our results prove that both cyclic and aromatic moieties are not necessary for lipophilic ions to effectively permeate through lipid membranes.


Assuntos
Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Oniocompostos/química , Compostos Organofosforados/química , Compostos de Tritil/química , Permeabilidade
19.
Molecules ; 25(23)2020 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33291596

RESUMO

The unprecedented Nazarov cyclization of a model divinyl ketone using phosphonium-based Deep Eutectic Solvents as sustainable non-innocent reaction media is described. A two-level full factorial Design of Experiments was conducted for elucidating the effect of the components of the eutectic mixture and optimizing the reaction conditions in terms of temperature, time, and substrate concentration. In the presence of the Deep Eutectic Solvent (DES) triphenylmethylphosphonium bromide/ethylene glycol, it was possible to convert more than 80% of the 2,4-dimethyl-1,5-diphenylpenta-1,4-dien-3-one, with a specific conversion, into the cyclopentenone Nazarov derivative of 62% (16 h, 60 °C). For the reactions conducted in the DES triphenylmethylphosphonium bromide/acetic acid, quantitative conversions were obtained with percentages of the Nazarov product above 95% even at 25 °C. Surface Responding Analysis of the optimized data furnished a useful tool to determine the best operating conditions leading to quantitative conversion of the starting material, with complete suppression of undesired side-reactions, high yields and selectivity. After optimization, it was possible to convert more than 90% of the model substrate into the desired cyclopentenone with cis percentages up to 77%. Experimental validation of the implemented model confirmed the robustness and the suitability of the procedure, leading to possible further extension to this specific combination of experimental designs to other substrates or even to other synthetic processes of industrial interest.


Assuntos
Solventes/química , Ácido Acético/química , Ciclização , Ciclopentanos/química , Oniocompostos/química , Temperatura , Compostos de Tritil/química
20.
Int J Mol Sci ; 21(20)2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-33081375

RESUMO

Y-27632 is known as a selective Rho-associated coiled coil-forming kinase (ROCK) inhibitor. Y-27632 has been shown to induce neurite outgrowth in several neuronal cells. However, the precise molecular mechanisms linking neurite outgrowth to Y-27632 are not completely understood. In this study, we examined the ability of Y-27632 to induce neurite outgrowth in PC12 cells and evaluated the signaling cascade. The effect of Y-27632 on the neurite outgrowth was inhibited by reactive oxygen species (ROS) scavengers such as N-acetyl cysteine (NAC) and trolox. Furthermore, Y-27632-induced neurite outgrowth was not triggered by NADPH oxidase 1 (NOX1) knockdown or diphenyleneiodonium (DPI), a NOX inhibitor. Suppression of the Rho-family GTPase Rac1, which is under the negative control of ROCK, with expression of the dominant negative Rac1 mutant (Rac1N17) prevented Y-27632-induced neurite outgrowth. Moreover, the Rac1 inhibitor NSC23766 prevented Y-27632-induced AKT and p21-activated kinase 1 (PAK1) activation. AKT inhibition with MK2206 suppressed Y-27632-induced PAK1 phosphorylation and neurite outgrowth. In conclusion, our results suggest that Rac1/NOX1-dependent ROS generation and subsequent activation of the AKT/PAK1 cascade contribute to Y-27632-induced neurite outgrowth in PC12 cells.


Assuntos
Amidas/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Transdução de Sinais , Acetilcisteína/farmacologia , Animais , Cromanos/farmacologia , Sequestradores de Radicais Livres/farmacologia , NADPH Oxidase 1/metabolismo , Oniocompostos/farmacologia , Células PC12 , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
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