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1.
Int J Mol Sci ; 25(9)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38731933

RESUMO

Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using Saccharomyces cerevisiae as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.


Assuntos
Compostos de Cádmio , Endocitose , Pontos Quânticos , Saccharomyces cerevisiae , Compostos de Selênio , Sulfetos , Compostos de Zinco , Pontos Quânticos/toxicidade , Pontos Quânticos/química , Endocitose/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Compostos de Cádmio/toxicidade , Compostos de Selênio/toxicidade , Sulfetos/toxicidade , Sulfetos/metabolismo , Compostos de Zinco/toxicidade , Vacúolos/metabolismo , Vacúolos/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos
2.
J Hazard Mater ; 470: 134172, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38569340

RESUMO

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.


Assuntos
Boehmeria , Cádmio , Parede Celular , Vacúolos , Cádmio/toxicidade , Cádmio/metabolismo , Parede Celular/metabolismo , Parede Celular/efeitos dos fármacos , Boehmeria/metabolismo , Boehmeria/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/efeitos dos fármacos , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Polissacarídeos/metabolismo , Oxilipinas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucanos/metabolismo , Xilanos/metabolismo , Estresse Fisiológico/efeitos dos fármacos
3.
J Nat Prod ; 87(4): 1197-1202, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38503712

RESUMO

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C-N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey's method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin-independent manner in tobacco BY-2 cells.


Assuntos
Nicotiana , Poríferos , Vacúolos , Animais , Estrutura Molecular , Poríferos/química , Nicotiana/química , Vacúolos/efeitos dos fármacos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Biologia Marinha , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Oligopeptídeos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Triptofano/química , Triptofano/farmacologia
4.
Int J Mol Sci ; 23(3)2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-35163447

RESUMO

Botrytis cinerea is considered an important plant pathogen and is responsible for significant crop yield losses. With the frequent application of commercial fungicides, B. cinerea has developed resistance to many frequently used fungicides. Therefore, it is necessary to develop new kinds of fungicides with high activity and new modes of action to solve the increasingly serious problem of resistance. During our screening of fungicide candidates, one novel sulfonamide compound, N-(2-trifluoromethyl-4-chlorphenyl)-2-oxocyclohexyl sulfonamide (L13), has been found to exhibit good fungicidal activity against B. cinerea. In this work, the mode of action of L13 against B. cinerea and the field control effect on tomato gray mold was studied. L13 had good control against B. cinerea resistant to carbendazim, diethofencarb, and iprodione commercial fungicides in the pot culture experiments. SEM and TEM observations revealed that L13 could cause obvious morphological and cytological changes to B. cinerea, including excessive branching, irregular ramification or abnormal configuration, and the decomposition of cell wall and vacuole. L13 induced more significant electrolyte leakage from hyphae than procymidone as a positive control. L13 had only a minor effect on the oxygen consumption of intact mycelia, with 2.15% inhibition at 50 µg/mL. In two locations over 2 years, the field control effect of L13 against tomato gray mold reached 83% at a rate of 450 g ai ha-1, better than the commercial fungicide of iprodione. Moreover, toxicological tests demonstrated the low toxicological effect of L13. This research seeks to provide technical support and theoretical guidance for L13 to become a real commercial fungicide.


Assuntos
Botrytis/crescimento & desenvolvimento , Fungicidas Industriais/farmacologia , Doenças das Plantas/prevenção & controle , Solanum lycopersicum/crescimento & desenvolvimento , Sulfonamidas/farmacologia , Administração Cutânea , Administração Oral , Animais , Botrytis/efeitos dos fármacos , Botrytis/metabolismo , Parede Celular/efeitos dos fármacos , Farmacorresistência Fúngica , Fungicidas Industriais/administração & dosagem , Fungicidas Industriais/efeitos adversos , Solanum lycopersicum/microbiologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Coelhos , Ratos , Pele/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
5.
Int J Mol Sci ; 22(23)2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34884427

RESUMO

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.


Assuntos
Beta vulgaris/crescimento & desenvolvimento , Chumbo/farmacologia , Canais de Potássio/metabolismo , Vacúolos/metabolismo , Beta vulgaris/efeitos dos fármacos , Beta vulgaris/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Patch-Clamp , Proteínas de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Canais de Potássio/efeitos dos fármacos , Vacúolos/efeitos dos fármacos
6.
Nutrients ; 13(10)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34684361

RESUMO

Lactoferrin (LF) was used at first as a vehicle to deliver non-soluble active compounds to the body, including the central nervous system (CNS). Nonetheless, it soon became evident that, apart from acting as a vehicle, LF itself owns active effects in the CNS. In the present study, the effects of LF are assessed both in baseline conditions, as well as to counteract methamphetamine (METH)-induced neurodegeneration by assessing cell viability, cell phenotype, mitochondrial status, and specific autophagy steps. In detail, cell integrity in baseline conditions and following METH administration was carried out by using H&E staining, Trypan blue, Fluoro Jade B, and WST-1. Western blot and immuno-fluorescence were used to assess the expression of the neurofilament marker ßIII-tubulin. Mitochondria were stained using Mito Tracker Red and Green and were further detailed and quantified by using transmission electron microscopy. Autophagy markers were analyzed through immuno-fluorescence and electron microscopy. LF counteracts METH-induced degeneration. In detail, LF significantly attenuates the amount of cell loss and mitochondrial alterations produced by METH; and mitigates the dissipation of autophagy-related proteins from the autophagy compartment, which is massively induced by METH. These findings indicate a protective role of LF in the molecular mechanisms of neurodegeneration.


Assuntos
Autofagia , Lactoferrina/farmacologia , Metanfetamina/toxicidade , Mitocôndrias/metabolismo , Substâncias Protetoras/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Catepsina D/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lactoferrina/administração & dosagem , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Metanfetamina/administração & dosagem , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Células PC12 , Fenótipo , Ratos , Fatores de Tempo , Tubulina (Proteína)/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
7.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502558

RESUMO

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Germinação/efeitos dos fármacos , Hordeum/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Cloreto de Sódio/farmacologia , Vacúolos/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Endosperma/citologia , Endosperma/metabolismo , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Proteômica/métodos , Sementes/citologia , Sementes/metabolismo , Vacúolos/metabolismo
8.
Mol Neurobiol ; 58(12): 6077-6091, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34449046

RESUMO

Following spinal cord injury (SCI), reactive astrocytes in the glial scar produce high levels of chondroitin sulfate proteoglycans (CSPGs), which are known to inhibit axonal regeneration. Transforming growth factor beta (TGFß) is a well-known factor that induces the production of CSPGs, and in this study, we report a novel mechanism underlying TGFß's effects on CSPG secretion in primary rat astrocytes. We observed increased TGFß-induced secretion of the CSPGs neurocan and brevican, and this occurred simultaneously with inhibition of autophagy flux. In addition, we show that neurocan and brevican levels are further increased when TGFß is administered in the presence of an autophagy inhibitor, Bafilomycin-A1, while they are reduced when cells are treated with a concentration of rapamycin that is not sufficient to induce autophagy. These findings suggest that TGFß mediates its effects on CSPG secretion through autophagy pathways. They also represent a potential new approach to reduce CSPG secretion in vivo by targeting autophagy pathways, which could improve axonal regeneration after SCI.


Assuntos
Astrócitos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Astrócitos/metabolismo , Autofagia/fisiologia , Brevicam/metabolismo , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Macrolídeos/farmacologia , Neurocam/metabolismo , Ratos , Ratos Long-Evans , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
9.
FASEB J ; 35(9): e21742, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34403506

RESUMO

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.


Assuntos
Adesão Celular , Inibição de Contato , Pinocitose , Receptores de Lipoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Toxinas Bacterianas/farmacologia , Sítios de Ligação , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fenótipo , Pinocitose/efeitos dos fármacos , Transporte Proteico , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
10.
Toxicol Appl Pharmacol ; 430: 115680, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34411581

RESUMO

Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.


Assuntos
Creatinina/análogos & derivados , Testes de Toxicidade Crônica , Administração Oral , Animais , Creatinina/administração & dosagem , Creatinina/farmacocinética , Creatinina/toxicidade , Cães , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Rim/efeitos dos fármacos , Rim/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Pneumopatias/induzido quimicamente , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/patologia , Miocárdio/patologia , Nível de Efeito Adverso não Observado , Medição de Risco , Fatores de Tempo , Toxicocinética , Vacúolos/efeitos dos fármacos , Vacúolos/patologia
11.
Toxicology ; 459: 152855, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252479

RESUMO

Cadmium (Cd) is a toxic heavy metal that accumulates in the brain and causes a series of histopathological changes. Selenium (Se) exerts a crucial function in protecting damage caused by toxic heavy metals, but its potential mechanism is rarely studied. The main purpose of this study is to explore the protective effects of Se on Cd-induced oxidative stress and autophagy in rabbit cerebrum. Forty rabbits were randomly divided into four groups and treated as follows: Control group, Cd (1 mg/kg⋅BW) group, Se (0.5 mg/kg⋅BW) group and Cd (1 mg/kg⋅BW)+Se (0.5 mg/kg⋅BW) group, with 30 days feeding management. Our results suggested that Se treatment significantly suppressed the Cd-induced degenerative changes including cell necrosis, vacuolization, and atrophic neurons. In addition, Se decreased the contents of MDA and H2O2 and increased the activities of CAT, SOD, GST, GSH and GSH-Px, alleviating the imbalance of the redox system induced by Cd. Furthermore, Cd caused the up-regulation of the mRNA levels of autophagy-related genes (ATG3, ATG5, ATG7, ATG12 and p62), AMPK (Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag2, Prkag3) and Nrf2 (Nrf2, HO-1 and NQO1) signaling pathway, and the expression levels of LC3II/LC3I, p-AMPK/AMPK, Beclin-1, Nrf2 and HO-1 proteins, which were alleviated by Se, indicated that Se inhibited Cd-induced autophagy and Nrf2 signaling pathway activation. In conclusion, our study found that Se antagonized Cd-induced oxidative stress and autophagy in the brain by generating crosstalk between AMPK and Nrf2 signaling pathway.


Assuntos
Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cádmio/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Selênio/farmacologia , Animais , Antioxidantes/uso terapêutico , Encéfalo/metabolismo , Encéfalo/patologia , Intoxicação por Cádmio/tratamento farmacológico , Intoxicação por Cádmio/patologia , Relação Dose-Resposta a Droga , Necrose , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Coelhos , Receptor Cross-Talk/efeitos dos fármacos , Selênio/uso terapêutico , Selenito de Sódio/farmacologia , Selenito de Sódio/uso terapêutico , Vacúolos/efeitos dos fármacos
12.
J Mol Histol ; 52(5): 919-928, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34309809

RESUMO

Lipopolysaccharides (LPS), which are components of the cell wall of Gram-negative bacteria, are among the important factors that induce inflammation, including pulpitis. Autophagy in human dental pulp cells (hDPCs) acts as a protective mechanism that promotes cell survival under adverse conditions through different signaling pathways. In this study, we examined whether LPS increases autophagy in hDPCs and investigated the role of mitogen-activated protein kinases signaling and nuclear factor κB (NF-κB) in this process. We found that stimulation of hDPCs with 0.1 µg/mL LPS increased the protein and mRNA levels of autophagy markers, beclin1 and microtubule associated protein light chain 3II (LC3II). In addition, acridine orange staining and transmission electron microscopy demonstrated the induction of autophagy upon the treatment of LPS. Furthermore, LPS affected phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and the nuclear translocation of NF-κB. While p38 inhibitor suppressed the LPS-induced increase in protein levels of beclin1 and LC3-II. Our results suggest that LPS induced autophagy in hDPCs and affected the phosphorylation of p38, ERK, and JNK, as well as the nuclear translocation of NF-κB. Phosphorylation of p38 may be involved in LPS-induced autophagy in hDPCs.


Assuntos
Autofagia , Polpa Dentária/citologia , Lipopolissacarídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adolescente , Adulto , Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Polpa Dentária/ultraestrutura , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Adulto Jovem
13.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281286

RESUMO

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane ß2-adrenergic receptors (ARs). Evidence indicates that ß2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.


Assuntos
Metanfetamina/antagonistas & inibidores , Metanfetamina/toxicidade , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Norepinefrina/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Adrenérgicos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/antagonistas & inibidores , Estimulantes do Sistema Nervoso Central/toxicidade , Desipramina/farmacologia , Relação Dose-Resposta a Droga , Metanfetamina/administração & dosagem , Microscopia Eletrônica de Transmissão , Modelos Neurológicos , Neurônios/ultraestrutura , Fármacos Neuroprotetores/farmacologia , Norepinefrina/metabolismo , Células PC12 , Ratos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
15.
Cell Mol Gastroenterol Hepatol ; 12(3): 943-981, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34102314

RESUMO

BACKGROUND & AIMS: The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. METHODS: Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. RESULTS: We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. CONCLUSIONS: Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning.


Assuntos
Amoxicilina/administração & dosagem , Antibacterianos/administração & dosagem , Redes Reguladoras de Genes/efeitos dos fármacos , Intestinos/metabolismo , Metronidazol/administração & dosagem , Vancomicina/administração & dosagem , Amoxicilina/efeitos adversos , Animais , Animais Recém-Nascidos , Antibacterianos/efeitos adversos , Modelos Animais de Doenças , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Metronidazol/efeitos adversos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Permeabilidade/efeitos dos fármacos , Cuidado Pós-Natal , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vancomicina/efeitos adversos
16.
Trop Biomed ; 38(2): 40-47, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33973571

RESUMO

The reduced efficacy of the mainstay antimalarial drugs due to the widespread of drugresistant Plasmodium falciparum has necessitated efforts to discover new antimalarial drugs with new targets. Quercus infectoria (Olivier) has long been used to treat various ailments including fever. The acetone extract of the plant galls has recently been reported to have a promising antimalarial activity in vitro. This study was aimed to determine the effect of the Q. infectoria gall acetone crude extract on pH of the digestive vacuole of Plasmodium falciparum. A ratiometric fluorescent probe, fluorescein isothiocyanate-dextran (FITC-dextran) was used to facilitate a quantitative measurement of the digestive vacuole pH by flow cytometry. Mid trophozoite stage malaria parasites grown in resealed erythrocytes containing FITC-dextran were treated with different concentrations of the acetone extract based on the 50% inhibitory concentration (IC50). Saponin-permeabilized parasites were analyzed to obtain the ratio of green/yellow fluorescence intensity (Rgy) plotted as a function of pH in a pH calibration curve of FITC-dextran. Based on the pH calibration curve, the pH of the digestive vacuole of the acetone extract-treated parasites was significantly altered (pH values ranged from 6.35- 6.71) in a concentration-dependent manner compared to the untreated parasites (pH = 5.32) (p < 0.001). This study provides a valuable insight into the potential of the Q. infectoria galls as a promising antimalarial candidate with a novel mechanism of action.


Assuntos
Antimaláricos , Extratos Vegetais/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Quercus , Vacúolos/efeitos dos fármacos , Acetona , Antimaláricos/farmacologia , Concentração de Íons de Hidrogênio , Quercus/química , Vacúolos/química
17.
Biomed Res Int ; 2021: 6625952, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33880372

RESUMO

The liver is the primary target organ for perfluorooctane sulphonate (PFOS), a recently discovered persistent organic pollutant. However, the mechanisms mediating hepatotoxicity remain unclear. Herein, we explored the relationship between reactive oxygen species (ROS) and autophagy and apoptosis induced by PFOS in L-02 cells, which are incubated with different concentrations of PFOS (0, 50, 100, 150, 200, or 250 µmol/L) for 24 or 48 hrs at 37°C. The results indicated that PFOS exposure decreased cell activities, enhanced ROS levels in a concentration-dependent manner, decreased mitochondrial membrane potential (MMP), and induced autophagy and apoptosis. Compared with the control, 200 µmol/L PFOS increased ROS levels; enhanced the expression of Bax, cleaved-caspase-3, and LC3-II; induced autophagy; decreased MMP; and lowered Bcl-2, p62, and Bcl-2/Bax ratio. The antioxidant N-acetyl cysteine (NAC) protected MMP against PFOS-induced changes and diminished apoptosis and autophagy. Compared with 200 µmol/L PFOS treatment, NAC pretreatment reversed the increase in ROS, Bax, and cleaved-caspase-3 protein caused by PFOS, lowered the apoptosis rate increased by PFOS, and increased the levels of MMP and Bcl-2/Bax ratio decreased by PFOS. The autophagy inhibitor 3-methyladenine and chloroquine decreased apoptosis and cleaved-caspase-3 protein level and increased the Bcl-2/Bax ratio. In summary, our results suggest that ROS-triggered autophagy is involved in PFOS-induced apoptosis in L-02 cells.


Assuntos
Ácidos Alcanossulfônicos/farmacologia , Apoptose , Autofagia , Embrião de Mamíferos/patologia , Fluorocarbonos/farmacologia , Fígado/embriologia , Fígado/patologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cadaverina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
18.
Biochem J ; 478(9): 1705-1732, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33843972

RESUMO

Autophagy, a lysosome-dependent degradative process, does not appear to be a major degradative process in malaria parasites and has a limited repertoire of genes. To better understand the autophagy process, we investigated Plasmodium falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 bind PI3P and play an essential role in autophagosome formation. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is likely a shared feature. Interaction of PfAtg18 with the food vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. Interestingly, wild type ScAtg18 interacted with PI3P, but its expression in P. falciparum showed complete cytoplasmic localization, indicating additional requirement for food vacuole localization. The food vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the immunoprecipitates of PfAtg18 and P. berghei Atg18, and also interacted with PfAtg18. In contrast with PfAtg18, ScAtg18 did not interact with MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine caused cytoplasmic localization of PfAtg18, suggesting that these target PfAtg18 transport pathway. Thus, PI3P and MDR1 are critical mediators of PfAtg18 localization.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Vacúolos/metabolismo , Amodiaquina/farmacologia , Animais , Antimaláricos/farmacologia , Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Transporte Biológico , Cloroquina/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Humanos , Malária/parasitologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/efeitos dos fármacos
19.
Nat Commun ; 12(1): 2183, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846348

RESUMO

Here we show that FTO as an N6-methyladenosine (m6A) RNA demethylase is degraded by selective autophagy, which is impaired by low-level arsenic exposure to promote tumorigenesis. We found that in arsenic-associated human skin lesions, FTO is upregulated, while m6A RNA methylation is downregulated. In keratinocytes, chronic relevant low-level arsenic exposure upregulated FTO, downregulated m6A RNA methylation, and induced malignant transformation and tumorigenesis. FTO deletion inhibited arsenic-induced tumorigenesis. Moreover, in mice, epidermis-specific FTO deletion prevented skin tumorigenesis induced by arsenic and UVB irradiation. Targeting FTO genetically or pharmacologically inhibits the tumorigenicity of arsenic-transformed tumor cells. We identified NEDD4L as the m6A-modified gene target of FTO. Finally, arsenic stabilizes FTO protein through inhibiting p62-mediated selective autophagy. FTO upregulation can in turn inhibit autophagy, leading to a positive feedback loop to maintain FTO accumulation. Our study reveals FTO-mediated dysregulation of mRNA m6A methylation as an epitranscriptomic mechanism to promote arsenic tumorigenicity.


Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Arsênio/toxicidade , Autofagia , Carcinogênese/genética , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Sequência de Bases , Carcinogênese/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Epiderme/metabolismo , Ontologia Genética , Células HEK293 , Células HaCaT , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Sequestossoma-1/metabolismo , Transcriptoma/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
20.
Toxins (Basel) ; 13(2)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673235

RESUMO

We are investigating plant species from the Canadian prairie ecological zone by phenotypic cell assays to discover toxins of biological interest. We provide the first report of the effects of extracts prepared from the shrub Symphoricarpos occidentalis in several human cell lines. S. occidentalis (Caprifoliaceae) extracts are cytotoxic, and, strikingly, treated cells undergo light-dependent vacuolation near the nucleus. The range of irradiation is present in standard ambient light and lies in the visible range (400-700 nm). Vacuolization in treated cells can be induced with specific wavelengths of 408 or 660 nm at 1 J/cm2 energies. Vacuolated cells show a striking phenotype of a large perinuclear vacuole (nuclear associated vacuole, NAV) that is distinct from vesicles observed by treatment with an autophagy-inducing agent. Treatment with S. occidentalis extracts and light induces an intense lamin A/C signal at the junction of a nuclear vacuole and the nucleus. Further study of S. occidentalis extracts and vacuolation provide chemical tools that may contribute to the understanding of nuclear envelope organization and human cell biology.


Assuntos
Núcleo Celular/efeitos dos fármacos , Extratos Vegetais/toxicidade , Plantas Tóxicas/toxicidade , Symphoricarpos/toxicidade , Toxinas Biológicas/toxicidade , Vacúolos/efeitos dos fármacos , Células A549 , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Núcleo Celular/efeitos da radiação , Células HT29 , Humanos , Lamina Tipo A/metabolismo , Luz , Extratos Vegetais/isolamento & purificação , Toxinas Biológicas/isolamento & purificação , Vacúolos/metabolismo , Vacúolos/patologia , Vacúolos/efeitos da radiação
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