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2.
Anticancer Res ; 39(9): 4721-4728, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519571

RESUMO

BACKGROUND/AIM: Recent research has identified the transcription factors NFATc2 and Sp1 as key regulators in the carcinogenesis of pancreatic carcinoma. This study aimed to examine the effect of clinically achievable dosages of analgesics including ketamine, s-ketamine, metamizole, and paracetamol as well as that of sufentanil, ropicavaine, and lidocaine on pancreatic carcinoma cells and the expression of NFATc2 and Sp1. MATERIALS AND METHODS: The effects of analgesics on the expression of NFATc2 and Sp1 were investigated with immunoblotting. Cell proliferation was measured with the ELISA BrdU assay. RESULTS: In PaTu8988t pancreatic carcinoma cells, 48 h stimulation with ketamine and s-ketamine significantly inhibited proliferation and decreased expression of NFATc2 in the nucleus. The addition of metamizole and lidocaine reduced proliferation of PaTu8988t cells after 48 h. CONCLUSION: New treatment concepts target specific signaling and transcription pathways. The extent to which drugs influence these mechanisms in pancreatic carcinoma cells needs to be investigated in future studies.


Assuntos
Analgésicos/farmacologia , Anestésicos Locais/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fator de Transcrição Sp1/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pancreáticas/patologia , Transcrição Genética
3.
Pestic Biochem Physiol ; 158: 54-60, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378361

RESUMO

Extensive planting of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has spurred increasingly rapid evolution of resistance in pests. In the pink bollworm, Pectinophora gossypiella, a devastating global pest, resistance to Bt toxin Cry1Ac produced by transgenic cotton is linked with mutations in a gene (PgCad1) encoding a cadherin protein that binds Cry1Ac in the larval midgut. We previously reported a long non-coding RNA (lncRNA) in intron 20 of cadherin alleles associated with both resistance and susceptibility to Cry1Ac. Here we tested the hypothesis that reducing expression of this lncRNA decreases transcription of PgCad1 and susceptibility to Cry1Ac. Quantitative RT-PCR showed that feeding susceptible neonates small interfering RNAs (siRNAs) targeting this lncRNA but not PgCad1 decreased the abundance of transcripts of both the lncRNA and PgCad1. Moreover, neonates fed the siRNAs had lower susceptibility to Cry1Ac. The results imply that the lncRNA increases transcription of PgCad1 and susceptibility of pink bollworm to Cry1Ac. The results suggest that disruption of lncRNA expression could be a novel mechanism of pest resistance to Bt toxins.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Caderinas/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Mariposas/efeitos dos fármacos , RNA Longo não Codificante/genética , Transcrição Genética/genética , Animais , Bacillus thuringiensis/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mariposas/genética , Mariposas/metabolismo , Controle Biológico de Vetores
4.
BMC Plant Biol ; 19(1): 367, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429697

RESUMO

BACKGROUND: Adaptation to abiotic stresses is crucial for the survival of perennial plants in a natural environment. However, very little is known about the underlying mechanisms. Here, we adopted a liquid culture system to investigate plant adaptation to repeated salt stress in Populus trees. RESULTS: We first evaluated phenotypic responses and found that plants exhibit better stress tolerance after pre-treatment of salt stress. Time-course RNA sequencing (RNA-seq) was then performed to profile changes in gene expression over 12 h of salt treatments. Analysis of differentially expressed genes (DEGs) indicated that significant transcriptional reprogramming and adaptation to repeated salt treatment occurred. Clustering analysis identified two modules of co-expressed genes that were potentially critical for repeated salt stress adaptation, and one key module for salt stress response in general. Gene Ontology (GO) enrichment analysis identified pathways including hormone signaling, cell wall biosynthesis and modification, negative regulation of growth, and epigenetic regulation to be highly enriched in these gene modules. CONCLUSIONS: This study illustrates phenotypic and transcriptional adaptation of Populus trees to salt stress, revealing novel gene modules which are potentially critical for responding and adapting to salt stress.


Assuntos
Adaptação Fisiológica/genética , Regulação da Expressão Gênica de Plantas , Populus/genética , Estresse Salino/genética , Transcrição Genética , Ontologia Genética , Redes Reguladoras de Genes , Genoma de Planta , Fenótipo , Populus/fisiologia , RNA de Plantas , Análise de Sequência de RNA , Transcriptoma , Árvores/genética , Árvores/fisiologia
6.
An Acad Bras Cienc ; 91(3): e20180646, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31411259

RESUMO

The hepatoprotective effects of the ethanolic extracts of propolis (EEP) on alcohol-induced liver steatosis were investigated in Wistar rats. Chronic alcoholic fatty liver was induced by administration of 52% alcohol to male Wistar rats at the dose of 1% body weight for 7 weeks. Then animals were simultaneously treated with 50% ethanol solutions of EEP or normal saline at the dose of 0.1% body weight for 4 further weeks. Serological analyses and liver histopathology studies were performed to investigate the development of steatosis. Microarray analysis was conducted to investigate the alterations of hepatic gene expression profiling. Our results showed that 4-week treatment of EEP helped to restore the levels of various blood indices, liver function enzymes and the histopathology of liver tissue to normal levels. Results from the microarray analysis revealed that the hepatic expressions of genes involved in lipogenesis were significantly down-regulated by EEP treatment, while the transcriptional expressions of functional genes participating in fatty acids oxidation were markedly increased. The ability of EEP to reduce the negative effects of alcohol on liver makes propolis a potential natural product for the alternative treatment of alcoholic fatty liver.


Assuntos
Fígado Gorduroso Alcoólico/metabolismo , Hepatopatias Alcoólicas/metabolismo , Extratos Vegetais/metabolismo , Própole/metabolismo , Substâncias Protetoras/metabolismo , Alanina Transaminase/metabolismo , Animais , Apiterapia/métodos , Aspartato Aminotransferases/metabolismo , Colesterol/metabolismo , Modelos Animais de Doenças , Etanol , Ácidos Graxos/biossíntese , Fígado Gorduroso Alcoólico/tratamento farmacológico , Fígado Gorduroso Alcoólico/genética , Fígado Gorduroso Alcoólico/patologia , Hepatopatias Alcoólicas/tratamento farmacológico , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/patologia , Masculino , Oxirredução , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Própole/química , Própole/uso terapêutico , Substâncias Protetoras/química , Substâncias Protetoras/uso terapêutico , Ratos Wistar , Análise Serial de Tecidos/métodos , Transcrição Genética/genética , Triglicerídeos/metabolismo
7.
Dokl Biochem Biophys ; 486(1): 163-167, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367812

RESUMO

The treatment of Arabidopsis thaliana plants with exogenous cytokinin (CK) followed by heat shock (HS) activated the expression of the genes for the plastid transcription machinery but adversely affected the plant viability. Abscisic acid (ABA), conversely, promoted maintaining the resistance to HS and had differentially affected different components of the plastid transcriptional complex. This hormone suppressed the accumulation of transcripts of PEP genes and the genes encoding PAP proteins, which are involved in DNA-RNA metabolism. However, it had no effect or activated the expression of NEP genes and PAP genes, which are involved in the redox regulation, as well as the genes encoding the stress-inducible trans-factor (SIG5) and the plastid transcription Ser/Thr protein kinase (cpCK2). Thus, for the adaptation of plants to elevated temperatures, both increase and decrease in the expression of the genes for the plastid transcriptional machinery with the involvement of various regulatory systems, including phytohormones, are equally significant.


Assuntos
Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Citocininas/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Resposta ao Choque Térmico/genética , Plastídeos/genética , Transcrição Genética/efeitos dos fármacos , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/fisiologia , Resposta ao Choque Térmico/efeitos dos fármacos , Plastídeos/efeitos dos fármacos
8.
World J Microbiol Biotechnol ; 35(9): 138, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451937

RESUMO

Monascus azaphilone pigments, including red, orange, and yellow, are world-famous food colorants. However, the pigments produced by different Monascus species vary in yields and compositions. The underlying mechanism is unclear. In this study, four wild-type Monascus strains, namely M. anka M7, M. purpureus M9, M. ruber C100, and M. aurantiacus M15, were selected as research objects according to the diversification of their pigments fermented in the same mediums and conditions. Twenty-three 3 kbp segments (300 bp overlap with adjacent segments) of the pigment gene cluster were amplified, sequenced, and assembled into the DNA sequences of the clusters. The DNA sequences of pigment biosynthetic gene clusters of the four strains showed 99.94% similarity according to the results of multiple alignment. The expression levels of 17 pigment biosynthetic genes of four strains were determined by using real-time quantitative PCR. The transcriptional regulation contributed more than the DNA sequence variation in Monascus pigments metabolism. Our result gives insight into the study of Monascus pigment biosynthesis.


Assuntos
Monascus/genética , Monascus/metabolismo , Pigmentos Biológicos/biossíntese , Transcrição Genética , Sequência de Aminoácidos , Sequência de Bases , Cor , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Variação Genética , Monascus/química , Monascus/classificação , Família Multigênica , Filogenia , Pigmentos Biológicos/química
9.
BMC Plant Biol ; 19(1): 343, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387524

RESUMO

BACKGROUND: European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance to P. viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance. RESULTS: In this study, Rpv3-mediated defense responses were investigated in Rpv3+ and Rpv3- grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3+ and avrRpv3-, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-1-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-1-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses. CONCLUSION: This study used histochemical, transcriptomic and metabolomic analyses of Rpv3+ and susceptible cultivars inoculated with avirulent and virulent P. viticola isolates to investigate mechanism underlying the Rpv3-1-mediated resistance response. We demonstrated a strong correlation between the expressions of stilbene biosynthesis related genes, the accumulation of fungi-toxic stilbenes, pathogen growth inhibition and PCD.


Assuntos
Resistência à Doença/genética , Genes de Plantas/fisiologia , Estilbenos/metabolismo , Vitis/genética , Regulação da Expressão Gênica de Plantas , Metaboloma , Oomicetos/patogenicidade , Doenças das Plantas/microbiologia , Transcrição Genética , Transcriptoma , Vitis/imunologia , Vitis/microbiologia
10.
Chem Biol Interact ; 311: 108786, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31401087

RESUMO

Naturally occurring oleanolic acid (OA) possesses a hepatoprotective activity and ability to inhibit proliferation of human hepatocellular carcinoma cells. Both properties might be related to its anti-inflammatory activity. Its low bioavailability justifies the search for more hydrophilic OA derivatives. The aim of this study was the design and synthesis of four novel OA oxime derivatives conjugated with succinic acid at the C-3 position of oleanane skeleton structure and evaluation of their effect on NF-κB and STATs expression and activation in HepG2 cells. The expression of NF-κB and cyclooxygenase-2 (COX-2), STAT5A/B and STAT3 with its target genes: BAX, BCL-XL and MYC was evaluated after 24 h treatment with tested compounds. The comparison of the levels of cytosolic and nuclear NF-κB subunits p50, p65 and STATs proteins was used as the measure of their activation. The results pointed out the 3-succinyloxyiminoolean-12-en-28-oic acid morpholide (SMAM) as the most potent modulator of NF-κB and STAT3. SMAM significantly reduced the expression and activation of NF-κB as well as its nuclear protein level of p65 subunit. This compound also reduced the expression and activation of STAT3 and STAT5A/B. Combined effect of SMAM on these transcription factors resulted in reduced expression of COX-2, MYC and anti-apoptotic BCL-XL genes. Simultaneously, the increased expression of pro-apoptotic BAX gene was observed. In the cells treated with 3-succinyloxyiminoolean-12-en-28-oic acid (SMAA) the increased expression of BAX was also found. The effects of 3-succinyloxyiminoolean-12-en-28-oic acid benzyl ester (SMAEB) and 3-succinyloxyiminoolean-12-en-28-oic acid methyl ester (SMAEM) were moderate and ambiguous in relation to the tested factors. Moreover, the coordinated action of SMAM on NF-κB and STAT3 confirms their close association in HepG2 cells. We conclude that SMAM efficiently downregulates the key elements of signaling pathways involved in inflammatory driven HCC. Thus, may be considered as a potential chemopreventive or therapeutic agent in this type of cancer.


Assuntos
NF-kappa B/metabolismo , Ácido Oleanólico/análogos & derivados , Oximas/farmacologia , Fatores de Transcrição STAT/metabolismo , Ácido Succínico/química , Carcinoma Hepatocelular , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas , NF-kappa B/genética , Oximas/química , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição STAT/genética , Transcrição Genética/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
11.
Int J Nanomedicine ; 14: 5017-5032, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371944

RESUMO

Background: Epigallocatechin gallate (EGCG), the major anti-inflammatory compound in green tea, has been shown to suppress osteoclast (OC) differentiation. However, the low aqueous solubility of EGCG always leads to poor bioavailability, adverse effects, and several drawbacks for clinical applications. Purpose: In this study, we synthesized EGCG-capped gold nanoparticles (EGCG-GNPs) to solve the drawbacks for clinical uses of EGCG in bone destruction disorders by direct reduction of HAuCl4 in EGCG aqueous solution. Methods and Results: The obtained EGCG-GNPs were negatively charged and spherical. Theoretical calculation results suggested that EGCG was released from GNPs in an acidic environment. Cellular uptake study showed an obviously large amount of intracellular EGCG-GNPs without cytotoxicity. EGCG-GNPs exhibited better effects in reducing intracellular reactive oxygen species levels than free EGCG. A more dramatic anti-osteoclastogenic effect induced by EGCG-GNPs than free EGCG was observed in lipopolysaccharide (LPS)-stimulated bone marrow macrophages, including decreased formation of TRAP-positive multinuclear cells and actin rings. Meanwhile, EGCG-GNPs not only suppressed the mRNA expression of genetic markers of OC differentiation but also inhibited MAPK signaling pathways. Furthermore, we confirmed that EGCG-GNPs greatly reversed bone resorption in the LPS-induced calvarial bone erosion model in vivo, which was more effective than applying free EGCG, specifically in inhibiting the number of OCs, improving bone density, and preventing bone loss. Conclusion: EGCG-GNPs showed better anti-osteoclastogenic effect than free EGCG in vitro and in vivo, indicating their potential in anti-bone resorption treatment strategy.


Assuntos
Catequina/análogos & derivados , Ouro/farmacologia , Nanopartículas Metálicas/química , Osteogênese/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/patologia , Catequina/farmacologia , Morte Celular/efeitos dos fármacos , Teoria da Densidade Funcional , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos BALB C , Modelos Biológicos , Ligante RANK/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Crânio/patologia , Transcrição Genética/efeitos dos fármacos
12.
Gene ; 714: 143985, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31330236

RESUMO

In all eukaryotes, the response to heat stress (HS) is dependent on the activity of HS transcription factors (Hsfs). Plants contain a large number of Hsfs, however, only members of the HsfA1 subfamily are considered as master regulators of stress response and thermotolerance. In Solanum lycopersicum, among the four HsfA1 members, only HsfA1a has been proposed to possess a master regulator function. We performed a comparative analysis of HsfA1a, HsfA1b, HsfA1c and HsfA1e at different levels of regulation and function. HsfA1a is constitutively expressed under control and stress conditions, while the other members are induced in specific tissues and stages of HS response. Despite that all members are localized in the nucleus when expressed in protoplasts, only HsfA1a shows a wide range of basal activity on several HS-induced genes. In contrast, HsfA1b, HsfA1c, and HsfA1e show only high activity for specific subsets of genes. Domain swapping mutants between HsfA1a and HsfA1c revealed that the variation in that transcriptional transactivation activity is due to differences in the DNA binding domain (DBD). Specifically, we identified a conserved arginine (R107) residue in the turn of ß3 and ß4 sheet in the C-terminus of the DBD of HsfA1a that is highly conserved in plant HsfA1 proteins, but is replaced by leucine and cysteine in tomato HsfA1c and HsfA1e, respectively. Although not directly involved in DNA interaction, R107 contributes to DNA binding and consequently the activity of HsfA1a. Thus, we demonstrate that this variation in DBD in part explains the functional diversification of tomato HsfA1 members.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Choque Térmico/genética , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Temperatura Alta , Domínios Proteicos/genética , Protoplastos/fisiologia , Temperatura Ambiente , Termotolerância/genética , Transcrição Genética/genética , Ativação Transcricional/genética
13.
Nat Commun ; 10(1): 2921, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266943

RESUMO

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.


Assuntos
Regulação da Expressão Gênica , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon/metabolismo , Interferons/metabolismo , Fator de Transcrição STAT2/metabolismo , Animais , Feminino , Humanos , Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Células RAW 264.7 , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Transcrição Genética
14.
Nat Commun ; 10(1): 2925, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266960

RESUMO

Bacteriophage Q protein engages σ-dependent paused RNA polymerase (RNAP) by binding to a DNA site embedded in late gene promoter and renders RNAP resistant to termination signals. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact Q-engaged arrested complex. The structure reveals key interactions responsible for σ-dependent pause, Q engagement, and Q-mediated transcription antitermination. The structure shows that two Q protomers (QI and QII) bind to a direct-repeat DNA site and contact distinct elements of the RNA exit channel. Notably, QI forms a narrow ring inside the RNA exit channel and renders RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Because the RNA exit channel is conserved among all multisubunit RNAPs, it is likely to serve as an important contact site for regulators that modify the elongation properties of RNAP in other organisms, as well.


Assuntos
Bacteriófagos/enzimologia , Códon de Terminação/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófagos/química , Bacteriófagos/genética , Códon de Terminação/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Regiões Promotoras Genéticas , Proteínas Virais/genética
15.
Chem Biol Interact ; 311: 108773, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351048

RESUMO

Hemangioma (HA) is tumor formed by hyper-proliferation of vascular endothelial cells. However, the potential effects of mono-(2-ethylhexyl) phthalate (MEHP) on the progression of HA are not well illustrated. Our present study revealed that MEHP exposure can significantly increase the in vitro proliferation of hemangioma-derived endothelial cells (HemECs). MEHP treatment can activate yes-associated protein (YAP), a key effector of Hippo pathway, by inhibiting its phosphorylation. The dephosphorylation of YAP induced by MEHP can promote the nuclear accumulation of YAP. Knockdown of YAP or its inhibitor can block MEHP triggered cell proliferation. MEHP can increase the levels of precursor and mature mRNA of YAP in HemECs. As well, MEHP extended the half-life of YAP protein. Mechanistically, MEHP can decrease the phosphorylation of YAP via suppressing the activity of large tumor suppressor kinase 1/2 (LATS1/2) to inhibit it induced degradation of YAP. Further, MEHP increased the expression of interferon regulatory factor 1 (IRF1), which can bind to the promoter of YAP to initiate its transcription. Collectively, we revealed that Hippo-YAP signal is involved in MEHP-induced proliferation of HA cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Dietilexilftalato/análogos & derivados , Hemangioma/patologia , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Dietilexilftalato/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Hemangioma/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Estabilidade Proteica/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Genética/efeitos dos fármacos
16.
Nat Commun ; 10(1): 3004, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285436

RESUMO

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.


Assuntos
Elementos Facilitadores Genéticos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Rabdomiossarcoma/genética , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Rabdomiossarcoma/patologia , Análise de Sequência de RNA , Transcrição Genética/efeitos dos fármacos
17.
Nature ; 571(7765): 419-423, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292545

RESUMO

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.


Assuntos
Regulação da Expressão Gênica/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única , Transcrição Genética/genética , Alquilação , Animais , Ciclo Celular , Citomegalovirus/fisiologia , Metilação de DNA , Fibroblastos/metabolismo , Fibroblastos/virologia , Cinética , Camundongos , Regiões Promotoras Genéticas/genética , RNA/análise , RNA/química , Compostos de Sulfidrila/química
18.
Cell Mol Biol Lett ; 24: 33, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31160892

RESUMO

Background: Induction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB. Methods: Expression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes. Results: Our results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes. Conclusions: Collectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Epitélio Pigmentado da Retina/patologia , Estresse Fisiológico , Adulto , Cloreto de Amônio/farmacologia , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Estresse Fisiológico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
19.
Biochemistry (Mosc) ; 84(4): 426-434, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228934

RESUMO

The bacterium Escherichia coli has seven σ subunits that bind core RNA polymerase and are necessary for promoter recognition. It was previously shown that the σ70 and σ38 subunits can also interact with the transcription elongation complex (TEC) and stimulate pausing by recognizing DNA sequences similar to the -10 element of promoters. In this study, we analyzed the ability of the σ32, σ28, and σ24 subunits to induce pauses in reconstituted TECs containing corresponding -10 consensus elements. It was found that the σ24 subunit can induce a transcriptional pause depending on the presence of the -10 element. Pause formation is suppressed by the Gre factors, suggesting that the paused complex adopts a backtracked conformation. Some natural promoters contain potential signals of σ24-dependent pauses in the initially transcribed regions, suggesting that such pauses may have regulatory functions in transcription.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Transcrição Genética/fisiologia , Sequência de Bases , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo
20.
Chem Pharm Bull (Tokyo) ; 67(6): 505-518, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155555

RESUMO

Nucleic acid therapeutics such as antisense and small interfering RNA (siRNA) have attracted increasing attention as innovative medicines that interfere with and/or modify gene expression systems. We have developed new functional oligonucleotides that can target DNA and RNA with high efficiency and selectivity. This review summarizes our achievements, including (1) the formation of non-natural triplex DNA for sequence-specific inhibition of transcription; (2) artificial receptor molecules for 8-oxidized-guanosine nucleosides; and (3) reactive oligonucleotides with a cross-linking agent or a functionality-transfer nucleoside for RNA pinpoint modification.


Assuntos
DNA/química , RNA/química , Reagentes para Ligações Cruzadas/química , DNA/metabolismo , DNA/farmacologia , Humanos , Nucleosídeos/análogos & derivados , Nucleosídeos/farmacologia , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Polietilenoglicóis/química , RNA/metabolismo , Telomerase/genética , Telomerase/metabolismo , Transcrição Genética/efeitos dos fármacos
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