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1.
Nat Commun ; 11(1): 4284, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855409

RESUMO

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.


Assuntos
Arabidopsis/metabolismo , Citocininas/metabolismo , Proteínas Recombinantes/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Líquido Extracelular/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Mutação , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Transdução de Sinais
2.
J Nanosci Nanotechnol ; 20(12): 7311-7323, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32711596

RESUMO

We started a study on the molecular docking of six potential pharmacologically active inhibitors compounds that can be used clinically against the COVID-19 virus, in this case, remdesivir, ribavirin, favipiravir, galidesivir, hydroxychloroquine and chloroquine interacting with the main COVID-19 protease in complex with a COVID-19 N3 protease inhibitor. The highest values of affinity energy found in order from highest to lowest were chloroquine (CHL), hydroxychloroquine (HYC), favipiravir (FAV), galidesivir (GAL), remdesivir (REM) and ribavirin (RIB). The possible formation of hydrogen bonds, associations through London forces and permanent electric dipole were analyzed. The values of affinity energy obtained for the hydroxychloroquine ligands was -9.9 kcal/mol and for the chloroquine of -10.8 kcal/mol which indicate that the coupling contributes to an effective improvement of the affinity energies with the protease. Indicating that, the position chosen to make the substitutions may be a pharmacophoric group, and cause changes in the protease.


Assuntos
Antivirais/química , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Adenina/administração & dosagem , Adenina/análogos & derivados , Adenina/química , Adenina/farmacologia , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Amidas/administração & dosagem , Amidas/química , Amidas/farmacologia , Antivirais/administração & dosagem , Sítios de Ligação , Cloroquina/administração & dosagem , Cloroquina/química , Cloroquina/farmacologia , Interações Medicamentosas , Humanos , Ligação de Hidrogênio , Hidroxicloroquina/administração & dosagem , Hidroxicloroquina/química , Hidroxicloroquina/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Nanotecnologia , Pandemias , Inibidores de Proteases/administração & dosagem , Pirazinas/administração & dosagem , Pirazinas/química , Pirazinas/farmacologia , Pirrolidinas/administração & dosagem , Pirrolidinas/química , Pirrolidinas/farmacologia , Ribavirina/administração & dosagem , Ribavirina/química , Ribavirina/farmacologia , Eletricidade Estática
3.
Int. j. morphol ; 38(3): 530-535, June 2020. graf
Artigo em Inglês | LILACS | ID: biblio-1098283

RESUMO

Dysregulated autophagy, whether excessive or downregulated, has been thought to be associated with neurodegenerative disorders including Parkinson's disease. Accordingly, the present study was carried out to investigate whether 3-methyladenine, an autophagy inhibitor, can modulate the effects of rotenone on dopaminergic neurons in primary mesencephalic cell culture. Cultures were prepared from embryonic mouse mesencephala at gestation day 14. Four groups of cultures were treated on the 10th DIV for 48 h as follows: the first was kept as an untreated control, the second was treated with 3-methyladenine alone (1, 10, 100, 200 mM), the third was treated with 20 nM rotenone and the fourth was co-treated with 20 nM rotenone and 3-methyladenine (1, 10, 100, 200 mM). On the 12th DIV, cultured cells were stained immunohistochemically against tyrosine hydroxylase and culture media were used to measure the levels of lactate dehydrogenase. 3methyladenine had no effects on both the survival of dopaminergic neurons and the release of lactate dehydrogenase. Rotenone significantly decreased the number of dopaminergic neurons and increased the levels of lactate dehydrogenase in the culture media. When cultures concomitantly treated with 3-methyladenine and rotenone, 3-methyladenine had no effect against rotenone-induced dopaminergic cell damage and lactate dehydrogenase release into the culture medium. In conclusion, the autophagy inhibitor 3-methyladenine could not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.


Se estima que la autofagia desregulada, ya sea excesiva o con baja regulación, está asociada con trastornos neurodegenerativos, incluyendo la enfermedad de Parkinson. En consecuencia, el se realizó este estudio para investigar si la 3metiladenina, un inhibidor de la autofagia,puede modular los efectos de la rotenona en las neuronas dopaminérgicas en el cultivo primario de células mesencefálicas. Los cultivos se prepararon a partir de mesencéfalo de ratón embrionario el día 14 de gestación. Cuatro grupos de cultivos se trataron en el 10º DIV durante 48 h de la siguiente manera: el primer grupo se mantuvo como un control no tratado, el segundo se trató con 3-metiladenina sola (1, 10, 100, 200 mM), el tercer grupo se trató con rotenona 20 nM y el cuarto se trató conjuntamente con rotenona 20 nM y 3-metiladenina (1, 10, 100, 200 mM). En el 12º DIV; las células cultivadas fueron tratadas mediante tinción inmunohistoquímica en tirosina hidroxilasa y se usaron medios de cultivo para medir los niveles de lactato deshidrogenasa. La 3-metiladenina no tuvo efectos tanto en la supervivencia de las neuronas dopaminérgicas como en la liberación de lactato deshidrogenasa. La rotenona disminuyó significativamente el número de neuronas dopaminérgicas y se observó un aumento de los niveles de lactato deshidrogenasa en los medios de cultivo. Cuando los cultivos tratados concomitantemente con 3-metiladenina y rotenona, la 3metiladenina no tuvo efecto contra el daño celular dopaminérgico inducido por la rotenona y la liberación de lactato deshidrogenasa en el medio de cultivo. En conclusión, el inhibidor de la autofagia 3-metiladenina no moduló el daño celular dopaminérgico inducido por la rotenona en el cultivo celular mesencefálico primario.


Assuntos
Animais , Camundongos , Doença de Parkinson , Rotenona/toxicidade , Adenina/análogos & derivados , Autofagia , Mesencéfalo , Adenina/farmacologia , Células Cultivadas , Morte Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , L-Lactato Desidrogenase/análise
4.
Arterioscler Thromb Vasc Biol ; 40(6): 1510-1522, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32349535

RESUMO

OBJECTIVE: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1-deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr-/- and Cav-1-/-Ldlr-/- mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1-/- mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis. CONCLUSIONS: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1-deficient mice attenuates the atheroprotection observed in Cav-1-/- mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.


Assuntos
Aterosclerose/prevenção & controle , Autofagia/fisiologia , Caveolina 1/deficiência , Endotélio Vascular/fisiopatologia , Vasculite/prevenção & controle , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Aorta/patologia , Aorta/fisiopatologia , Aorta/ultraestrutura , Aterosclerose/etiologia , Autofagia/efeitos dos fármacos , Caveolina 1/análise , Caveolina 1/fisiologia , Dieta Ocidental , Células Endoteliais/química , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Endotélio Vascular/química , Endotélio Vascular/ultraestrutura , Feminino , Humanos , Masculino , Microdomínios da Membrana/química , Microdomínios da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Receptores de LDL/deficiência
5.
Scand J Immunol ; 91(6): e12880, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32219875

RESUMO

Synthetic Toll-like receptor (TLR) 7 agonists have been suggested as immune modulators in a range of conditions. In contrast, self-derived TLR7 activators, such as RNA-containing immune complexes (RNA-IC), can contribute to autoimmune diseases due to endogenous immune activation. The exact difference in immune cell response between synthetic and endogenous TLR7 triggers is only partly known. An understanding of these differences could aid in the development of new therapeutic agents and provide insights into autoimmune disease mechanisms. We therefore compared the stimulatory capacity of two TLR7 agonists, RNA-IC and a synthetic small molecule DSR-6434, on blood leucocytes, plasmacytoid dendritic cells (pDCs) and B cells from healthy individuals. IFN-α, IL-6, IL-8 and TNF levels were measured by immunoassays, and gene expression in pDCs was analysed by an expression array. DSR-6434 triggered 20-fold lower levels of IFN-α by pDCs, but higher production of IL-6, IL-8 and TNF, compared to RNA-IC. Furthermore, IFN-α and TNF production were increased with exogenous IFN-α2b priming, whereas IL-8 synthesis by B cells was reduced for both stimuli. Cocultivation of pDCs and B cells increased the RNA-IC-stimulated IFN-α and TNF levels, while only IL-6 production was enhanced in the DSR-6434-stimulated cocultures. When comparing pDCs stimulated with RNA-IC and DSR-6434, twelve genes were differentially expressed (log2 fold change >2, adjusted P-value <.05). In conclusion, RNA-IC, which mimics an endogenous TLR7 stimulator, and the synthetic TLR7 agonist DSR-6434 trigger distinct inflammatory profiles in immune cells. This demonstrates the importance of using relevant stimuli when targeting the TLR7 pathway for therapeutic purposes.


Assuntos
Adenina/farmacologia , Complexo Antígeno-Anticorpo/farmacologia , Linfócitos B/imunologia , Células Dendríticas/imunologia , Complexos Multiproteicos/farmacologia , RNA/farmacologia , Receptor 7 Toll-Like/metabolismo , Adenina/análogos & derivados , Adenina/química , Complexo Antígeno-Anticorpo/química , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Estrutura Molecular , Complexos Multiproteicos/química , RNA/química , Receptor 7 Toll-Like/agonistas
6.
J Nat Med ; 74(3): 525-532, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32207025

RESUMO

Autophagy is a catabolic process that degrades dysfunctional proteins and organelles and plays critical roles in cancer development. Our preliminary screening identified that extracts of the fruits of Arctium lappa and the fruits of Forsythia suspensa notably suppressed the proliferation of hepatocellular carcinoma HepG2 cells and downregulated the autophagy. In this study, we explored the effect of arctigenin (ARG), a bioactive lignan in both extracts, on cell proliferation and autophagy-related proteins in HepG2 cells. ARG inhibited the proliferation of HepG2 cells. Analysis of autophagy-related proteins demonstrated that ARG might block the autophagy that leads to sequestosome 1/p62 (p62) accumulation. The stage of inhibition in autophagy by ARG differed from those by the autophagy inhibitors 3-methyladenine (3-MA) or chloroquine (CQ). ARG could also inhibit starvation-induced autophagy. Further analysis of apoptosis-related proteins indicated that ARG did not affect caspase-3 activation and PARP cleavage, suggesting that the antiproliferative effect of ARG can occur independently of apoptosis. In summary, our study showed that ARG suppresses cell proliferation and inhibits autophagy, and might lead to the development of agents for autophagy research and cancer chemoprevention.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Furanos/farmacologia , Lignanas/farmacologia , Neoplasias Hepáticas/patologia , Adenina/análogos & derivados , Adenina/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Arctium/química , Linhagem Celular Tumoral , Cloroquina/farmacologia , Forsythia/química , Células Hep G2 , Humanos , Proteína Sequestossoma-1
7.
Artigo em Inglês | MEDLINE | ID: mdl-32186399

RESUMO

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.


Assuntos
Caspase 1/genética , AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Caspase 1/metabolismo , Proliferação de Células/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/agonistas , AMP Cíclico/antagonistas & inibidores , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Dinoprostona/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Inflamassomos/efeitos dos fármacos , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Cultura Primária de Células , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Ratos , Rolipram/farmacologia , Análise de Célula Única
8.
Transfusion ; 60(5): 1042-1049, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32187700

RESUMO

BACKGROUND: Some jurisdictions require leukoreduction of cellular blood components. The only whole blood collection set with a platelet-saving filter uses citrate-phosphate-dextrose (CPD) as storage solution. Substituting CPD with citrate-phosphate-dextrose-adenine (CPDA-1) increases shelf life from 21 to 35 days. This would simplify prehospital and rural resupply and reduce wastage. We investigated in vitro quality and hemostatic properties of CPDA-1 whole blood leukoreduced with a platelet-saving filter. STUDY DESIGN AND METHODS: CPDA-1 whole blood was leukoreduced using a platelet-saving filter and stored 35 days. EDQM requirements, hematology, metabolic parameters, thromboelastography, light transmission aggregometry, fibrinogen, factor VIII, and interleukin-6 were measured on Days 0, 1, 14, 21, and 35 and compared to non-leukoreduced blood. RESULTS: All units met EDQM requirements. Leukoreduction yielded residual white blood cell count <1 × 106 and 87% platelet recovery on Day 1. It caused reduction in thromboelastography parameters, but not aggregometry response. No hemolysis >0.8% was observed. Factor VIII was higher on Day 35 in the leukoreduced group, 37.9 (95% CI: 26.0, 49.8) versus 13.8 (9.4, 18.2) IU/dL. In both groups, aggregation was significantly reduced by Day 14. Thromboelastography showed remaining platelet activity on Day 35, MA 46.9 (42.1, 51.7) in the leukoreduced and 44.3 (39.6, 49.0) mm in the non-leukoreduced group. Fibrinogen was within reference ranges at Day 35 (>2 g/dL). Interleukin-6 was not detectable. CONCLUSION: Leukoreducing CPDA-1 whole blood with a platelet-saving filter did not compromise hemostatic properties. We encourage development of a single bag CPDA-1 whole blood collection set with in-line platelet-saving filter.


Assuntos
Adenina/química , Preservação de Sangue/métodos , Coleta de Amostras Sanguíneas/métodos , Citratos/química , Temperatura Baixa , Glucose/química , Procedimentos de Redução de Leucócitos/métodos , Fosfatos/química , Adenina/farmacologia , Sangue/efeitos dos fármacos , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Preservação de Sangue/normas , Coleta de Amostras Sanguíneas/normas , Citratos/farmacologia , Filtração/métodos , Glucose/farmacologia , Hemólise/efeitos dos fármacos , Hemostasia/efeitos dos fármacos , Humanos , Técnicas In Vitro , Procedimentos de Redução de Leucócitos/normas , Fosfatos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Controle de Qualidade , Refrigeração/métodos
9.
Biomed Pharmacother ; 122: 109698, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31918272

RESUMO

AIM: We aim to explore the effects of nucleos(t)ide analogues (NUCs) on the changes of HBsAg in chronic hepatitis B (CHB) patients. METHODS: A total of 264 CHB patients were enrolled in our study. All of them were treated with NUCs for at least three years. Quantification of HBsAg levels were measured by Elecsys HBsAg II. RESULTS: Although HBsAg levels were significantly higher in HBeAg seropositive CHB patients at baseline than in HBeAg seronegative CHB patients (3.84 ±â€¯0.82 vs 3.21 ±â€¯0.59 IU/mL), HBsAg levels declined more rapidly in the HBeAg seropositive group (P < 0.001). In HBeAg-positive CHB patients, HBsAg level in the telbivudine (LDT)-treated group was 3.68 ±â€¯0.56 IU/mL after 52-week of treatment, which was significantly higher than that in lamivudine (LAM)-treated group (P = 0.009). Multivariable analyses showed that baseline HBV DNA viral load (OR = 0.75, P = 0.018), baseline ALT level (OR = 0.99, P = 0.015), and baseline HBsAg level (OR = 0.188, P < 0.001) were independent factors that affected HBsAg decline in HBeAg seropositive CHB patients. For HBeAg seronegative CHB patients, the average of serum HBsAg levels in LAM-, LdT-, adefovir (ADV)-, and entecavir (ETV)-treated groups at baseline, 52 weeks, 104 weeks, and 156 weeks were similar. Multivariable analyses showed that only baseline HBV DNA level (OR = 0.56, P = 0.020) and baseline HBsAg level (OR = 0.57, P = 0.012) were independent factors that affected HBsAg decline in HBeAg seronegative patients with CHB. Baseline HBV DNA level (OR = 0.72, P = 0.010) and baseline HBsAg level (OR = 0.19, P < 0.001) were independent factors that affected all CHB patients. CONCLUSIONS: CHB Patients who had received NUCs antiviral treatment showed a slow but significant decrease in serum HBsAg level. Long-term monitoring and continuous antiviral treatment are necessary, especially for those patients with risk factors associated with HBsAg decline.


Assuntos
Antivirais/farmacologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Nucleotidases/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Adulto , DNA Viral/efeitos dos fármacos , Feminino , Guanina/análogos & derivados , Guanina/farmacologia , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/sangue , Humanos , Masculino , Organofosfonatos/farmacologia , Carga Viral/efeitos dos fármacos
10.
DNA Cell Biol ; 39(3): 368-378, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31971825

RESUMO

Nuclear protein 1 (NUPR1) is a stress-related small molecule and plays important roles in various tumors, including multiple myeloma (MM). Autophagy is essential for maintaining cellular homoeostasis in response to stress and, together with apoptosis, determines cell fate. Previous studies indicate that NUPR1 is involved in cancer progression of MM, but the underlying mechanisms have not been elucidated. In this study, we confirmed that NUPR1 and basal autophagy markers were highly expressed in the bone marrow of MM patients. The overexpression of NUPR1 was correlated with staging (both by Revised International Staging System [RISS] and Durie-Salmon [D-S] Staging System), levels of hemoglobin and calcium, and bone marrow plasma cell ratio in the MM patients. NUPR1 silencing reduced autophagy activities and induced apoptosis in U266 and RPMI 8226. We further observed a decrease in NUPR1 silencing-induced apoptosis in the presence of rapamycin, while an increase in apoptosis after chloroquine and 3-methyladenine treatment. Analysis of the mechanism indicated that PI3K/AKT/mTOR pathway was involved in autophagy-mediated apoptosis upon NUPR1 knockdown. In summary, our results demonstrate that NUPR1 silencing suppresses autophagy activities and induces autophagy-mediated apoptosis in MM cells through the PI3K/AKT/mTOR pathway, which exhibits potential as a treatment strategy for MM.


Assuntos
Apoptose , Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/genética , Transdução de Sinais , Adenina/análogos & derivados , Adenina/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Inativação Gênica , Humanos , Mieloma Múltiplo/genética , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
11.
Chem Biol Interact ; 317: 108943, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31926917

RESUMO

Epidemiological studies have shown that cigarette smoking is beneficial in ulcerative colitis and that nicotine may be responsible for this effect. However, the mechanism remains unclear. In a previous study, nicotine was found to induce autophagy in intestinal cells. Here, we evaluated the effect of nicotine-induced autophagy in a dextran sodium sulfate (DSS)-induced colitis mouse model. C57BL/6 adult male mice drank DSS water solution freely for seven consecutive days, and then tap water was administered. The effect of nicotine treatment was examined in the DSS model, including colon length, disease severity, histology of the colon tissue, and inflammation levels. Moreover, autophagy levels were detected by Western blot analysis (LC3II/LC3I, p62, and beclin-1). The levels of DSS-induced colitis were significantly decreased following nicotine treatment. The disease activity score, body weight, histologic damage scores, and the level of colonic inflammatory factors of nicotine-treated mice all decreased compared to those of the control mice. Additionally, nicotine enhanced the expression of LC3II/LC3I and beclin-1 but decreased the p62 protein level. Inhibiting autophagy by 3-MA attenuated the protective effects of nicotine on colitis. Additionally, both in vitro and in vivo experiments showed changes in AMPK-mTOR-P70S6K during this process. These results suggest that nicotine improved colitis by regulating autophagy and provided a protective effect against DSS-induced colitis.


Assuntos
Adenilato Quinase/metabolismo , Autofagia/efeitos dos fármacos , Colite/prevenção & controle , Nicotina/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adenilato Quinase/genética , Animais , Colite/induzido quimicamente , Sulfato de Dextrana/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/genética
12.
Theriogenology ; 142: 320-327, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711691

RESUMO

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ±â€¯1.2 vs. 100% ±â€¯0.0), presence of first polar body (65.9% ±â€¯1.2 vs. 70.5% ±â€¯1.8), nuclear status in second metaphase (62.5% ±â€¯11.6 vs. 68.4% ±â€¯4.9), cytoplasmic maturation (100.0% ±â€¯0.7 vs. 75.0% ±â€¯0.7), reactive oxygen species levels (0.5 ±â€¯0.2 vs. 0.3 ±â€¯0.1), and mitochondrial membrane potential (1.1 ±â€¯0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.


Assuntos
Adenina/análogos & derivados , Artiodáctilos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Ionomicina/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Adenina/farmacologia , Animais , Artiodáctilos/embriologia , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologia , Partenogênese/fisiologia
13.
Hypertension ; 75(1): 109-118, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31786976

RESUMO

c-Kit+ progenitor smooth muscle cells (P-SMCs) can develop into SMCs that contribute to injury-induced neointimal thickening. Here, we investigated whether adenosine reduces P-SMC migration and proliferation and whether this contributes to adenosine's inhibitory actions on neointima formation. In human P-SMCs, 2-chloroadenosine (stable adenosine analogue) and BAY60-6583 (A2B agonist) inhibited P-SMC proliferation and migration. Likewise, increasing endogenous adenosine by blocking adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl) adenine (inhibits adenosine deaminase) and 5-iodotubercidin (inhibits adenosine kinase) attenuated P-SMC proliferation and migration. Neither N6-cyclopentyladenosine (A1 agonist), CGS21680 (A2A agonist), nor N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (A3 agonist) affected P-SMC proliferation or migration. 2-Chloroadenosine increased cyclic AMP, reduced Akt phosphorylation (activates cyclin D expression), and reduced levels of cyclin D1 (promotes cell-cycle progression). Moreover, 2-chloroadenosine inhibited expression of Skp2 (promotes proteolysis of p27Kip1) and upregulated levels of p27Kip1 (negative cell-cycle regulator). A2B receptor knockdown prevented the effects of 2-chloroadenosine on cyclic AMP production and P-SMC proliferation and migration. Likewise, inhibition of adenylyl cyclase and protein kinase A rescued P-SMCs from the inhibitory effects of 2-chloroadenosine. The inhibitory effects of adenosine were similar in male and female P-SMCs. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 µmol/L for 7 days) reduced neointimal hyperplasia by 64.5% (P<0.05; intima/media ratio: control, 1.4±0.02; treated, 0.53±0.012) and reduced neointimal c-Kit+ cells. Adenosine inhibits P-SMC migration and proliferation via the A2B receptor/cyclic AMP/protein kinase A axis, which reduces cyclin D1 expression and activity via inhibiting Akt phosphorylation and Skp2 expression and upregulating p27kip1 levels. Adenosine attenuates neointima formation in part by inhibiting infiltration and proliferation of c-Kit+ P-SMCs.


Assuntos
2-Cloroadenosina/farmacologia , Proliferação de Células/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Receptor A2B de Adenosina/metabolismo , Adenina/farmacologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Aminopiridinas/farmacologia , Movimento Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Fenetilaminas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo
14.
J Cell Physiol ; 235(1): 267-280, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31206674

RESUMO

Studies have shown autophagy participation in the immunopathology of inflammatory diseases. However, autophagy role in asthma and in eosinophil extracellular traps (EETs) release is poorly understood. Here, we attempted to investigate the autophagy involvement in EETs release and in lung inflammation in an experimental asthma model. Mice were sensitized with ovalbumin (OVA), followed by OVA challenge. Before the challenge with OVA, mice were treated with an autophagy inhibitor, 3-methyladenine (3-MA). We showed that 3-MA treatment decreases the number of eosinophils, eosinophil peroxidase (EPO) activity, goblet cells hyperplasia, proinflammatory cytokines, and nuclear factor kappa B (NFκB) p65 immunocontent in the lung. Moreover, 3-MA was able to improve oxidative stress, mitochondrial energy metabolism, and Na+ , K+ -ATPase activity. We demonstrated that treatment with autophagy inhibitor 3-MA reduced EETs formation in the airway. On the basis of our results, 3-MA treatment can be an interesting alternative for reducing lung inflammation, oxidative stress, mitochondrial damage, and EETs formation in asthma.


Assuntos
Adenina/análogos & derivados , Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Autofagia/imunologia , Armadilhas Extracelulares/imunologia , Adenina/farmacologia , Animais , Asma/induzido quimicamente , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Peroxidase de Eosinófilo/metabolismo , Eosinófilos/imunologia , Feminino , Células Caliciformes/patologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Ovalbumina , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo
15.
Acta Biochim Pol ; 66(4): 585-588, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804790

RESUMO

Fluorescent tri-cyclic purine analogs, derivatives of isoguanine and adenine, were examined as potential substrates of purine-nucleoside phosphorylase. It was found previously that etheno- derivatives of both compounds are ribosylated in phosphate-free media, but ribosylation places in some instances differ from purine N9. New ribosides are examined as potential substrates of human blood PNP and indicators of this enzyme. Of these, N6-riboside of 1,N6-etheno-adenine was found the most promising.


Assuntos
Adenina/química , Glicosídeos/química , Guanina/química , Purina-Núcleosídeo Fosforilase/sangue , Adenina/farmacologia , Fluorescência , Guanina/farmacologia , Humanos , Cinética , Purina-Núcleosídeo Fosforilase/isolamento & purificação , Purinas/química
16.
Int J Mol Sci ; 20(23)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31816893

RESUMO

OBJECTIVE: To investigate the process by which quercetin suppresses atherosclerosis by upregulating MST1-mediated autophagy in RAW264.7 macrophages. METHODS: An in vitro foam cell model was established by culturing RAW264.7 macrophages with oxidized low-density lipoprotein (ox-LDL). The cells were treated with quercetin alone or in combination with the autophagy inhibitor, 3-methyladenine, and autophagy agonist, rapamycin. Cell viability was detected with a CCK-8 kit. Lipid accumulation was detected by oil red O staining, senescence was detected by SA-ß-gal (senescence-associated ß-galactosidase) staining, reactive oxygen species were detected by ROS assay kit. Autophagosomes and mitochondria were detected by transmission electron microscope (TEM), and expression of MST1, LC3-II/I, Beclin1, Bcl-2, P21, and P16 were detected by immunofluorescence and Western blot. RESULTS: Ox-LDL induced RAW264.7 macrophage-derived foam cell formation, reduced survival, aggravated cell lipid accumulation, and induced a senescence phenotype. This was accompanied by decreased formation of autophagosome; increased expression of P53, P21, and P16; and decreased expression of LC3-II/I and Beclin1. After intervention with quercetin, the cell survival rate was increased, and lipid accumulation and senescence phenotype were reduced. Furthermore, the expression of LC3-II/I and Beclin1 were increased, which was consistent with the ability of quercetin to promote autophagy. Ox-LDL also increased the expression of MST1, and this increase was blocked by quercetin, which provided a potential mechanism by which quercetin may protect foam cells against age-related detrimental effects. CONCLUSION: Quercetin can inhibit the formation of foam cells induced by ox-LDL and delay senescence. The mechanism may be related to the regulation of MST1-mediated autophagy of RAW264.7 cells.


Assuntos
Aterosclerose/patologia , Autofagia/efeitos dos fármacos , Progressão da Doença , Células Espumosas/metabolismo , Células Espumosas/patologia , Fator de Crescimento de Hepatócito/metabolismo , Lipoproteínas LDL/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Quercetina/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Espumosas/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Células RAW 264.7 , Sirolimo/farmacologia , Regulação para Cima/efeitos dos fármacos
17.
Infez Med ; 27(4): 365-373, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31846985

RESUMO

The fourth HIV strand-transfer integrase inhibitor (INSTI) has been released into the market as part of a single-tablet-regimen (STR) consisting of bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF). The newest component is thus BIC, a booster-free INSTI with pharmacological characteristics similar to those of dolutegravir (DTG), including high intrinsic antiretroviral potency. The BIC-containing STR underwent clinical development in both treatment-naive and virologically suppressed patients and was found non-inferior to DTG-based comparator arms. In the currently evolving therapeutic scenario, the BIC/FTC/TAF STR regimen represents the smartest response on the side of triple conventional regimens, while new 2-drug regimens have received regulatory approval and nowadays epitomize the search for simpler and lighter antiretroviral regimens. The overall characteristics of BIC/FTC/TAF, however, make this therapeutic option quite comparable in terms of simplicity to the newly approved dual regimens, and the main reasons (e.g., toxicity) accounting in the past for the search of regimens consisting of less than three drugs are no longer in place.


Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacologia , Emtricitabina/administração & dosagem , Emtricitabina/farmacologia , Infecções por HIV/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Tenofovir/administração & dosagem , Tenofovir/farmacologia , Adenina/administração & dosagem , Adenina/farmacologia , Ensaios Clínicos como Assunto , Combinação de Medicamentos , Humanos , Comprimidos
18.
Int J Dev Biol ; 63(8-9-10): 395-405, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31840778

RESUMO

In the last few decades, we have learned a considerable amount about how eukaryotic cells communicate with each other, and what it is the cells are telling each other. The simplicity of Dictyostelium discoideum, and the wide variety of available tools to study this organism, makes it the equivalent of a hydrogen atom for cell and developmental biology. Studies using Dictyostelium have pioneered a good deal of our understanding of eukaryotic cell communication. In this review, we will present a brief overview of how Dictyostelium cells use extracellular signals to attract each other, repel each other, sense their local cell density, sense whether the nearby cells are starving or stressed, count themselves to organize the formation of structures containing a regulated number of cells, sense the volume they are in, and organize their multicellular development. Although we are probably just beginning to learn what the cells are telling each other, the elucidation of Dictyostelium extracellular signals has already led to the development of possible therapeutics for human diseases.


Assuntos
Comunicação Celular , Fatores Quimiotáticos/metabolismo , Dictyostelium/fisiologia , Adenina/análogos & derivados , Adenina/farmacologia , Trifosfato de Adenosina/metabolismo , Amônia/farmacologia , Meios de Cultivo Condicionados , AMP Cíclico/metabolismo , Dictyostelium/genética , Morfogênese , Policetídeos/metabolismo , Polifosfatos/metabolismo
19.
J Neuroinflammation ; 16(1): 217, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31722730

RESUMO

BACKGROUND: Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain. However, whether it contributes to the mechanism of CM remains unknown. Thus, the present study investigated the precise details of microglial P2Y12R involvement in CM. METHODS: Mice subjected to recurrent nitroglycerin (NTG) treatment were used as the CM model. Hyperalgesia were assessed by mechanical withdrawal threshold to electronic von Frey and thermal withdrawal latency to radiant heat. Western blot and immunohistochemical analyses were employed to detect the expression of P2Y12R, Iba-1, RhoA, and ROCK2 in the trigeminal nucleus caudalis (TNC). To confirm the role of P2Y12R and RhoA/ROCK in CM, we systemically administered P2Y12R antagonists (MRS2395 and clopidogrel) and a ROCK2 inhibitor (fasudil) and investigated their effects on microglial activation, c-fos, and calcitonin gene-related peptide (CGRP) expression in the TNC. To further confirm the effect of P2Y12R on microglial activation, we preincubated lipopolysaccharide (LPS)-treated BV-2 microglia with MRS2395 and clopidogrel. ELISA was used to evaluate the levels of inflammatory cytokines. RESULTS: The protein levels of P2Y12R, GTP-RhoA, ROCK2, CGRP, c-fos, and inducible nitric oxide synthase (iNOS) in the TNC were increased after recurrent NTG injection. A double labeling study showed that P2Y12R was restricted to microglia in the TNC. MRS2395 and clopidogrel attenuated the development of tactile allodynia and suppressed the expression of CGRP, c-fos, and GTP-RhoA/ROCK2 in the TNC. Furthermore, fasudil also prevented hyperalgesia and suppressed the expression of CGRP in the TNC. In addition, inhibiting P2Y12R and ROCK2 activities suppressed NTG-induced microglial morphological changes (process retraction) and iNOS production in the TNC. In vitro, a double labeling study showed that P2Y12R was colocalized with BV-2 cells, and the levels of iNOS, IL-1ß, and TNF-α in LPS-stimulated BV-2 microglia were reduced by P2Y12R inhibitors. CONCLUSIONS: These data demonstrate that microglial P2Y12R in the TNC plays a critical role in the pathogenesis of CM by regulating microglial activation in the TNC via RhoA/ROCK pathway.


Assuntos
Microglia/metabolismo , Transtornos de Enxaqueca/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Núcleos do Trigêmeo/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Clopidogrel/farmacologia , Modelos Animais de Doenças , Camundongos , Microglia/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Núcleos do Trigêmeo/efeitos dos fármacos , Valeratos/farmacologia
20.
Int J Mol Sci ; 20(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744172

RESUMO

Although studies have shown the concomitant occurrence of autophagic and programmed cell death (PCD) in plants, the relationship between autophagy and PCD and the factors determining this relationship remain unclear. In this study, seedlings of the wheat cultivar Jimai 22 were used to examine the occurrence of autophagy and PCD during polyethylene glycol (PEG)-8000-induced drought stress. Autophagy and PCD occurred sequentially, with autophagy at a relatively early stage and PCD at a much later stage. These findings suggest that the duration of drought stress determines the occurrence of PCD following autophagy. Furthermore, the addition of 3-methyladenine (3-MA, an autophagy inhibitor) and the knockdown of autophagy-related gene 6 (ATG6) accelerated PEG-8000-induced PCD, respectively, suggesting that inhibition of autophagy also results in PCD under drought stress. Overall, these findings confirm that wheat seedlings undergo autophagic survival under mild drought stress, with subsequent PCD only under severe drought.


Assuntos
Apoptose , Autofagia , Secas , Triticum/crescimento & desenvolvimento , Adenina/análogos & derivados , Adenina/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/antagonistas & inibidores , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Polietilenoglicóis/toxicidade , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Triticum/metabolismo
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