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1.
Arch Virol ; 165(10): 2259-2277, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32699981

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely disseminated, macrophage-tropic arterivirus that exhibits profound genetic and pathogenic heterogeneity. The present study was conducted to determine the complete genome sequences of two novel Korean lineage 1 PRRSV-2 strains, KNU-1901 and KNU-1902, which were isolated from vaccinated pig farms experiencing unusually high morbidity and mortality. Both isolates contained notable discontinuous 423-nucleotide deletions (DELs) within the genes encoding nonstructural protein 2 (nsp2) and GP3 when compared with the prototype strain VR-2332. In particular, the nsp2 DEL viruses had unique quadripartite discontinuous DEL signatures (111-1-19-9) in nsp2; this is an expanded version of the tripartite 111-1-19 DEL previously identified in virulent lineage 1 PRRSV-2 strains. Phylogenetic analysis revealed that both novel nsp2 DEL viruses belong to the Korean clade (KOR C) of lineage 1 isolates based on ORF5 but cluster with lineage KOR A strains based on the nsp2 or complete genome sequence. Recombination detection analysis suggested that both novel isolates are recombinants and may have evolved via natural inter-lineage recombination between circulating KOR A and KOR C strains. Interestingly, compared with the prototype VR-2332 virus, the novel nsp2 DEL variants were less efficient at promoting the expression of immune response genes in porcine alveolar macrophage culture. Taken together, we conclude that KNU-1901 and KNU-1902 are recently evolved recombinant variants of the virulent lineage 1 family that caused the regional severe PRRS outbreaks.


Assuntos
Citocinas/genética , Genoma Viral , Filogenia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Transformada , Citocinas/imunologia , Evolução Molecular , Expressão Gênica , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Fases de Leitura Aberta , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/patologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Recombinação Genética , República da Coreia/epidemiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Suínos , Virulência
2.
J Vis Exp ; (161)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716395

RESUMO

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.


Assuntos
Autofagia/fisiologia , Bioensaio/métodos , Condrócitos/patologia , Citometria de Fluxo/métodos , Osteoartrite/patologia , Linhagem Celular Transformada , Condrócitos/efeitos dos fármacos , Descoberta de Drogas/métodos , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/farmacologia , Proteínas Associadas aos Microtúbulos/uso terapêutico , Osteoartrite/tratamento farmacológico , Osteoartrite/fisiopatologia
3.
Nat Commun ; 11(1): 3158, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572033

RESUMO

Efficient repair of DNA double-strand breaks (DSBs) requires a coordinated DNA Damage Response (DDR), which includes phosphorylation of histone H2Ax, forming γH2Ax. This histone modification spreads beyond the DSB into neighboring chromatin, generating a DDR platform that protects against end disassociation and degradation, minimizing chromosomal rearrangements. However, mechanisms that determine the breadth and intensity of γH2Ax domains remain unclear. Here, we show that chromosomal contacts of a DSB site are the primary determinants for γH2Ax landscapes. DSBs that disrupt a topological border permit extension of γH2Ax domains into both adjacent compartments. In contrast, DSBs near a border produce highly asymmetric DDR platforms, with γH2Ax nearly absent from one broken end. Collectively, our findings lend insights into a basic DNA repair mechanism and how the precise location of a DSB may influence genome integrity.


Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Animais , Linhagem Celular Transformada , Cromatina/metabolismo , Camundongos , Fosforilação
4.
Toxicol Appl Pharmacol ; 401: 115091, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32525019

RESUMO

Prostate cancer (PCa) incidence is surging in United States and other parts of the world. Synthetic and natural compounds have been explored as potential modulators of PI3K/Akt signaling that is known to drive PCa growth. Here, we evaluated the efficacy of a series of triphenyltin (IV) carboxylate derivatives against PCa. From this library, triphenylstannyl 2-(benzylcarbamoyl)benzoate (Ch-319) resulted in reduced viability and induction of cell cycle arrest in PTEN-/- PC3M and PTEN+/- DU145 cells. In parallel, downregulation of PI3K p85/p110 subunits, dephosphorylation of Akt-1 and increase in FOXO3a expression were observed. In silico studies indicated binding interactions of Ch-319 within the ATP binding site of Akt-1 at Met281, Phe442 and Glu234 residues. Elevated po-pulation of apoptotic cells, activation of Bax and reduced Bcl2 expression indicated apoptosis by Ch-319. Pre-sensitization of PCa cells with Ch-319 augmented the effect of cabazitaxel, a commonly used taxane in patients with castration-resistant PCa. Next, in a prostate-specific PTENp-/- mice, Ch-319 showed reduced weights of genitourinary apparatus as compared to DMSO treated controls. Histological studies indicated absence of neoplastic foci in Ch-319 treated prostates. Consistently, dephosphorylation of Akt-1, reduced expression of PRAS40 and androgen receptor and increase in FOXO3a were observed in treated group. Notably, no overt organ toxicity was noted in Ch-319 treated animals. Our studies identify Akt/FOXO3a signaling as a target of triphenyltin (IV) carboxylate Ch-319 and provide a molecular basis of its growth inhibitory effect in PCa cells. We propose that Ch-319 has the potential to be developed as an anticancer agent against PCa.


Assuntos
Progressão da Doença , Proteína Forkhead Box O3/biossíntese , Compostos Orgânicos de Estanho/química , Compostos Orgânicos de Estanho/farmacologia , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Compostos Orgânicos de Estanho/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
5.
Life Sci ; 256: 117964, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32534036

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. MATERIALS AND METHODS: Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. KEY FINDINGS: Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SIGNIFICANCE: In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. CHEMICAL COMPOUNDS STUDIED IN THIS STUDY: HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Osteoblastos/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inativação Gênica/efeitos dos fármacos , Cinética , Miócitos de Músculo Liso/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteopontina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Mutat Res ; 852: 503169, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265043

RESUMO

The phycotoxins, okadaic acid (OA) and dinophysistoxins 1 and 2 (DTX-1 and -2), are protein phosphatase PP2A and PP1 inhibitors involved in diarrhetic shellfish poisoning (DSP) in humans. Data on the in vivo acute toxicity of the OA-group toxins show some differences and the European Food Safety Authority (EFSA) has determined toxicity equivalent factors (TEFs) of one for the reference toxin, OA, as well as for DTX-1 and 0.6 for DTX-2. However, recent in vitro studies indicated that DTX-1 seems to be more toxic than OA. As OA was described as apoptotic and aneugenic compound, we analyzed the DNA damage responses induced by the 3 toxins through γH2AX and pH3 biomarkers on proliferative HepaRG cells using High Content Analysis. We quantitatively examined the responses for γH2AX and pH3 by benchmark dose analyzing (BMD) using PROAST software. We found that the three toxins increased both γH2AX- and pH3-positive cells populations in a concentration-dependent manner. The 3 toxins induced mitotic arrest, characteristic of aneugenic compounds, as well as DNA strand-breaks concomitantly to cytotoxicity. BMD analysis showed that DTX-1 is the most potent inducer of DNA damage, followed by OA and DTX-2. The quantitative genotoxic data provided in this study are additional findings for reconsidering the estimated TEFs of this group of phycotoxins.


Assuntos
Inibidores Enzimáticos/toxicidade , Histonas/genética , Mutagênicos/toxicidade , Ácido Okadáico/toxicidade , Piranos/toxicidade , Benchmarking , Biomarcadores/metabolismo , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Histonas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mitose/efeitos dos fármacos , Testes de Mutagenicidade , Fosforilação/efeitos dos fármacos , Software
7.
PLoS Pathog ; 16(3): e1008371, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130281

RESUMO

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50-70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma.


Assuntos
Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Vesículas Extracelulares/metabolismo , Vírus JC/metabolismo , Leucoencefalopatia Multifocal Progressiva/metabolismo , Neuroglia/metabolismo , Receptores Virais/metabolismo , Linhagem Celular Transformada , Plexo Corióideo/patologia , Plexo Corióideo/virologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Vesículas Extracelulares/patologia , Vesículas Extracelulares/virologia , Humanos , Leucoencefalopatia Multifocal Progressiva/patologia , Neuroglia/patologia , Neuroglia/virologia
8.
J Med Chem ; 63(7): 3763-3783, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32189500

RESUMO

The immunoproteasome (iP), an inducible proteasome variant harboring three immunosubunits, low molecular mass polypeptide-2 (LMP2), multicatalytic endopeptidase complex subunit-1, and low molecular mass polypeptide-7 (LMP7), is involved in multiple facets of inflammatory responses. We recently reported that YU102, a dual inhibitor of the iP subunit LMP2 and the constitutive proteasome catalytic subunit ß1, ameliorates cognitive impairments in mouse models of Alzheimer's disease (AD) independently of amyloid deposits. To investigate whether inhibition of LMP2 is sufficient to improve the cognitive functions of AD mice, here we prepared 37 YU102 analogues and identified a potent LMP2 inhibitor DB-310 (28) (IC50: 80.6 nM) with improved selectivity and permeability in cells overexpressing ABCB1 transporters. We show that DB-310 induces suppression of IL-1α production in microglia cells and improves cognitive functions in the Tg2576 transgenic mouse model of AD. This study supports that inhibition of LMP2 is a promising therapeutic strategy for treatment of AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/uso terapêutico , Nootrópicos/uso terapêutico , Oligopeptídeos/uso terapêutico , Animais , Linhagem Celular Transformada , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Interleucina-1alfa/metabolismo , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Estrutura Molecular , Nootrópicos/síntese química , Nootrópicos/toxicidade , Oligopeptídeos/síntese química , Oligopeptídeos/toxicidade , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Bibliotecas de Moléculas Pequenas/toxicidade , Relação Estrutura-Atividade
9.
Prep Biochem Biotechnol ; 50(2): 204-214, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935152

RESUMO

Biotechnology through plant cell cultures in bioreactors is a tool that allows increasing the production of secondary metabolites of commercial interest. The hydrodynamic characterization, in addition to the transfer (OTR) and uptake (OUR) of oxygen through the dynamic method with different aeration rate, were used to see their influence on the production of biomass and saponins. The culture poisoning technique was used to determine the antifungal activity of the SC-2 and SC-3 saponins in vitro. Likewise, the shear or hydrodynamic stress of 273.6 mN/m2 were calculated based on the Reynolds Number. The oxygen supply (OTR) was always greater than the demand (OUR) for all the aeration rate evaluated. Dry weight values of 8.6 gDW/L and a concentration of 2.7 mg/L and 187.3 mg/L of the saponins SC-2 and SC-3 respectively were obtained with an air flow of 0.1 vvm. In addition, it was possible to inhibit the growth of phytopathogenic fungi in vitro by up to 93%, while in vivo it was possible to reduce the infections of strawberry seeds inoculated with phytopathogens, obtaining up to 94% of germinated seeds. This information will facilitate the rational operation of the bioreactor culture system that produces secondary metabolites.


Assuntos
Antifúngicos/síntese química , Reatores Biológicos , Fragaria/microbiologia , Saponinas/síntese química , Saponinas/farmacologia , Solanum/química , Antifúngicos/farmacologia , Linhagem Celular Transformada
10.
Medicine (Baltimore) ; 99(2): e18626, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31914044

RESUMO

Detection of the chronic kidney disease (CKD) progression can begin early intervention to improve the prognosis of severe non-alcoholic fatty liver disease (NAFLD). This bi-directional cross-sectional study evaluates the roles of fatty acid-binding protein (FABP) and retinol binding protein (RBP4), which are produced from inflamed liver, adipose tissue and immune cells, for the prediction of CKD progression in severe NAFLD. Ninety severe NAFLD patients with hypertension and proteinuria (NAFLDHTN) were enrolled and divided into CKD (n = 39) and non-CKD groups (n = 51). Among 39 NAFLDHTN patients, 18 cases were categorized as CKD progression group. In comparison with CKD stable group (n = 21), the positive correlation between fold change values of hepatic fibrotic score (KPa), urinary FABP4 or urinary RBP4 versus severity of albuminuria were noted among CKD progression group. On multivariate analysis, high body mass index (BMI, >25 kg/m), high hepatic fibrosis score (>9.5 KPa), high urinary level of vascular cell adhesion molecule-1 (VCAM-1, >2239 µg/g cr), high urinary level of FABP4 (>115 ng/g cr) and high urinary level of RBP4 (>33.5 mg/g cr) are 5 independent predictors for progressive CKD during 24 months of follow-up. Synergetic effect was noted among these 5 risk factors for the prediction of CKD progression in NAFLDHTN patients. The in vitro experiments revealed that both FABP4 and RBP4 directly enhanced albumin-induced ER stress and apoptosis of human renal tubular epithelial cell line HK-2 cells and human podocytes cell lines. Through clinical and experimental approaches, this study revealed new 5 synergetic predictors including high BMI, hepatic fibrosis score, urinary level of VCAM-1, urinary level of FABP4 and RBP4, for the CKD progression in severe NAFLD patients with hypertension and proteinuria.


Assuntos
Ácidos Graxos/urina , Hipertensão/epidemiologia , Hipertensão/fisiopatologia , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Proteínas de Ligação ao Retinol/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminúria/epidemiologia , Índice de Massa Corporal , Linhagem Celular Transformada , Estudos Transversais , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/epidemiologia , Índice de Gravidade de Doença , Molécula 1 de Adesão de Célula Vascular/urina , Adulto Jovem
11.
Toxicol Lett ; 322: 12-19, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31899212

RESUMO

Benzene exposure is a risk factor of acute myeloid leukemia (AML), during such carcinogenesis long non-coding RNAs (lncRNAs) are important epigenetic regulators. HOTAIRM1 (HOXA transcript antisense RNA, myeloid-specific 1) plays an indispensable role in the development of AML. Hydroquinone (HQ) is one major metabolite of benzene and its ideal replacement in toxicology research. But the influence of benzene or HQ on HOTAIRM1 expression in AML associated pathway is still unclear. In the TK6 cells with short-term exposure to HQ (HQ-ST cells) or long term HQ exposure induced malignant transformed TK6 cells (HQ-MT cells), the relationship between DNMT3b and HOTAIRM1 was explored. Comparing to counterparts, HOTAIRM1 expression was increased firstly and then decreased in HQ-ST cells, and definitely decreased in HQ-MT cells; while the expression change tendency of DNMT3b was in contrast to that of HOTAIRM1. Moreover, the average HOTAIRM1 expression of 17 paired workers being exposed to benzene within 1.5 years was increased, but that of the remaining 92 paired workers with longer exposure time was decreased. Furthermore, in 5-AzaC (DNA methyltransferase inhibitor) or TSA (histone deacetylation inhibitor) treated HQ-MT cells, the expression of HOTAIRM1 was restored by reduced DNA promoter methylation levels. HQ-MT cells with DNMT3b knockout by CRISPR/Cas9 displayed the promoter hypomethylation and the increase of HOTAIRM1, also confirmed in benzene exposure workers. These suggest that long term exposure to HQ or benzene might induce the increase of DNMT3b expression and the promoter hypermethylation to silence the expression of HOTAIRM1, a possible tumor-suppressor in the AML associated carcinogenesis pathway.


Assuntos
Benzeno/efeitos adversos , DNA (Citosina-5-)-Metiltransferases/biossíntese , Metilação de DNA/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Hidroquinonas/toxicidade , Leucemia Mieloide Aguda/induzido quimicamente , MicroRNAs/metabolismo , Doenças Profissionais/induzido quimicamente , Exposição Ocupacional/efeitos adversos , Estudos de Casos e Controles , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/genética , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Doenças Profissionais/enzimologia , Doenças Profissionais/genética , Regiões Promotoras Genéticas , Medição de Risco
12.
PLoS One ; 15(1): e0220348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935221

RESUMO

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.


Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Células Epiteliais/enzimologia , Fibroblastos/enzimologia , Acetiltransferases/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Quebra Cromossômica , Segregação de Cromossomos , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Ectromelia/enzimologia , Ectromelia/genética , Ectromelia/patologia , Células Epiteliais/patologia , Fibroblastos/patologia , Expressão Gênica , Humanos , Hipertelorismo/enzimologia , Hipertelorismo/genética , Hipertelorismo/patologia , Camundongos , Mitose , Modelos Biológicos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
13.
Am J Physiol Gastrointest Liver Physiol ; 318(3): G464-G478, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984785

RESUMO

The frequency of esophageal adenocarcinoma is rising despite widespread use of proton pump inhibitors (PPIs), which heal reflux esophagitis but do not prevent reflux of weakly acidic gastric juice and bile in Barrett's esophagus patients. We aimed to determine if weakly acidic (pH 5.5) bile salt medium (WABM) causes DNA damage in Barrett's cells. Because p53 is inactivated frequently in Barrett's esophagus and p38 can assume p53 functions, we explored p38's role in DNA damage response and repair. We exposed Barrett's cells with or without p53 knockdown to WABM, and evaluated DNA damage, its response and repair, and whether these effects are p38 dependent. We also measured phospho-p38 in biopsies of Barrett's metaplasia exposed to deoxycholic acid (DCA). WABM caused phospho-H2AX increases that were blocked by a reactive oxygen species (ROS) scavenger. WABM increased phospho-p38 and reduced bromodeoxyuridine incorporation (an index of S phase entry). Repair of WABM-induced DNA damage proceeded through p38-mediated base excision repair (BER) associated with reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease I (Ref-1/APE1). Cells treated with WABM supplemented with ursodeoxycholic acid (UDCA) exhibited enhanced p38-mediated responses to DNA damage. All of these effects were observed in p53-intact and p53-deficient Barrett's cells. In patients, esophageal DCA perfusion significantly increased phospho-p38 in Barrett's metaplasia. WABM exposure generates ROS, causing oxidative DNA damage in Barrett's cells, a mechanism possibly underlying the rising frequency of esophageal adenocarcinoma despite PPI usage. p38 plays a central role in oxidative DNA damage response and Ref-1/APE1-associated BER, suggesting potential chemopreventive roles for agents like UDCA that increase p38 activity in Barrett's esophagus.NEW & NOTEWORTHY We found that weakly acidic bile salt solutions, with compositions similar to the refluxed gastric juice of gastroesophageal reflux disease patients on proton pump inhibitors, cause oxidative DNA damage in Barrett's metaplasia that could contribute to the development of esophageal adenocarcinoma. We also have elucidated a critical role for p38 in Barrett's metaplasia in its response to and repair of oxidative DNA damage, suggesting a potential chemopreventive role for agents like ursodeoxycholic acid that increase p38 activity in Barrett's esophagus.


Assuntos
Esôfago de Barrett/enzimologia , Dano ao DNA , Reparo do DNA , Ácido Desoxicólico/toxicidade , Células Epiteliais/efeitos dos fármacos , Mucosa Esofágica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Mucosa Esofágica/enzimologia , Mucosa Esofágica/patologia , Feminino , Histonas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Masculino , Fosforilação , Cultura Primária de Células , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ácido Ursodesoxicólico/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética
14.
Mol Med Rep ; 21(2): 777-785, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31974614

RESUMO

Choline is used to synthesize phospholipids and a lack of choline induces a number of liver­related diseases, including non­alcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N­4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcription­quantitative polymerase chain reaction (PCR) revealed choline transporter­like protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N­4 cells. [Methyl­3H]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+­independent but pH­dependent manner, which was similar to CTL1. Hemicholinium­3 (HC­3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase­3 and ­7 activities, and increased fluorescein isothiocyanate­Annexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl­3H]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol­12­myristate 13­acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N­4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism.


Assuntos
Antígenos CD/metabolismo , Membrana Celular/metabolismo , Fígado/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteína Quinase C/metabolismo , Antígenos CD/genética , Apoptose , Transporte Biológico , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/genética , Colina/metabolismo , Humanos , Proteínas de Transporte de Cátions Orgânicos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
FASEB J ; 34(2): 2882-2895, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908022

RESUMO

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non-genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3',5'-cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real-time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane- impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid-stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein-coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non-genomic actions of budesonide were analyzed by RNA-Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs . A 140-gene budesonide signature was identified, of which 48 genes represent a non-genomic signature that requires Gαs signaling. Collectively, this non-genomic cAMP signaling modality contributes to one-third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.


Assuntos
Cromograninas/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Miócitos de Músculo Liso/metabolismo , Sistema Respiratório/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Linhagem Celular Transformada , Cromograninas/genética , AMP Cíclico/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Humanos , Miócitos de Músculo Liso/patologia , Sistema Respiratório/patologia , Sistemas do Segundo Mensageiro/genética
16.
Nucleic Acids Res ; 48(1): 231-248, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31722399

RESUMO

Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.


Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição/genética , Transcrição Genética , Ubiquitina/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Raios Ultravioleta
17.
Int Arch Allergy Immunol ; 181(1): 56-70, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31707382

RESUMO

INTRODUCTION: Phospholipases are enzymes that occur in many types of human cells, including mast cells, and play an important role in the molecular background of asthma pathogenesis, and the development of inflammation NF-κB activities that affect numerous biological processes has been reported in many inflammatory diseases including asthma. Vitamin D is a widely studied factor that affects many diseases, including asthma. The aim of this study is to assess the influence of 1,25-(OH)2D3 on regulation of chosen phospholipase-A2 (PLA2) expression-selected inflammation mediators. METHODS: LUVA mast cells were stimulated with 1,25(OH)2D3, and inhibitors of NF-κB p65 and ubiquitination. Expression analysis of phospholipases (PLA2G5, PLA2G10, PLA2G12, PLA2G15, PLA2G4A, PLA2G4B, PLA2G4C, PLAA, NF-κB p65, and UBC) was done utilizing real-time PCR and Western blot. Eicosanoid (LTC4, LXA4, 15[S]-HETE, and PGE2) levels and sPLA2 were also measured. RESULTS: We found that 1,25(OH)2D3 decreased the expression of PLA2G5, PLA2G15, PLA2G5,UBC, and NF-κB p65 but increased expression of PLAA and PLA2G4C (p < 0.05). Moreover, the expression of PLA2G5 and PLA2G15 decreased after inhibition of NF-κB p65 and UBC. Increased levels of released LXA4 and 15(S)-HETE, decreased levels of LTC4, and sPLA2s enzymatic activity in response to 1,25(OH)2D3 were also observed. Additionally, NF-κB p65 inhibition led to an increase in the LXA4 concentration. CONCLUSION: Future investigations will be needed to further clarify the role of 1,25(OH)2D3 in the context of asthma and the inflammatory process; however, these results confirm a variety of effects which can be caused by this vitamin. 1,25(OH)2D3-mediated action may result in the development of new therapeutic strategies for asthma treatment.


Assuntos
Asma/metabolismo , Colecalciferol/metabolismo , Inflamação/metabolismo , Mastócitos/metabolismo , NF-kappa B/metabolismo , Fosfolipases A2/metabolismo , Asma/genética , Linhagem Celular Transformada , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Lipoxinas/metabolismo , NF-kappa B/genética , Fosfolipases A2/genética , Transdução de Sinais , Ubiquitinação
18.
Food Chem Toxicol ; 135: 111044, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31830547

RESUMO

Hemistepsin A (HsA), isolated from Hemistepta lyrata (Bunge) Bunge, has the ability to ameliorate hepatitis in mice. However, the effects of H. lyrata and HsA on other types of liver disease have not been explored. In this report, we investigated the effects of H. lyrata and HsA on liver fibrosis and the underlying molecular mechanisms in activated hepatic stellate cells (HSCs). Based on cell viability-guided isolation, we found HsA was the major natural product responsible for H. lyrata-mediated cytotoxicity in LX-2 cells. HsA significantly decreased the viability of LX-2 cells and primary activated HSCs, increased the binding of Annexin V, and altered the expression of apoptosis-related proteins, suggesting that HsA induces apoptosis in activated HSCs. HsA reduced the phosphorylation of IKKε and the transactivation of nuclear factor-κB (NF-κB). Moreover, HsA decreased the phosphorylation of Akt and its downstream signaling molecules. Transfection experiments suggested that inhibition of NF-κB or Akt is essential for HsA-induced apoptosis of HSCs. In a CCl4-induced liver fibrosis model, HsA administration significantly decreased ALT and AST activities. Furthermore, HsA attenuated CCl4-mediated collagen deposits and profibrogenic genes expression in hepatic tissue. Thus, HsA may serve as a natural product for managing liver fibrosis through inhibition of NF-κB/Akt-dependent signaling.


Assuntos
Apoptose/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Lactonas/farmacologia , Cirrose Hepática/prevenção & controle , Sesquiterpenos/farmacologia , Animais , Linhagem Celular Transformada , Clorofórmio/farmacologia , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Biomed Pharmacother ; 121: 109605, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31706102

RESUMO

Bladder cancer (BC) brings a heavy burden to afflicted patients worldwide. In order to find new diagnostic markers and therapeutic targets for this disease, we investigated the role of a novel lncRNA, AC114812.8, in bladder cancer progression. Clone formation and CCK-8 assays were used to detect the proliferative capacity of the cells, and the transwell assay was used to explore their invasion and migration abilities. Wound healing experiments were also used to detect cell migration. Luciferase reporter assays were used to investigate the interactions between lncRNA, target gene and miRNA. The expression of FUT4 and marker genes related to epithelial-mesenchymal transition was explored through western blot analysis. Our findings revealed that AC114812.8 was significantly upregulated in BC and could markedly facilitate the proliferation, migration, and invasion of bladder cancer cells both in vitro and in vivo. Furthermore, duel-luciferase reporter assay revealed that AC114812.8 could regulate the FUT4 expression level by sponging miR-371b-5p to facilitate BC progression. We detected the levels of EMT-related biomarkers in AC114812.8-overexpressing BC cells by western blot analysis and found that AC114812.8 could promote EMT process. Rescue experiments showed that miR-371b-5p could rescue the effect of AC114812.8 on proliferation and metastasis of BC. Our results suggest that AC114812.8 could be a novel prognostic biomarker and therapeutic target for bladder cancer.


Assuntos
Progressão da Doença , Fucosiltransferases/biossíntese , MicroRNAs/biossíntese , RNA Longo não Codificante/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Fucosiltransferases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Longo não Codificante/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
20.
Eur J Med Chem ; 188: 112009, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31883488

RESUMO

SRO-91 is a non-natural ribofuranosyl-1,2,3-triazole C-nucleoside obtained by a synthetic sequence involving a C-alkynyl glycosylation mediated by metallic indium and a Huisgen cycloaddition for the construction of the triazole. Its structure is close to the one of ribavirin, a drug presenting a broad-spectrum against viral infections. SRO-91 antitumor activities were investigated on 9 strains of tumor cells and IC50 of the order of 1 µM were obtained on A431 epidermoid carcinoma cells and B16F10 skin melanoma cells. In addition, studies of ovarian tumor cell inhibitions show an interesting activity in regard to the need for new drugs for this pathology. Finally, cytotoxicity and mouse toxicity studies reveal a favorable therapeutic index for SRO-91.


Assuntos
Antineoplásicos/farmacologia , Ribavirina/análogos & derivados , Ribavirina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Camundongos , Ribavirina/toxicidade
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