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1.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
2.
FASEB J ; 35(9): e21778, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383971

RESUMO

As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits ß-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to ß-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on ß-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin-glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting ß-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.


Assuntos
Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Glicerol/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Estilbenos/farmacologia , Transcrição Genética/efeitos dos fármacos , beta-Glucanas/farmacologia , Caspofungina/farmacologia , Parede Celular/genética , Parede Celular/metabolismo , Quitina/farmacologia , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Concentração Osmolar , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Genética/genética
3.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445254

RESUMO

Nitrogen forms (nitrate (NO3-) or ammonium (NH4+)) are vital to plant growth and metabolism. In stevia (Stevia rebaudiana), it is important to assess whether nitrogen forms can influence the synthesis of the high-value terpene metabolites-steviol glycosides (SGs), together with the underlying mechanisms. Field and pot experiments were performed where stevia plants were fertilized with either NO3- or NH4+ nutrition to the same level of nitrogen. Physiological measurements suggested that nitrogen forms had no significant impact on biomass and the total nitrogen content of stevia leaves, but NO3--enhanced leaf SGs contents. Transcriptomic analysis identified 397 genes that were differentially expressed (DEGs) between NO3- and NH4+ treatments. Assessment of the DEGs highlighted the responses in secondary metabolism, particularly in terpenoid metabolism, to nitrogen forms. Further examinations of the expression patterns of SGs synthesis-related genes and potential transcription factors suggested that GGPPS and CPS genes, as well as the WRKY and MYB transcription factors, could be driving N form-regulated SG synthesis. We concluded that NO3-, rather than NH4+, can promote leaf SG synthesis via the NO3--MYB/WRKY-GGPPS/CPS module. Our study suggests that insights into the molecular mechanism of how SG synthesis can be affected by nitrogen forms.


Assuntos
Amônia/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucosídeos/biossíntese , Nitratos/metabolismo , Stevia/metabolismo , Transcrição Genética/efeitos dos fármacos , Diterpenos do Tipo Caurano , Perfilação da Expressão Gênica , Glucosídeos/genética , Nitratos/farmacologia , Stevia/genética
4.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360903

RESUMO

Despite the fact that many studies have examined the effectiveness of different gaseous postharvest treatments applied at low temperature to maintain table grape quality, the use of ethanol vapor has hardly been investigated. Thus, this work has studied the effectiveness of ethanol vapor-generating sachets in the maintenance of It 681-30 table grape quality, a new cultivar, during storage at low temperature and after the shelf-life period at 20 °C. To this end, various quality assessments have been carried out and the effect of the ethanol treatment on the expression of different genes (phenylpropanoids, transcription factors, PRs, and aquaporins) was determined. The results indicated that the application of ethanol vapor reduced the total decay incidence, weight loss, and the rachis browning index in It 681-30 grapes stored at 0 °C and after the shelf-life period at 20 °C, as compared to non-treated samples. Moreover, the modulation of STS7 and the different PR genes analyzed seems to play a part in the molecular mechanisms activated to cope with fungal attacks during the postharvest of It 681-30 grapes, and particularly during the shelf-life period at 20 °C. Furthermore, the expression of aquaporin transcripts was activated in samples showing higher weight loss. Although further work is needed to elucidate the role of ethanol in table grape quality, the results obtained in this work provide new insight into the transcriptional regulation triggered by ethanol treatment.


Assuntos
Temperatura Baixa , Etanol/farmacologia , Conservação de Alimentos/métodos , Conservantes de Alimentos/farmacologia , Qualidade dos Alimentos , Armazenamento de Alimentos/métodos , Frutas/efeitos dos fármacos , Gases/farmacologia , Vitis/efeitos dos fármacos , Aquaporinas/genética , Frutas/genética , Expressão Gênica/efeitos dos fármacos , Reação de Maillard/efeitos dos fármacos , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Transcrição Genética/efeitos dos fármacos , Vitis/genética , Volatilização
5.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443374

RESUMO

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.


Assuntos
Neoplasias Encefálicas/patologia , Calcineurina/metabolismo , Glioma/patologia , Fatores de Transcrição NFATC/metabolismo , Oligopeptídeos/farmacologia , Transdução de Sinais , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Glioma/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição NFATC/química , Oligopeptídeos/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
6.
Science ; 373(6557)2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34301855

RESUMO

Stochastic fluctuations in gene expression ("noise") are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean expression levels. This amplified expression noise originates from shorter-duration, higher-intensity transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we refer to as "discordant transcription through repair" ("DiThR," which is pronounced "dither"), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease.


Assuntos
Diferenciação Celular , Reprogramação Celular , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/química , Expressão Gênica , Transcrição Genética , Animais , Células Cultivadas , Simulação por Computador , DNA/genética , DNA/metabolismo , Células-Tronco Embrionárias , Expressão Gênica/efeitos dos fármacos , Idoxuridina/metabolismo , Idoxuridina/farmacologia , Camundongos , Modelos Genéticos , Proteína Homeobox Nanog/genética , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Processos Estocásticos , Timidina Quinase/genética , Timidina Quinase/metabolismo , Transcrição Genética/efeitos dos fármacos
7.
Curr Opin Hematol ; 28(5): 356-363, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34267079

RESUMO

PURPOSE OF REVIEW: Comprehensive sequencing studies aimed at determining the genetic landscape of myeloid neoplasms have identified epigenetic regulators to be among the most commonly mutated genes. Detailed studies have also revealed a number of epigenetic vulnerabilities. The purpose of this review is to outline these vulnerabilities and to discuss the new generation of drugs that exploit them. RECENT FINDINGS: In addition to deoxyribonucleic acid-methylation, novel epigenetic dependencies have recently been discovered in various myeloid neoplasms and many of them can be targeted pharmacologically. These include not only chromatin writers, readers, and erasers but also chromatin movers that shift nucleosomes to allow access for transcription. Inhibitors of protein-protein interactions represent a novel promising class of drugs that allow disassembly of oncogenic multiprotein complexes. SUMMARY: An improved understanding of disease-specific epigenetic vulnerabilities has led to the development of second-generation mechanism-based epigenetic drugs against myeloid neoplasms. Many of these drugs have been introduced into clinical trials and synergistic drug combination regimens have been shown to enhance efficacy and potentially prevent drug resistance.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas , Transtornos Mieloproliferativos , Transcrição Genética/efeitos dos fármacos , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Humanos , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo
8.
Nat Commun ; 12(1): 4563, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315897

RESUMO

The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen in P. falciparum to identify genes allowing parasites to survive febrile temperatures. Screening identifies more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants are more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin include highly essential, conserved pathways associated with protein-folding, heat shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the ancestral genome of the parasite's algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general are upregulated in response to heat shock, as are other Plasmodium genes with orthologs in plant and algal genomes. Plasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiont-derived genes in the parasite's genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors.


Assuntos
Apicoplastos/metabolismo , Artemisininas/farmacologia , Resistência a Medicamentos , Febre/parasitologia , Malária Falciparum/parasitologia , Parasitos/fisiologia , Animais , Apicoplastos/efeitos dos fármacos , Resistência a Medicamentos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Resposta ao Choque Térmico/efeitos dos fármacos , Mutação/genética , Parasitos/efeitos dos fármacos , Fenótipo , Plasmodium falciparum/genética , Transdução de Sinais/efeitos dos fármacos , Temperatura , Terpenos/metabolismo , Transcrição Genética/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
9.
Front Immunol ; 12: 648815, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34305888

RESUMO

Multiple lines of evidence have demonstrated that cigarette smoke or Chronic Obstructive Pulmonary Disease upregulates angiotensin-converting enzyme 2, the cellular receptor for the entry of the severe acute respiratory syndrome coronavirus 2, which predisposes individuals to develop severe Coronavirus disease 2019. The reason for this observation is unknown. We recently reported that the loss of function of Miz1 in the lung epithelium in mice leads to a spontaneous COPD-like phenotype, associated with upregulation of angiotensin-converting enzyme 2. We also reported that cigarette smoke exposure downregulates Miz1 in lung epithelial cells and in mice, and Miz1 is also downregulated in the lungs of COPD patients. Here, we provide further evidence that Miz1 directly binds to and represses the promoter of angiotensin-converting enzyme 2 in mouse and human lung epithelial cells. Our data provide a potential molecular mechanism for the upregulation of angiotensin-converting enzyme 2 observed in smokers and COPD patients, with implication in severe Coronavirus disease 2019.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Receptores Virais/genética , Transcrição Genética , Células Epiteliais Alveolares/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Domínio BTB-POZ , Linhagem Celular , Fumar Cigarros/efeitos adversos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Virais/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Transcrição Genética/efeitos dos fármacos , Fatores de Necrose Tumoral/farmacologia , Internalização do Vírus
10.
BMC Plant Biol ; 21(1): 308, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193032

RESUMO

BACKGROUND: Rice (Oryza sativa L.) Chalkiness, the opaque part in the kernel endosperm formed by loosely piled starch and protein bodies. Chalkiness is a complex quantitative trait regulated by multiple genes and various environmental factors. Phytohormones play important roles in the regulation of chalkiness formation but the underlying molecular mechanism is still unclear at present. RESULTS: In this research, Xiangzaoxian24 (X24, pure line of indica rice with high-chalkiness) and its origin parents Xiangzaoxian11 (X11, female parent, pure line of indica rice with high-chalkiness) and Xiangzaoxian7 (X7, male parent, pure line of indica rice with low-chalkiness) were used as materials. The phenotype, physiological and biochemical traits combined with transcriptome analysis were conducted to illustrate the dynamic process and transcriptional regulation of rice chalkiness formation. Impressively, phytohormonal contents and multiple phytohormonal signals were significantly different in chalky caryopsis, suggesting the involvement of phytohormones, particularly ABA and auxin, in the regulation of rice chalkiness formation, through the interaction of multiple transcription factors and their downstream regulators. CONCLUSION: These results indicated that chalkiness formation is a dynamic process associated with multiple genes, forming a complex regulatory network in which phytohormones play important roles. These results provided informative clues for illustrating the regulatory mechanisms of chalkiness formation in rice.


Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/genética , Oryza/fisiologia , Reguladores de Crescimento de Plantas/farmacologia , Transcrição Genética/efeitos dos fármacos , Endosperma/efeitos dos fármacos , Endosperma/metabolismo , Endosperma/ultraestrutura , Perfilação da Expressão Gênica , Ontologia Genética , Oryza/efeitos dos fármacos , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reprodutibilidade dos Testes , Amido/metabolismo , Amido/ultraestrutura , Sacarose/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208198

RESUMO

The role of auxin in the fruit-ripening process during the early developmental stages of commercial strawberry fruits (Fragaria x ananassa) has been previously described, with auxin production occurring in achenes and moving to the receptacle. Additionally, fruit softening is a consequence of the depolymerization and solubilization of cell wall components produced by the action of a group of proteins and enzymes. The aim of this study was to compare the effect of exogenous auxin treatment on the physiological properties of the cell wall-associated polysaccharide contents of strawberry fruits. We combined thermogravimetric (TG) analysis with analyses of the mRNA abundance, enzymatic activity, and physiological characteristics related to the cell wall. The samples did not show a change in fruit firmness at 48 h post-treatment; by contrast, we showed changes in the cell wall stability based on TG and differential thermogravimetric (DTG) analysis curves. Less degradation of the cell wall polymers was observed after auxin treatment at 48 h post-treatment. The results of our study indicate that auxin treatment delays the cell wall disassembly process in strawberries.


Assuntos
Biopolímeros/metabolismo , Parede Celular/metabolismo , Fragaria/metabolismo , Frutas/metabolismo , Ácidos Indolacéticos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Fragaria/efeitos dos fármacos , Fragaria/genética , Frutas/efeitos dos fármacos , Frutas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Temperatura , Termogravimetria , Transcrição Genética/efeitos dos fármacos , Ácidos Tri-Iodobenzoicos/farmacologia
12.
BMC Plant Biol ; 21(1): 310, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210277

RESUMO

BACKGROUND: The ability of chickpea to obtain sufficient nitrogen via its symbiotic relationship with Mesorhizobium ciceri is of critical importance in supporting growth and grain production. A number of factors can affect this symbiotic relationship including abiotic conditions, plant genotype, and disruptions to host signalling/perception networks. In order to support improved nodule formation in chickpea, we investigated how plant genotype and soil nutrient availability affect chickpea nodule formation and nitrogen fixation. Further, using transcriptomic profiling, we sought to identify gene expression patterns that characterize highly nodulated genotypes. RESULTS: A study involving six chickpea varieties demonstrated large genotype by soil nitrogen interaction effects on nodulation and further identified agronomic traits of genotypes (such as shoot weight) associated with high nodulation. We broadened our scope to consider 29 varieties and breeding lines to examine the relationship between soilborne disease resistance and the number of nodules developed and real-time nitrogen fixation. Results of this larger study supported the earlier genotype specific findings, however, disease resistance did not explain differences in nodulation across genotypes. Transcriptional profiling of six chickpea genotypes indicates that genes associated with signalling, N transport and cellular localization, as opposed to genes associated with the classical nodulation pathway, are more likely to predict whether a given genotype will exhibit high levels of nodule formation. CONCLUSIONS: This research identified a number of key abiotic and genetic factors affecting chickpea nodule development and nitrogen fixation. These findings indicate that an improved understanding of genotype-specific factors affecting chickpea nodule induction and function are key research areas necessary to improving the benefits of rhizobial symbiosis in chickpea.


Assuntos
Cicer/genética , Resistência à Doença/efeitos dos fármacos , Nitrogênio/farmacologia , Nodulação/genética , Raízes de Plantas/fisiologia , Solo , Biomassa , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Genótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Nódulos Radiculares de Plantas/efeitos dos fármacos , Nódulos Radiculares de Plantas/metabolismo , Transcrição Genética/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
13.
Cardiovasc Ther ; 2021: 5554569, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34257705

RESUMO

Ginkgolide B (GB) is an active ingredient extracted from Ginkgo biloba leaves. However, the effects of GB on cardiac hypertrophy remain unclear. The study is aimed at determining whether GB could alleviate cardiac hypertrophy and exploring its underlying molecular mechanism. Rat cardiomyocyte cell line H9c2 cells were pretreated with GB and incubated with angiotensin II (Ang II) to simulate an in vitro cardiac hypertrophy model. Cell viability, cell size, hypertrophy markers, and autophagy were determined in H9c2 cells after Ang II treatment. Proteins involved in autophagy and the SIRT1 pathway were determined by western blot. Our data demonstrated that GB attenuated Ang II-induced cardiac hypertrophy and reduced the mRNA expressions of hypertrophy marker, atrial natriuretic peptide (ANP), and ß-myosin heavy chain (ß-MHC). GB further increased Ang II-induced autophagy in H9c2 cells and modulated expressions of autophagy-related proteins Beclin1 and P62. Modulation of autophagy using autophagy inhibitor 3-methyladenine (3-MA) could abrogate GB-downregulated transcription of NPPA. We then showed that GB attenuated Ang II-induced oxidative stress and reduction in SIRT1 and FoxO1 protein expression. Finally, the effect of GB on autophagy and cardiac hypertrophy could be reversed by SIRT1 inhibitor EX-527. GB inhibits Ang II-induced cardiac hypertrophy by enhancing autophagy via the SIRT1-FoxO1 signaling pathway and might be a potential agent in treating pathological cardiac hypertrophy.


Assuntos
Angiotensina II/toxicidade , Autofagia/efeitos dos fármacos , Ginkgolídeos/farmacologia , Lactonas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Sirtuína 1/metabolismo , Animais , Fator Natriurético Atrial/genética , Cardiomegalia/tratamento farmacológico , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Miócitos Cardíacos/patologia , Substâncias Protetoras/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Miosinas Ventriculares/genética
14.
Methods Mol Biol ; 2329: 323-335, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085233

RESUMO

The revolutionary CRISPR technology opens a new era of cell biology in mammalian cells. The InDel mutation is induced by CRISPR and results in the frameshift mutation of the gene. Owing to the nature of CRISPR induced knockout, the conditional knockout using CRISPR technology is not common. With the recent development of the small molecule-inducible degron system, an analogous system to the classical genetic conditional knockout has become feasible. By integrating CRISPR-knockout, the tetracycline-controlled transcriptional and auxin-induced degradation post-translational control of protein expression, a method imitating the conditional knockout is developed. We herein describe the detailed protocol for the generation of a conditional protein inactivation in human cancer cells. The system is especially useful to study essential gene function in aneuploidy cancer cells where gain in copy number is common.


Assuntos
Ciclina A2/genética , Ciclina A2/metabolismo , Técnicas de Inativação de Genes/métodos , Ácidos Indolacéticos/farmacologia , Tetraciclina/farmacologia , Sistemas CRISPR-Cas , Mutação da Fase de Leitura , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Proteólise , Retroviridae/genética , Transcrição Genética/efeitos dos fármacos
15.
J Endocrinol ; 250(2): 49-65, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34060475

RESUMO

Hyperandrogenemia (HA) is a hallmark of polycystic ovary syndrome (PCOS) and is an integral element of non-alcoholic fatty liver disease (NALFD) in females. Administering low-dose dihydrotestosterone (DHT) induced a normal weight PCOS-like female mouse model displaying NAFLD. The molecular mechanism of HA-induced NAFLD has not been fully determined. We hypothesized that DHT would regulate hepatic lipid metabolism via increased SREBP1 expression leading to NAFLD. We extracted liver from control and low-dose DHT female mice; and performed histological and biochemical lipid profiles, Western blot, immunoprecipitation, chromatin immunoprecipitation, and real-time quantitative PCR analyses. DHT lowered the 65 kD form of cytosolic SREBP1 in the liver compared to controls. However, DHT did not alter the levels of SREBP2 in the liver. DHT mice displayed increased SCAP protein expression and SCAP-SREBP1 binding compared to controls. DHT mice exhibited increased AR binding to intron-8 of SCAP leading to increased SCAP mRNA compared to controls. FAS mRNA and protein expression was increased in the liver of DHT mice compared to controls. p-ACC levels were unaltered in the liver. Other lipid metabolism pathways were examined in the liver, but no changes were observed. Our findings support evidence that DHT increased de novo lipogenic proteins resulting in increased hepatic lipid content via regulation of SREBP1 in the liver. We show that in the presence of DHT, the SCAP-SREBP1 interaction was elevated leading to increased nuclear SREBP1 resulting in increased de novo lipogenesis. We propose that the mechanism of action may be increased AR binding to an ARE in SCAP intron-8.


Assuntos
Di-Hidrotestosterona/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Síndrome do Ovário Policístico/metabolismo , Animais , Composição Corporal , Peso Corporal , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/induzido quimicamente , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Transcrição Genética/efeitos dos fármacos
16.
Methods Mol Biol ; 2326: 95-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34097263

RESUMO

Semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) is a simple and specific method for quantitative RNA in recent years. The relative quantity of a specific mRNA in the samples can be inferred by reverse transcription of mRNA into cDNA, and PCR amplification and determination of the quantity of PCR products. The semi-quantitative analysis is carried out under a fixed number of PCR cycles, and the total RNA concentration is kept in the exponential phase of the PCR. The method is to use a housekeeping gene (usually actin, GAPDH, and EF1α) as a reference standard in treated and control organisms to observe the expression of the interested genes (upregulated or downregulated) in toxicology. In this chapter, we describe a step-by-step method for determining the differential regulation of target genes in organisms exposed to environmental pollutants.


Assuntos
Poluentes Ambientais/toxicidade , Reação em Cadeia da Polimerase/métodos , Transcrição Genética/efeitos dos fármacos , Animais , Humanos , RNA/genética , RNA/isolamento & purificação , Testes de Toxicidade/métodos
17.
Nat Commun ; 12(1): 3962, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172723

RESUMO

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimer's disease and type II diabetes, identified a tripyridylamide, ADH-6, that abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53's transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment effectively shrinks xenografts harboring mutant p53, while exhibiting no toxicity to healthy tissue, thereby substantially prolonging survival. This study demonstrates the successful application of a bona fide small-molecule amyloid inhibitor as a potent anticancer agent.


Assuntos
Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Agregação Patológica de Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Amidas/química , Amidas/farmacologia , Amidas/uso terapêutico , Amiloide/química , Amiloide/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Agregação Patológica de Proteínas/tratamento farmacológico , Domínios Proteicos , Piridinas/química , Piridinas/farmacologia , Piridinas/uso terapêutico , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética
18.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063046

RESUMO

Receptor-like kinases (RLKs) constitute a large group of cell surface receptors that play crucial roles in multiple biological processes. However, the function of most RLKs in plants has not been extensively explored, and much less for the class of cell wall associated kinases (WAKs) and WAK-like kinases (WAKLs). In this study, analyses of developmental expression patterns uncovered a putative role of AtWAKL10 in modulating leaf senescence, which was further investigated at physiological and molecular levels. The expression level of AtWAKL10 increased with the developmental progression and was rapidly upregulated in senescing leaf tissues. The promoter of AtWAKL10 contains various defense and hormone responsive elements, and its expression could be significantly induced by exogenous ABA, JA and SA. Moreover, the loss-of-function atwakl10 mutant showed earlier senescence along the course of natural development and accelerated leaf senescence under darkness and hormonal stresses, while plants overexpressing AtWAKL10 showed an opposite trend. Additionally, some defense and senescence related WRKY transcription factors could bind to the promoter of AtWAKL10. In addition, deletion and overexpression of AtWAKL10 caused several specific transcriptional alterations, including genes involved in cell extension, cell wall modification, defense response and senescence related WRKYs, which may be implicated in regulatory mechanisms adopted by AtWAKL10 in controlling leaf senescence. Taken together, these results revealed that AtWAKL10 negatively regulated leaf senescence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Parede Celular/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Parede Celular/efeitos dos fármacos , Escuridão , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação/genética , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Frações Subcelulares/metabolismo , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
19.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067160

RESUMO

Puccinia striiformis f. sp. tritici (Pst) is an important pathogen of wheat (Triticum aestivum L.) stripe rust, and the effector protein secreted by haustoria is a very important component involved in the pathogenic process. Although the candidate effector proteins secreted by Pst haustoria have been predicted to be abundant, few have been functionally validated. Our study confirmed that chitin and flg22 could be used as elicitors of the pathogenic-associated molecular pattern-triggered immune (PTI) reaction in wheat leaves and that TaPr-1-14 could be used as a marker gene to detect the PTI reaction. In addition, the experimental results were consistent in wheat protoplasts. A rapid and efficient method for screening and identifying the effector proteins of Pst was established by using the wheat protoplast transient expression system. Thirty-nine Pst haustorial effector genes were successfully cloned and screened for expression in the protoplast. We identified three haustorial effector proteins, PSEC2, PSEC17, and PSEC45, that may inhibit the response of wheat to PTI. These proteins are localized in the somatic cytoplasm and nucleus of wheat protoplasts and are highly expressed during the infection and parasitism of wheat.


Assuntos
Proteínas Fúngicas/metabolismo , Imunidade , Padrões Moleculares Associados a Patógenos/metabolismo , Protoplastos/microbiologia , Puccinia/fisiologia , Triticum/imunologia , Triticum/microbiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quitina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Imunidade/efeitos dos fármacos , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Protoplastos/efeitos dos fármacos , Puccinia/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transcrição Genética/efeitos dos fármacos , Triticum/efeitos dos fármacos , Triticum/genética
20.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068728

RESUMO

To mimic more realistic lung tissue conditions, co-cultures of epithelial and immune cells are one comparatively easy-to-use option. To reveal the impact of immune cells on the mode of action (MoA) of CuO nanoparticles (NP) on epithelial cells, A549 cells as a model for epithelial cells have been cultured with or without differentiated THP-1 cells, as a model for macrophages. After 24 h of submerged incubation, cytotoxicity and transcriptional toxicity profiles were obtained and compared between the cell culture systems. Dose-dependent cytotoxicity was apparent starting from 8.0 µg/cm2 CuO NP. With regard to gene expression profiles, no differences between the cell models were observed concerning metal homeostasis, oxidative stress, and DNA damage, confirming the known MoA of CuO NP, i.e., endocytotic particle uptake, intracellular particle dissolution within lysosomes with subsequent metal ion deliberation, increased oxidative stress, and genotoxicity. However, applying a co-culture of epithelial and macrophage-like cells, CuO NP additionally provoked a pro-inflammatory response involving NLRP3 inflammasome and pro-inflammatory transcription factor activation. This study demonstrates that the application of this easy-to-use advanced in vitro model is able to extend the detection of cellular effects provoked by nanomaterials by an immunological response and emphasizes the use of such models to address a more comprehensive MoA.


Assuntos
Epitélio/efeitos dos fármacos , Nanopartículas Metálicas/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Estresse Oxidativo/genética , Transcrição Genética/efeitos dos fármacos , Células A549 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Cobre/química , Cobre/farmacologia , Dano ao DNA/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
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