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1.
Exp Parasitol ; 212: 107886, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32209315

RESUMO

In aquaculture of the swimming crab Portunus trituberculatus, massive deaths have been recorded in the winter months due to infection with a novel emerging parasite, Mesanophrys sp. However, no information was available regarding the prevention and control of this particular parasite. Therefore, the present study was conducted to evaluate the anti-parasitic efficacy and toxicity of formalin against the Mesanophrys sp. In vitro results showed that the anti-parasitic efficacy of formalin improved with concentration increasing from 0.0 to 20.0 ppm within 24 h. In particular, when treated with formalin at 16.0, 15.0, 11.0, 10.0, 9.0, and 6.0 ppm for 0.5, 1, 2, 4, 6, 12, and 24 h respectively, the Mesanophrys sp. mortality rate reached 100%. To gain insights into the effects the formalin treatment had on the parasite, cell micro- and ultra-structure were investigated. It was determined that the cells contracted gradually and became rounded, intracellular vacuoles were observed at early time points (Ф≤4.83 ± 1.26 µm) and then disappeared. Cilia were shed and macronuclear chromatin became condensed and agglutinated. Small holes and bubbles appeared on surface of the parasites. In an in vivo trial, formalin was applied prior to Mesanophrys sp. artificial infection as prophylaxis to P. trituberculatus. The results showed that formalin prophylactic treatment effectively prevented P. trituberculatus from Mesanophrys sp. infection, thus remarkably reducing the mortality of crabs compared with the non-formalin-exposed and infected crabs. Furthermore, the normal behavior and survival of P. trituberculatus were not impacted by the prophylactic treatment.


Assuntos
Antiparasitários/farmacologia , Braquiúros/parasitologia , Desinfetantes/farmacologia , Formaldeído/farmacologia , Oligoimenóforos/efeitos dos fármacos , Análise de Variância , Animais , Aquicultura , Braquiúros/crescimento & desenvolvimento , Cromatina/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cílios/ultraestrutura , DNA de Protozoário/efeitos dos fármacos , DNA de Protozoário/isolamento & purificação , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Hemolinfa/parasitologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Interferência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oligoimenóforos/genética , Oligoimenóforos/patogenicidade , Oligoimenóforos/ultraestrutura , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestrutura
2.
PLoS One ; 15(1): e0227116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31951625

RESUMO

In this study we aimed to explore the potential biological effect of ethanol exposure on healthy colon epithelial cells using normal human colon 3D organoid "mini-gut" cultures. In numerous published studies ethanol use has been shown to be an environmental risk factor for colorectal cancer (CRC) development; however, the influence of ethanol exposure on normal colon epithelial cell biology remains poorly understood. We investigated the potential molecular effects of ethanol exposure in normal colon 3D organoids in a small pilot study (n = 3) using RNA-seq and ATAC-seq. We identify 1965 differentially expressed genes and 2217 differentially accessible regions of chromatin in response to ethanol treatment. Further, by cross-referencing our results with previously published analysis in colorectal cancer cell lines, we have not only validated a number of reported differentially expressed genes, but also identified several novel candidates for future investigation. In summary, our data highlights the potential importance for the use of normal colon 3D organoid models as a novel tool for the investigation of the relationship between the effects of environmental risk factors associated with colorectal cancer and the molecular mechanisms through which they confer this risk.


Assuntos
Montagem e Desmontagem da Cromatina , Colo/efeitos dos fármacos , Etanol/farmacologia , Organoides/efeitos dos fármacos , Transcriptoma , Adulto , Linhagem Celular Tumoral , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Colo/citologia , Colo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Organoides/metabolismo
3.
Exp Parasitol ; 209: 107810, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31801691

RESUMO

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.


Assuntos
Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Curcumina/farmacologia , Fasciola/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Benzoquinonas/química , Búfalos , Cromatina/efeitos dos fármacos , Curcumina/química , Dano ao DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Eletroforese em Gel de Ágar , Inibidores Enzimáticos/farmacologia , Fasciola/citologia , Fasciola/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Modelos Moleculares , Simulação de Acoplamento Molecular , Espécies Reativas de Oxigênio/metabolismo
4.
PLoS One ; 14(12): e0216515, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887110

RESUMO

The HIV genome is rich in A but not G or U and deficient in C. This nucleotide bias controls HIV phenotype by determining the highly unusual composition of all major HIV proteins. The bias is also responsible for the high frequency of narrow DNA minor groove sites in the double-stranded HIV genome as compared to cellular protein coding sequences and the bulk of the human genome. Since drugs that bind in the DNA minor groove disrupt nucleosomes on sequences that contain closely spaced oligo-A tracts which are prevalent in HIV DNA because of its bias, it was of interest to determine if these drugs exert this selective inhibitory effect on HIV chromatin. To test this possibility, nucleosomes were reconstituted onto five double-stranded DNA fragments from the HIV-1 pol gene in the presence and in the absence of several minor groove binding drugs (MGBDs). The results demonstrated that the MGBDs inhibited the assembly of nucleosomes onto all of the HIV-1 segments in a manner that was proportional to the A-bias, but had no detectable effect on the formation of nucleosomes on control cloned fragments or genomic DNA from chicken and human. Nucleosomes preassembled onto HIV DNA were also preferentially destabilized by the drugs as evidenced by enhanced nuclease accessibility in physiological ionic strength and by the preferential loss of the histone octamer in hyper-physiological salt solutions. The drugs also selectively disrupted HIV-containing nucleosomes in yeast as revealed by enhanced nuclease accessibility of the in vivo assembled HIV chromatin and reductions in superhelical densities of plasmid chromatin containing HIV sequences. A comparison of these results to the density of A-tracts in the HIV genome indicates that a large fraction of the nucleosomes that make up HIV chromatin should be preferred in vitro targets for the MGBDs. These results show that the MGBDs preferentially disrupt HIV-1 chromatin in vitro and in vivo and raise the possibility that non-toxic derivatives of certain MGBDs might serve as a novel class of anti-HIV agents.


Assuntos
Cromatina/efeitos dos fármacos , Cromatina/genética , HIV/genética , Sequência de Bases , Sítios de Ligação/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Biologia Computacional/métodos , DNA/efeitos dos fármacos , DNA/genética , Genes pol/genética , HIV/metabolismo , Infecções por HIV/genética , Humanos
5.
Nat Med ; 25(11): 1721-1727, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31700186

RESUMO

Anthracyclines are a highly effective component of curative breast cancer chemotherapy but are associated with substantial morbidity1,2. Because anthracyclines work in part by inhibiting topoisomerase-II (TOP2) on accessible DNA3,4, we hypothesized that chromatin regulatory genes (CRGs) that mediate DNA accessibility might predict anthracycline response. We studied the role of CRGs in anthracycline sensitivity in breast cancer through integrative analysis of patient and cell line data. We identified a consensus set of 38 CRGs associated with anthracycline response across ten cell line datasets. By evaluating the interaction between expression and treatment in predicting survival in a metacohort of 1006 patients with early-stage breast cancer, we identified 54 CRGs whose expression levels dictate anthracycline benefit across the clinical subgroups; of these CRGs, 12 overlapped with those identified in vitro. CRGs that promote DNA accessibility, including Trithorax complex members, were associated with anthracycline sensitivity when highly expressed, whereas CRGs that reduce accessibility, such as Polycomb complex proteins, were associated with decreased anthracycline sensitivity. We show that KDM4B modulates TOP2 accessibility to chromatin, elucidating a mechanism of TOP2 inhibitor sensitivity. These findings indicate that CRGs mediate anthracycline benefit by altering DNA accessibility, with implications for the stratification of patients with breast cancer and treatment decision making.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Cromatina/genética , DNA Topoisomerases Tipo II/genética , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Adulto , Idoso , Antraciclinas/administração & dosagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Humanos , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas do Grupo Polycomb/genética , Inibidores da Topoisomerase II/administração & dosagem
6.
Nat Genet ; 51(10): 1494-1505, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570894

RESUMO

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.


Assuntos
Linfócitos B/imunologia , Cromatina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Elementos de Resposta/genética , Linfócitos T/imunologia , Desequilíbrio Alélico , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/imunologia , Epigênese Genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Polissacarídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transcriptoma
7.
Nucleic Acids Res ; 47(16): 8502-8520, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616951

RESUMO

Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling enzyme with an emerging role in DNA damage response (DDR), but the underlying mechanism remains largely unknown. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1), a key chromatin-associated enzyme responsible for the synthesis of poly(ADP-ribose) (PAR) polymers in mammalian cells, interacts with and PARylates MORC2 at two residues within its conserved CW-type zinc finger domain. Following DNA damage, PARP1 recruits MORC2 to DNA damage sites and catalyzes MORC2 PARylation, which stimulates its ATPase and chromatin remodeling activities. Mutation of PARylation residues in MORC2 results in reduced cell survival after DNA damage. MORC2, in turn, stabilizes PARP1 through enhancing acetyltransferase NAT10-mediated acetylation of PARP1 at lysine 949, which blocks its ubiquitination at the same residue and subsequent degradation by E3 ubiquitin ligase CHFR. Consequently, depletion of MORC2 or expression of an acetylation-defective PARP1 mutant impairs DNA damage-induced PAR production and PAR-dependent recruitment of DNA repair proteins to DNA lesions, leading to enhanced sensitivity to genotoxic stress. Collectively, these findings uncover a previously unrecognized mechanistic link between MORC2 and PARP1 in the regulation of cellular response to DNA damage.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Reparo do DNA , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Acetilação/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dano ao DNA , Células HEK293 , Humanos , Mutação , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismo , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteólise/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
8.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470631

RESUMO

Metal cations are associated with many biological processes. The effects of these cations on nucleic acids and chromatin were extensively studied in the early stages of nucleic acid and chromatin research. The results revealed that some monovalent and divalent metal cations, including Mg2+, profoundly affect the conformations and stabilities of nucleic acids, the folding of chromatin fibers, and the extent of chromosome condensation. Apart from these effects, there have only been a few reports on the functions of these cations. In 2007 and 2013, however, Mg2+-implicated novel phenomena were found: Mg2+ facilitates or enables both self-assembly of identical double-stranded (ds) DNA molecules and self-assembly of identical nucleosomes in vitro. These phenomena may be deeply implicated in the heterochromatin domain formation and chromatin-based phase separation. Furthermore, a recent study showed that elevation of the intranuclear Mg2+ concentration causes unusual differentiation of mouse ES (embryonic stem) cells. All of these phenomena seem to be closely related to one another. Mg2+ seems to be a key regulator of chromatin dynamics and chromatin-based biological processes.


Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Magnésio/farmacologia , Nucleossomos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Cromatina/química , Cromatina/metabolismo , Humanos , Magnésio/metabolismo , Camundongos , Conformação Molecular/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Nucleossomos/metabolismo
9.
Pharmacol Rev ; 71(4): 520-538, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31530573

RESUMO

Chromosome conformation capture methods have revealed the dynamics of genome architecture which is spatially organized into topologically associated domains, with gene regulation mediated by enhancer-promoter pairs in chromatin space. New evidence shows that endogenous hormones and several xenobiotics act within circumscribed topological domains of the spatial genome, impacting subsets of the chromatin contacts of enhancer-gene promoter pairs in cis and trans Results from the National Institutes of Health-funded PsychENCODE project and the study of chromatin remodeling complexes have converged to provide a clearer understanding of the organization of the neurogenic epigenome in humans. Neuropsychiatric diseases, including schizophrenia, bipolar spectrum disorder, autism spectrum disorder, attention deficit hyperactivity disorder, and other neuropsychiatric disorders are significantly associated with mutations in neurogenic transcriptional networks. In this review, we have reanalyzed the results from publications of the PsychENCODE Consortium using pharmacoinformatics network analysis to better understand druggable targets that control neurogenic transcriptional networks. We found that valproic acid and other psychotropic drugs directly alter these networks, including chromatin remodeling complexes, transcription factors, and other epigenetic modifiers. We envision a new generation of CNS therapeutics targeted at neurogenic transcriptional control networks, including druggable parts of chromatin remodeling complexes and master transcription factor-controlled pharmacogenomic networks. This may provide a route to the modification of interconnected gene pathways impacted by disease in patients with neuropsychiatric and neurodegenerative disorders. Direct and indirect therapeutic strategies to modify the master regulators of neurogenic transcriptional control networks may ultimately help extend the life span of CNS neurons impacted by disease.


Assuntos
Redes Reguladoras de Genes/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/fisiologia , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Genoma Humano/efeitos dos fármacos , Humanos , Receptores de Neurotransmissores/agonistas , Receptores de Neurotransmissores/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
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
11.
Theriogenology ; 138: 77-83, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31302434

RESUMO

This study evaluated the effect of leptin on the in vitro culture of isolated sheep early antral follicles. Early antral follicles (300-450 µm) were isolated and cultured for 12 days in tissue culture medium 199 (TCM 199) supplemented with glutamine, hypoxanthine, transferrin, insulin, selenium, ascorbic acid, bovine serum albumin (BSA) and recombinant follicle stimulating hormone (rFSH) (TCM 199+: control medium) or TCM 199+ supplemented with 2 or 10 ng/mL leptin. After culture, oocytes were subjected to in vitro maturation (IVM). The parameters analyzed were morphology, extrusion rate, follicular diameter, growth and fully-grown oocytes (oocytes ≥110 µm) rates. After IVM, reactive oxygen species (ROS) levels, mitochondrial activity, meiotic stages and meiotic resumption rates were also analyzed. After 12 days of culture, the concentration of 2 ng/mL of leptin showed a higher percentage of morphologically normal follicles, fully-grown oocytes (≥110 µm), active mitochondria and meiotic resumption compared to the control medium (TCM 199+; P < 0.05) but did not differ when compared to leptin concentration of 10 ng/mL (P > 0.05). After culturing, no significant differences existed among treatments in terms of the follicle diameter and ROS levels. In conclusion, the addition of 2 ng/mL leptin to the base culture medium is capable of improving follicular survival, oocyte growth, mitochondrial activity and meiotic resumption after the in vitro culture of isolated sheep early antral follicles.


Assuntos
Leptina/farmacologia , Mitocôndrias/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Animais , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Mitocôndrias/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Folículo Ovariano/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ovinos
12.
Biosci Biotechnol Biochem ; 83(10): 1912-1923, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31187696

RESUMO

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G2/M phase arrest. CTD increased the production of reactive oxygen species and Ca2+, and elevated the activities of caspase-3 and -9, but decreased the level of mitochondrial membrane potential. Furthermore, CTD increased the ROS- and ER stress-associated protein expressions and increased the levels of pro-apoptosis-associated proteins, but decreased that of anti-apoptosis-associated proteins. Based on these observations, we suggested that CTD decreased cell number through G2/M phase arrest and the induction of cell apoptosis in U-2 OS cells and CTD could be a potential candidate for osteosarcoma treatments.


Assuntos
Apoptose/efeitos dos fármacos , Cantaridina/farmacologia , Divisão Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Osteossarcoma/patologia , Cálcio/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Dano ao DNA , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Osteossarcoma/enzimologia , Osteossarcoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
Andrologia ; 51(8): e13313, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31179568

RESUMO

Reproductive dysfunction is one of the diabetes complications. Resveratrol, a polyphenol compound, shows antidiabetic and antioxidant effects. The aim of the present study was to investigate the protective effects of resveratrol on sperm parameters and chromatin quality in experimentally induced type 2 diabetes by streptozotocin and nicotinamide. Forty male adult Wistar rats were grouped into normal control, diabetic control and resveratrol-treated diabetic groups (1, 5 and 10 mg/kg orally treated for 30 days). Type 2 diabetes was induced using a single dose of streptozotocin and nicotinamide by intraperitoneal injection. Then, the different parameters and chromatin condensation of the epididymal extracted spermatozoon were studied using aniline blue (AB), acridine orange (AO) and toluidine blue (TB) staining. The sperm parameters including count, motility and viability had significant reduction in diabetic rats (p < 0.05). Resveratrol increased count, motility and viable spermatozoa relative to the diabetic group (p < 0.05). The mean percentage of AB, AO and TB staining positive spermatozoa was increased in diabetic groups compared to control (p < 0.001) and decreased after treatment with 1 and 5 mg/kg resveratrol (p < 0.001). The results of AO and TB staining showed that resveratrol did not have any beneficial effect on chromatin condensation and denatured DNA at the dose of 10 mg/kg.


Assuntos
Antioxidantes/administração & dosagem , Diabetes Mellitus Tipo 2/complicações , Suplementos Nutricionais , Infertilidade Masculina/prevenção & controle , Resveratrol/administração & dosagem , Animais , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/induzido quimicamente , Relação Dose-Resposta a Droga , Humanos , Infertilidade Masculina/etiologia , Infertilidade Masculina/patologia , Masculino , Niacinamida/toxicidade , Ratos , Ratos Wistar , Contagem de Espermatozoides , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/patologia , Estreptozocina/toxicidade , Resultado do Tratamento
14.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180045

RESUMO

Recent research has shown that cell-free chromatin (cfCh) particles that are released from the billions of cells that die in the body everyday can enter into healthy cells, integrate into their genomes and induce dsDNA breaks and apoptotic responses. Genomic integration of cfCh activates NF κ B suggesting a novel mechanism of induction of systemic inflammation. Since DNA damage and inflammation are underlying pathologies in multiple devastating acute and chronic disease conditions, the discovery of agents that can inactivate cfCh may provide therapeutic possibilities.


Assuntos
Anti-Inflamatórios/farmacologia , Cromatina/patologia , Histonas/genética , NF-kappa B/genética , Animais , Anticorpos Neutralizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Transporte Biológico , Cromatina/química , Cromatina/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Desoxirribonuclease I/farmacologia , Espaço Extracelular/química , Regulação da Expressão Gênica , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Inflamação/prevenção & controle , Células Jurkat/transplante , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Resveratrol/análogos & derivados , Resveratrol/farmacologia , Transdução de Sinais
15.
Nucleic Acids Res ; 47(15): 7914-7928, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31216030

RESUMO

SIRT6 is critical for activating transcription of Nuclear factor (erythroid-derived 2)-like 2 (NRF2) responsive genes during oxidative stress. However, while the mechanism of SIRT6-mediated silencing is well understood, the mechanism of SIRT6-mediated transcriptional activation is unknown. Here, we employed SIRT6 separation of function mutants to reveal that SIRT6 mono-ADP-ribosylation activity is required for transcriptional activation. We demonstrate that SIRT6 mono-ADP-ribosylation of BAF170, a subunit of BAF chromatin remodeling complex, is critical for activation of a subset of NRF2 responsive genes upon oxidative stress. We show that SIRT6 recruits BAF170 to enhancer region of the Heme oxygenase-1 locus and promotes recruitment of RNA polymerase II. Furthermore, SIRT6 mediates the formation of the active chromatin 10-kb loop at the HO-1 locus, which is absent in SIRT6 deficient tissue. These results provide a novel mechanism for SIRT6-mediated transcriptional activation, where SIRT6 mono-ADP-ribosylates and recruits chromatin remodeling proteins to mediate the formation of active chromatin loop.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Heme Oxigenase-1/genética , Proteínas de Membrana/genética , Fator 2 Relacionado a NF-E2/genética , Sirtuínas/genética , Transcrição Genética , ADP-Ribosilação , Animais , Linhagem Celular , Cromatina/química , Cromatina/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos , Elementos Facilitadores Genéticos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Paraquat/farmacologia , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transdução de Sinais , Sirtuínas/deficiência
16.
J Exp Clin Cancer Res ; 38(1): 203, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101118

RESUMO

BACKGROUND: The VRK1 chromatin kinase regulates the organization of locally altered chromatin induced by DNA damage. The combination of ionizing radiation with inhibitors of DNA damage responses increases the accumulation of DNA damage in cancer cells, which facilitates their antitumor effect, a process regulated by VRK1. METHODS: Tumor cell lines with different genetic backgrounds were treated with olaparib to determine their effect on the activation of DNA repair pathways induced by ionizing radiation. The effect of combining olaparib with depletion of the chromatin kinase VRK1 was studied in the context of double-strand breaks repair pathway after treatment with ionizing radiation. The initiation and progression of DDR were studied by specific histone acetylation, as a marker of local chromatin relaxation, and formation of γH2AX and 53BP1 foci. RESULTS: In this work, we have studied the effect that VRK1 by itself or in collaboration with olaparib, an inhibitor of PARP, has on the DNA oxidative damage induced by irradiation in order to identify its potential as a new drug target. The combination of olaparib and ionizing radiation increases DNA damage permitting a significant reduction of their respective doses to achieve a similar amount of DNA damage detected by γH2AX and 53BP1 foci. Different treatment combinations of olaparib and ionizing radiation permitted to reach the maximum level of DNA damage at lower doses of both treatments. Furthermore, we have studied the effect that depletion of the VRK1 chromatin kinase, a regulator of DDR, has on this response. VRK1 knockdown impaired all steps in the DDR induced by these treatments, which were detected by a reduction of sequential markers such as H4K16 ac, γH2AX, NBS1 and 53BP1. Moreover, this effect of VRK1 is independent of TP53 or ATM, two genes frequently mutated in cancer. CONCLUSION: The protective DNA damage response induced by ionizing radiation is impaired by the combination of olaparib with depletion of VRK1, and can be used to reduce doses of radiation and their associated toxicity. Proteins implicated in DNA damage responses are suitable targets for development of new therapeutic strategies and their combination can be an alternative form of synthetic lethality.


Assuntos
Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Células A549 , Acetilação/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/radioterapia , Fosforilação/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Interferência de RNA , Radiação Ionizante , Mutações Sintéticas Letais/genética
17.
Nat Commun ; 10(1): 1935, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028249

RESUMO

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.


Assuntos
Regulação da Expressão Gênica , Leucemia Mielomonocítica Crônica/genética , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Corantes Fluorescentes/química , Edição de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Maleimidas/química , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais , Células THP-1 , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo
18.
Nat Commun ; 10(1): 1897, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015486

RESUMO

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Cromatina/química , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Animais , Butadienos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nitrilos/farmacologia , Piperazinas/farmacologia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
19.
Basic Clin Pharmacol Toxicol ; 125 Suppl 3: 44-57, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30801972

RESUMO

Obesity and associated disorders are now a global pandemic. The prevailing clinical model for obesity is overconsumption of calorie-dense food and diminished physical activity (the calories in-calories out model). However, this explanation does not account for numerous recent research findings demonstrating that a variety of environmental factors can be superimposed on diet and exercise to influence the development of obesity. The environmental obesogen model proposes that exposure to chemical obesogens during in utero and/or early life can strongly influence later predisposition to obesity. Obesogens are chemicals that inappropriately stimulate adipogenesis and fat storage, in vivo either directly or indirectly. Numerous obesogens have been identified in recent years and some of these elicit transgenerational effects on obesity as well as a variety of health end-points after exposure of pregnant F0 females. Prenatal exposure to environmental obesogens can produce lasting effects on the exposed animals and their offspring to at least the F4 generation. Recent results show that some of these transgenerational effects of obesogen exposure can be carried across the generations via alterations in chromatin structure and accessibility. That some chemicals can have permanent effects on the offspring of exposed animals suggests increased caution in the debate about whether and to what extent exposure to endocrine-disrupting chemicals and obesogens should be regulated.


Assuntos
Adipogenia/genética , Disruptores Endócrinos/efeitos adversos , Poluentes Ambientais/efeitos adversos , Obesidade/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Cromatina/efeitos dos fármacos , Cromatina/genética , Exposição Ambiental/efeitos adversos , Exposição Ambiental/legislação & jurisprudência , Exposição Ambiental/prevenção & controle , Política Ambiental/legislação & jurisprudência , Poluição Ambiental/efeitos adversos , Poluição Ambiental/legislação & jurisprudência , Poluição Ambiental/prevenção & controle , Epigênese Genética/efeitos dos fármacos , Feminino , Humanos , Obesidade/induzido quimicamente , Obesidade/epidemiologia , Pandemias/prevenção & controle , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/epidemiologia
20.
Nucleic Acids Res ; 47(8): 4068-4085, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799487

RESUMO

DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.


Assuntos
Cromatina/metabolismo , Genoma Humano , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Reparo de DNA por Recombinação , Proteína de Replicação A/genética , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína de Replicação A/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
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