Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21.668
Filtrar
1.
Anticancer Res ; 39(11): 5867-5877, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704811

RESUMO

BACKGROUND/AIM: The aim of this study was to examine clonal heterogeneity, to test the utility of liquid biopsy in monitoring disease progression and to evaluate the usefulness of ex vivo drug screening in a BRAF L597Q-mutated colorectal cancer (CRC) patient developing metastases during adjuvant therapy. MATERIALS AND METHODS: Next generation sequencing (NGS) and droplet digital PCR (ddPCR) were performed in samples from tumor tissues and liquid biopsies. Live cancer cells from a metastatic lesion were used in ex vivo drug sensitivity assays. RESULTS: We found evidence of continued dependence of MEK/MAPK pathway activation, but different activating mutations in primary tumor and metastases. Liquid biopsy based BRAF L597Q ddPCR testing was a sensitive personalized biomarker predicting the rise of clinically aggressive metastatic disease. Ex vivo drug sensitivity assays with BRAF L597Q mutated cells showed response to MEK/MAPK targeted therapies. CONCLUSION: The rare BRAF L597Q mutation may be associated with aggressive tumor behavior in CRC. Liquid biopsy can be used to capture clinically relevant tumor features.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/secundário , MAP Quinase Quinase 1/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Capecitabina/administração & dosagem , Evolução Clonal , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxaliplatina/administração & dosagem , Prognóstico
2.
J Agric Food Chem ; 67(44): 12191-12198, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31588747

RESUMO

Fermented black garlic has multiple beneficial biological activities, including cardiovascular protection, anticancer, hepatoprotective, and antibacterial properties. In this study, metabolic differences in the properties of black and fresh garlic were investigated via liquid chromatography quadrupole/time-of-flight-based metabolomics, leading to the identification of characteristic components. Fermented black garlic samples and their Amadori products (AC) promoted angiogenesis, prevented thrombus formation by rescuing chemical-induced vascular lesions in zebrafish, and inhibited H2O2-induced injury of endothelial cells, thus reducing the risk of cardiovascular disease. AC suppressed activation of the mitogen-activated protein kinase pathway through inhibition of p38 and ERK1/2 phosphorylation, in turn, increasing the availability of c-Fos/c-Jun or c-Jun/c-Jun complexes for apoptotic resistance. Clarification of the associated signaling pathways should therefore provide a solid foundation for optimization of black garlic-based therapies.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Alho/química , Extratos Vegetais/administração & dosagem , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Alimentos Fermentados/análise , Humanos , Peróxido de Hidrogênio/toxicidade , Masculino , Espectrometria de Massas , Metabolômica , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Extratos Vegetais/química , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra
3.
Cell Physiol Biochem ; 53(4): 656-686, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31573152

RESUMO

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for approaches that identify new melanoma targets. We have previously reported a discovery of novel anti-melanoma compound 2155-14 (Onwuha-Ekpete et al., J Med Chem. 2014 Feb 27; 57(4):1599-608). In the report presented herein we aim to identify its target(s) and mechanism of action. METHODS: We utilized biotinylated analog of 2155-14 to pull down its targets from melanoma cells. Proteomics in combination with western blot were used to identify the targets. Mechanism of action of 2155-14 was determined using flow cytometry, RT-PCR, microscopy, western blot, and enzymatic activity assays. Where applicable, one-way analysis of variance (ANOVA) was used followed by Dunnett post hoc test. RESULTS: In the present study, we identified ATP-dependent RNA helicase DDX1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) H1, H2 and A2/B1 as targets of anti-melanoma compound 215514. To the best of our knowledge, this is a first report suggesting that these proteins could be targeted for melanoma therapy. Mechanistic investigations showed that 2155-14 induces ER stress leading to potentiation of basal autophagy resulting in melanoma cell death in BRAF and NRAS mutated melanoma cells. CONCLUSION: Identification of mode of action of 2155-14 may provide insight into novel therapies against a broad range of melanoma subtypes. These studies were enabled by the novel probe derived from a mixture-based library, an important class of chemical biology tools for discovering novel targets.


Assuntos
Apoptose , Autofagia , RNA Helicases DEAD-box/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
Plant Mol Biol ; 101(3): 325-339, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31399934

RESUMO

KEY MESSAGE: Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins. Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and in identification of downstream substrates.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteômica , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Brassica napus/enzimologia , Engenharia Genética , Sistema de Sinalização das MAP Quinases , Fosforilação , Imunidade Vegetal , Folhas de Planta/enzimologia , Proteoma , Transdução de Sinais , Tabaco/enzimologia
5.
Ecotoxicol Environ Saf ; 183: 109508, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31408819

RESUMO

As a new type of antibacterial agent, nanosilver has attracted great attention in biomedical applications. However, the safety of nanosilver to humans and the environment has not been well elucidated. The objective of this study was to investigate the influence of nanosilver on novel effector mechanism of neutrophil extracellular traps (NETs), and its possible molecular mechanisms. In this study, nanosilver (10, 20 and 40 µg/mL) was incubated with neutrophils for 90 min. Then, nanosilver-induced the release of NETs was observed by laser confocal microscopy. Nanosilver-induced NETs release was also quantitatively detected by pico Green®. In addition, the role of NADPH oxidase, extracellular signal-regulated kinase (ERK) and p38 signaling pathways in nanosilver-induced NETs release were detected by the inhibitors and pico Green®. The results indicated that nanosilver significantly activated polymorphonuclear neutrophils (PMN) to release NETs, which was a DNA-based network structure modified with histones (H3) and neutrophil elastase (NE). The inhibitors of NADPH oxidase, ERK and p38 signaling pathways significantly inhibited the formation of nanosilver-induced NETs. Furthermore, nanosilver did not alter the extracellular lactate dehydrogenase (LDH) level of PMN cells. All these results showed that nanosilver significantly induced NETs release, and the potential molecular mechanisms were correlated with reactive oxygen species (ROS) production-dependent on NADPH oxidase, ERK and p38 signaling pathways, which might provide a new perspective on nanosilver-induced excess NETs release related to the host immune damage.


Assuntos
Antibacterianos/toxicidade , Armadilhas Extracelulares/metabolismo , Nanopartículas Metálicas/toxicidade , Neutrófilos/efeitos dos fármacos , Prata/toxicidade , Animais , Antibacterianos/química , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nanopartículas Metálicas/química , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Prata/química
6.
Biol Res ; 52(1): 41, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387634

RESUMO

BACKGROUND: Di-N-butyl-phthalate (DBP) is an endocrine disrupting substance. We investigated the adverse effect of DBP on testis of male rat and reveal its potential mechanism of MAPK signaling pathway involved this effect in vivo and in vitro. Gonadal hormone, sperm quality, morphological change and the activation status of JNK, ERK1/2 and p38 was determined in vivo. Primary Sertoli cell was established and cultivated with JNK, ERK1/2 inhibitors, then determine the cell viability, apoptosis and the expression of p-JNK, p-ERK1/2. Data in this study were presented as mean ± SD and determined by one-way analysis of variance (ANOVA) followed by Bonferroni's test. Difference was considered statistically significant at P < 0.05. RESULTS: In vivo experiment, DBP impaired the normal structure of testicular tissue, reduced testosterone levels in blood serum, decreased sperm count and increased sperm abnormality, p-ERK1/2 and p-JNK in rat testicular tissue increased in a dose-dependent manner. In vitro studies, DBP could decrease the viability of Sertoli cells and increase p-ERK1/2 and p-JNK. Cell apoptosis in SP600125 + DBP group was significantly lower than in DBP group (P < 0.05). p-JNK was not significantly decreased in SP600125 + DBP group, while p-ERK1/2 was significantly decreased in U0126 + DBP group. CONCLUSIONS: These results suggest that DBP can lead to testicular damage and the activation of ERK1/2 and JNK pathways, the JNK signaling pathway may be primarily associated with its effect.


Assuntos
Dibutilftalato/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais/fisiologia , Testículo/lesões , Testículo/metabolismo , Animais , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Ratos , Ratos Sprague-Dawley , Testículo/efeitos dos fármacos
7.
Zhonghua Fu Chan Ke Za Zhi ; 54(8): 541-547, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31461811

RESUMO

Objective: To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines, and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation, apoptosis as well as cell cycle of ovarian cancer cells. To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer. Methods: (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital, Dalian Medical University from January 2004 to December 2013 were selected. The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens, and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed. (2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3 treated with or without MEK inhibitor. The cellular proliferation, apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P>0.05) . However, a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma, mucinous carcinoma and endometrioid carcinoma (all P<0.05) . There was no significant correlation between the protein expression of p-ERK1/2 and patients' age, pathological stage of surgery, and preoperative serum CA(125) level (P>0.05). (2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3. After treatment with AZD6244 (5, 10 µmol/L), the level of p-ERK1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner. Additionally, we found a reduction of the expression level of cyclin D1, caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8, compared with control groups. MTT assays showed that OVCAR5, OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0, 2.5, 5, 10, 25, 50 and 100 µmol/L, all P<0.05). Further tested by flow cytometry, the results showed that AZD6244 (5, 10 µmol/L) was able to induce the apoptosis of OVCAR5, OVCAR8 and A2780S, as well as G(0)/G(1) phase arrest, both in a dose-dependent manner (P<0.05). Conclusions: As the main active and functional unit of MAPK/ERK signaling pathway, p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines. AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells, accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells. In conclusion, MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer, and may be provide a novel option for molecular targeted therapies of the disease.


Assuntos
Carcinoma Epitelial do Ovário , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Ovarianas , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Neoplasias Ovarianas/genética
8.
Adv Exp Med Biol ; 1155: 1001-1014, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468463

RESUMO

Batillaria multiformis (B. multiformis) belong to gastropods. They live generally in the sandpit of the lagoons and the estuaries of the intertidal zone. Most of them are distributed in Korea, Japan and China. In this study, we investigated the anti-inflammatory potential of B. multiformis water extracts (BMW). The results showed that the extracts significantly decreased the production of nitric oxide (NO) and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-induced RAW 264.7 macrophages. In addition, the extracts suppressed the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose dependent manner. Further investigation indicated that BMW suppressed phosphorylated c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase (ERK) and p38 through the MAPK signaling pathway and influenced the NF-κB signaling pathway by suppressing the IκBα degradation in LPS-induced RAW 264.7 macrophages.


Assuntos
Anti-Inflamatórios/farmacologia , Extratos Celulares/farmacologia , Gastrópodes/química , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Lipopolissacarídeos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Água
9.
Adv Exp Med Biol ; 1155: 1069-1081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468468

RESUMO

Scallops belong to cosmopolitan family of bivalves which are found in any oceans. They are one of the most important marine fishery resources in the world. The shell, meat and pearl layer have a high utilization value and a lot of scallops are eaten as food. In this study, we established anti-inflammatory effect of Scallops water extract in lipopolysaccharide (LPS) stimulated RAW 264.7 mononuclear macrophage. Our results indicated that Scallop water extract effectively reduced the synthesis of nitric oxide (NO). In addition, Scallop water extract suppressed the reactive oxygen species (ROS) generation and the expression of IL-6 and TNF-α. Further investigation indicated that anti-inflammatory effect of Scallop water extract via suppressing downregulation of MAPK (JNK, p38 and ERK) and NF-κB signaling.


Assuntos
Anti-Inflamatórios/farmacologia , Extratos Celulares/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Pectinidae/química , Animais , Citocinas/metabolismo , Inflamação , Lipopolissacarídeos , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
10.
Aquat Toxicol ; 215: 105261, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31419757

RESUMO

Harmful cyanobacteria and their production of microcystins (MCs) exert significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 µg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 µg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 µg/L MC-LR treatment of 4 h. In the oocytes exposed to 100 µg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels. Moreover, the MAPK inhibitors (10 µM) were applied to explore the role of MAPK subtypes during MC-LR influencing OM and results showed that ERK inhibitor U0126 and p38 inhibitor SB203580 mitigated the effects of 100 µg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes. In conclusion, the present study indicates that microcystins disrupt the meiotic maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation disorder in oocytes.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microcistinas/toxicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peixe-Zebra/fisiologia , Animais , Ciclina B/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Fator Promotor de Maturação/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/enzimologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/metabolismo , Vitelogeninas/metabolismo , Poluentes Químicos da Água/toxicidade
11.
Chemosphere ; 233: 786-795, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31340409

RESUMO

Microbial volatile organic compounds (mVCs) are formed in the metabolism of microorganisms and widely distributed in nature and pose threats to human health. However, the air pollution by microorganisms is a situation which is poorly understood. In this study, the cytotoxicity of E. aerogenes VCs was evaluated in the model organism Saccharomyces cerevisiae. E. aerogenes VCs inhibited the survival of yeast and triggered the formation of intracellular reactive oxygen species (ROS). The hypersensitive of MAP kinase mpk1/slt2 and 19S regulatory assembly chaperone adc17 mutants to the E. aerogenes VCs indicated cell wall integrity (CWI) pathway together with stress-inducible proteasome assembly regulation are essentially involved in mVCs tolerance mechanism. Furthermore, exposure to the mVCs resulted in the transcriptional upregulation of the CWI pathway, the regulatory particle assembly chaperones, and genes involved in proteasome regulations. Our research suggested that the ROS/MAPK signaling and proteasome regulatory pathway play pivotal roles in the integration and fine-tuning of the mVCs stress response. This study provides a molecular framework for future study of the effects of mVCs on more complex organisms, such as humans.


Assuntos
Enterobacter aerogenes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Compostos Orgânicos Voláteis/farmacologia , Poluentes Atmosféricos/análise , Poluição do Ar/análise , Parede Celular/metabolismo , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional
12.
Vet Microbiol ; 235: 127-135, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282370

RESUMO

Lawsonia intracellularis is an obligate intracellular Gram-negative bacterium that has been identified as the etiological agent of the contagious disease proliferative enteropathy (PE) in a wide range of animals, mainly pigs. The genome sequence of L. intracellularis indicates that this bacterium possess a type III secretion system (T3SS), which may assist the bacterium during cell invasion and host innate immune system evasion and could be a mechanism for inducing cellular proliferation. However, the effectors secreted by the T3SS (T3Es) of L. intracellularis have not been reported. T3Es often target conserved eukaryotic cellular processes, and yeast is an established and robust model system in which to reveal their function. By screening the growth inhibition of an ordered array of Saccharomyces cerevisiae strains expressing the hypothetical genes of L. intracellularis, LI1035 was identified as the first putative effector that inhibits yeast growth. The LI1035-induced growth inhibition was rescued in two of the 14 mitogen-activated protein kinase (MAPK) yeast haploid deletion strains, suggesting that LI1035 interacts with the components of the MAPK pathway in yeast. Phosphorylation assays confirmed that LI1035 inhibits MAPK signaling cascades in yeast and mammalian cells. Actin staining assays revealed that LI1035 regulates actin organization in yeast and mammalian cells. Taken together, these results indicate that LI1035 alters MAPK pathway activity and regulates actin organization in the host. These findings may contribute to the understanding the pathogenesis of L. intracellularis and support the use of yeast as a heterologous system for the functional analysis of pathogen-specific gene products in the laboratory.


Assuntos
Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Lawsonia (Bactéria)/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , Animais , Proteínas de Bactérias/genética , Proliferação de Células , Interações entre Hospedeiro e Microrganismos , Lawsonia (Bactéria)/genética , Fosforilação , Saccharomyces cerevisiae/genética , Sorbitol/farmacologia , Suínos , Temperatura Ambiente
13.
J Biochem ; 166(2): 175-185, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31329883

RESUMO

TMEM55B is first identified as phosphatidylinositol-4,5-P24-phosphatases (PtdIns-4,5-P24-phosphatases) that catalyse dephosphorylation of PtdIns-4,5-P2 to PtdIns-5-P. We demonstrate for the first time that TMEM55B is phosphorylated by Erk/MAPK and that this mechanism participates in regulation of lysosomal clustering. Exposure of RAW264.7 macrophages to various stimuli induces phosphorylation of TMEM55B on Ser76 and Ser169, sites corresponding to consensus sequences (PX(S/T)P) for phosphorylation by MAPK. Of these stimuli, Toll-like receptor ligands most strongly induce TMEM55B phosphorylation, and this is blocked by the MEK1/2 inhibitor U0126. However, phosphorylation does not impact intrinsic phosphatase activity of TMEM55B. TMEM55B has recently been implicated in starvation induced lysosomal translocation. Amino acid starvation induces perinuclear lamp1 clustering in RAW264.7 macrophages, which was attenuated by shRNA-mediated knock-down or CRISPR/Cas9-mediated knock-out of TMEM55B. Cells exposed to U0126 also exhibit attenuated lamp1 clustering. Overexpression of TMEM55B but not TMEM55A notably enhances lamp1 clustering, with TMEM55B mutants (lacking phosphorylation sites or mimicking the phosphorylated state) exhibiting lower and higher efficacies (respectively) than wild-type TMEM55B. Collectively, results suggest that phosphorylation of TMEM55B by Erk/MAPK impacts lysosomal dynamics.


Assuntos
Lisossomos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatases de Fosfoinositídeos/química , Fosfatases de Fosfoinositídeos/metabolismo , Animais , Camundongos , Fosforilação , Células RAW 264.7
14.
Life Sci ; 231: 116550, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185235

RESUMO

AIM: Acrylamide (AA) is a common heat-generated toxicant in some food. Inhibiting its formation with natural antioxidants is of great significance. The current study aims to investigate the alleviative effect and the underlying mechanism of allicin against AA-induced oxidative stress in BRL-3A cells. MAIN METHODS: BRL-3A cells were pretreated with allicin at different concentrations for 2 h, followed by AA treatment. Cell viability was determined by Cell Counting kit-8 (CCK-8). Intracellular reactive oxygen species (ROS) status was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method. Levels of oxidative stress markers were determined by measuring total superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and 8-hydroxy-desoxyguanosine (8-OHdG) using commercial kits. Expression of the mitogen-activated protein kinase (MAPK) pathway-related proteins was determined by Western blotting. KEY FINDINGS: Allicin markedly mitigated oxidative and DNA damage by increasing the activities of SOD and GSH-Px and decreasing the levels of ROS and 8-OHdG. Concomitant with these biochemical parameters, pretreatment with allicin reversed the impact of AA on the expression of p-JNK, p-ERK1/2 and p-p38. Allicin combined with SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor) enhanced cell viability in the presence of AA, as opposed to SCH772984 (ERK inhibitor). Notably, allicin ameliorated the expression of KGF, Gadd45a, c-Fos, Dusp5 and Phospholipase A2, which were related to liver injury. SIGNIFICANCE: Collectively, these findings demonstrate that allicin exerts protective effects against AA-induced oxidative stress by modulating the MAPK signaling pathway in BRL-3A cells.


Assuntos
Estresse Oxidativo/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Acrilamida/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
15.
J Microbiol Biotechnol ; 29(7): 1022-1032, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216608

RESUMO

Probiotics are known to provide the host with immune-modulatory effects and are therefore of remarkable interest for therapeutic and prophylactic applications against various disorders, including inflammatory diseases. Weissella cibaria JW15 (JW15) has been reported to possess probiotic and antioxidant properties. However, the effect of JW15 on inflammatory responses has not yet been reported. Therefore, the objective of the current study was to evaluate the anti-inflammatory potential of JW15 against lipopolysaccharide (LPS) stimulation. The production of pro-inflammatory factors and the cellular signaling pathways following treatment with heat-killed JW15 was examined in LPS-induced RAW 264.7 cells. Treatment with heat-killed JW15 decreased nitric oxide and prostaglandin E2 production via downregulation of the inducible nitric oxide synthase and cyclooxygenase-2. In addition, treatment with heat-killed JW15 suppressed the expression of pro-inflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α. The anti-inflammatory properties of treating with heat-killed JW15 were associated with mitogen-activated protein kinase signaling pathwaymediated suppression of nuclear factor-κB. These results indicated that JW15 possesses antiinflammatory potential and provide a molecular basis regarding the development of functional probiotic products.


Assuntos
Anti-Inflamatórios/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Probióticos/farmacologia , Fator de Transcrição RelA/metabolismo , Weissella/fisiologia , Animais , Sobrevivência Celular , Ciclo-Oxigenase 2/genética , Citocinas/genética , Citocinas/metabolismo , Dinoprostona/metabolismo , Alimentos Fermentados/microbiologia , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
16.
Food Chem Toxicol ; 131: 110583, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31220533

RESUMO

We investigated the anti-inflammatory activity of protopine (PTP) and sought to determine its mechanism of action in LPS-stimulated BV2 cells and a carrageenan (CA)-induced mouse model. Treatment with PTP (5, 10, and 20 µM) significantly suppresses the secretion of NO and PGE2 in a concentration-dependent manner without affecting cell viability by downregulating iNOS and COX-2 expression in LPS-induced BV2 cells. PTP also attenuates the production of pro-inflammatory chemokines, such as MCP-1, and cytokines, including TNF-α, IL-1ß and IL-6, and augments the expression of the anti-inflammatory cytokine IL-10. In addition, PTP suppresses the nuclear translocation of NF-κB by hindering the degradation of IκB and downregulating the expression of mitogen-activated protein kinases (MAPKs), including p38, ERK1/2 and JNK protein. Furthermore, PTP treatment significantly suppresses CA-induced paw oedema in mice compared to that seen in untreated mice. Expression of iNOS and COX-2 proteins is also abrogated by PTP (50 mg/kg) treatment in CA-induced mice. PTP treatment also abolishes IκB phosphorylation, which hinders the activation of NF-κB. Collectively, these results suggest PTP has potential for attenuating CA- and LPS-induced inflammatory symptoms through modulation of MAPKs/NF-κB signaling cascades.


Assuntos
Anti-Inflamatórios/uso terapêutico , Benzofenantridinas/uso terapêutico , Alcaloides de Berberina/uso terapêutico , Inflamação/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Anti-Inflamatórios/toxicidade , Benzofenantridinas/toxicidade , Alcaloides de Berberina/toxicidade , Carragenina , Linhagem Celular Transformada , Quimiocinas/metabolismo , Inflamação/induzido quimicamente , Lipopolissacarídeos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos
17.
Gen Physiol Biophys ; 38(4): 315-324, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31241043

RESUMO

Diosmin is an unsaturated flavonoid glycoside, presents in citrus fruits. The aim of this study is to investigate the molecular mechanism of diosmin with respect to the NF-κB and MAPKs signaling pathways. Firstly, 10, 20, 30, 40 and 50 µM diosmin were treated to lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The anti-inflammatory effects of diosmin was displayed via measuring prostaglandin E2 (PGE2), nitric oxide (NO), interleukines (IL-6, IL-12), tumor necrosis factor α (TNF-α) production, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6, IL-12, TNF-α mRNA levels, and phosphorylation levels of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκB-α) and mitogen-activated protein kinases (MAPKs); JNK, ERK, and p38 in LPS induced RAW264.7 macrophages. Our study showed that especially high concentrations of diosmin decreased NO, PGE2, IL-6, IL-12, TNF-α production and mRNA levels of these mediators (p < 0.05). The expression of phosphorylated-JNK was significantly suppressed by diosmin at 40 and 50 µM concentrations. Furthermore, diosmin significantly inhibited the expression of phosphorylated-ERK, p38, and p-IκB-α in a dose-dependent manner. Our results suggest that diosmin is a potent anti-inflammatory agent and has potential for development into a therapeutic agent for inflammation-associated disorders.


Assuntos
Diosmina/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos
18.
Mol Plant Microbe Interact ; 32(11): 1496-1507, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31251114

RESUMO

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1-silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.


Assuntos
Interações Hospedeiro-Patógeno , Proteínas Quinases Ativadas por Mitógeno , Doenças das Plantas , Transdução de Sinais , Interações Hospedeiro-Patógeno/fisiologia , Lycopersicon esculentum/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Pseudomonas syringae/enzimologia , Tabaco/enzimologia
19.
PLoS Genet ; 15(5): e1008192, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31150379

RESUMO

Quorum sensing (QS), a mechanism of microbial communication dependent on cell density, governs developmental decisions in many bacteria and in some pathogenic and non-pathogenic fungi including yeasts. In these simple eukaryotes this response is mediated by the release into the growth medium of quorum-sensing molecules (QSMs) whose concentration increases proportionally to the population density. To date the occurrence of QS is restricted to a few yeast species. We show that a QS mediated by the aromatic alcohols phenylethanol and tryptophol represses the dimorphic yeast to hypha differentiation in the fission yeast S. japonicus in response to an increased population density. In addition, the stress activated MAPK pathway (SAPK), which controls cell cycle progression and adaptation to environmental changes in this organism, constitutively represses yeast to hypha differentiation both at transcriptional and post-translational levels. Moreover, deletion of its main effectors Sty1 MAPK and Atf1 transcription factor partially suppressed the QS-dependent block of hyphal development under inducing conditions. RNAseq analysis showed that the expression of nrg1+, which encodes a putative ortholog of the transcription factor Nrg1 that represses yeast to hypha dimorphism in C. albicans, is downregulated both by QS and the SAPK pathway. Remarkably, Nrg1 may act in S. japonicus as an activator of hyphal differentiation instead of being a repressor. S. japonicus emerges as an attractive and amenable model organism to explore the QS mechanisms that regulate cellular differentiation in fungi.


Assuntos
Hifas/crescimento & desenvolvimento , Percepção de Quorum/fisiologia , Schizosaccharomyces/genética , Divisão Celular , Regulação Fúngica da Expressão Gênica/genética , Hifas/genética , Indóis/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Álcool Feniletílico/metabolismo , Densidade Demográfica , Processamento de Proteína Pós-Traducional , Percepção de Quorum/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição/metabolismo
20.
J Photochem Photobiol B ; 197: 111518, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31202076

RESUMO

Disclosure of ultraviolet (UV) radiation is the key feature from environment to cause redness of the skin, inflammation, photoaging and skin cancer. 6-Shogaol, a spicy compound secluded from ginger, which shows anti-inflammatory effects. Present study was demonstrated the role of 6-Shogaol on UVB induced oxidative stress and photoaging signaling in human epidermal keratinocytes (HaCaT cells). In this study, UVB-irratiation (180 mJ/cm2) significantly elevated the intracellular ROS levels, depletion of antioxidants resulted in apoptotic HaCaT cells. MAPKs signaling are concerned in oxidative stress; these signaling events are measured as differentiation. We found that 6-shogaol prevents over expression of MAPKs (ERK1, JNK1 & p38), in disclosure of UVB in HaCaT cells. Moreover, 6-shogaol infringed Bax and Bcl-2 in which 20 µg 6-shogaol influenced apoptosis in HaCaT cells by investigating augmented appearance of Bax and condensed appearance of Bcl-2 in contrast to control HaCaT cells. These results suggest that 6-shogaol could be a successful healing agent provides fortification against UVB-induced provocative and oxidative skin reimbursement.


Assuntos
Catecóis/farmacologia , Gengibre/química , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Raios Ultravioleta , Apoptose/efeitos dos fármacos , Catecóis/química , Catecóis/uso terapêutico , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Gengibre/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/prevenção & controle , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA