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1.
Einstein (Sao Paulo) ; 17(4): eAO4742, 2019 Sep 09.
Artigo em Inglês, Português | MEDLINE | ID: mdl-31508660

RESUMO

OBJECTIVE: To evaluate the induction of DNA damage in peripheral blood mononuclear cells of patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea. METHODS: The study subjects were divided into two groups: one group of 22 patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea, and a Control Group composed of 24 patients with sickle cell disease who were not treated with hydroxyurea. Peripheral blood samples were submitted to peripheral blood mononuclear cell isolation to assess genotoxicity by the cytokinesis-block micronucleus cytome assay, in which DNA damage biomarkers - micronuclei, nucleoplasmic bridges and nuclear buds - were counted. RESULTS: Patients with sickle cell disease treated with hydroxyurea had a mean age of 25.4 years, whereas patients with sickle cell disease not treated with hydroxyurea had a mean age of 17.6 years. The mean dose of hydroxyurea used by the patients was 12.8mg/kg/day, for a mean period of 44 months. The mean micronucleus frequency per 1,000 cells of 8.591±1.568 was observed in the Hydroxyurea Group and 10.040±1.003 in the Control Group. The mean frequency of nucleoplasmic bridges per 1,000 cells and nuclear buds per 1,000 cells for the hydroxyurea and Control Groups were 0.4545±0.1707 versus 0.5833±0.2078, and 0.8182±0.2430 versus 0.9583±0.1853, respectively. There was no statistically significant difference between groups. CONCLUSION: In the study population, patients with sickle cell disease treated with the standard dose of hydroxyurea treatment did not show evidence of DNA damage induction.


Assuntos
Anemia Falciforme/genética , Dano ao DNA/efeitos dos fármacos , Hidroxiureia/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Criança , Pré-Escolar , Citocinese , Dano ao DNA/genética , Feminino , Humanos , Hidroxiureia/efeitos adversos , Hidroxiureia/uso terapêutico , Masculino , Testes para Micronúcleos , Pessoa de Meia-Idade , Testes de Mutagenicidade , Mutação/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/efeitos adversos , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , Adulto Jovem
2.
Exp Parasitol ; 204: 107727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31344389

RESUMO

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Citocinese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Trypanosoma rangeli/citologia , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Citocinese/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Citometria de Fluxo , Imunofluorescência , Hidroxiureia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Fatores de Tempo , Trypanosoma rangeli/efeitos dos fármacos , Trypanosoma rangeli/enzimologia , Trypanosoma rangeli/genética , Tripanossomíase/parasitologia
3.
Future Microbiol ; 14: 175-184, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30644320

RESUMO

Cytomegalovirus (CMV) manifestations remain important complications after allogeneic hematopoietic stem cell transplantation (HSCT), even in the current era. Unfortunately, available anti-CMV agents, mainly viral polymerase inhibitors, have a substantial risk of myelosuppression and nephrotoxicity. Letermovir, a novel anti-CMV drug that targets the viral terminase complex, has recently been approved for the prevention of clinically significant CMV infection in adult CMV seropositive hematopoietic stem cell transplantation recipients. This molecule could become a paradigm-shifting drug in preventing CMV manifestations based on its novel mechanism of action, lack of cross-resistance with available drugs, proven efficacy in a large randomized clinical trial, and its beneficial toxicity and tolerability profile. Drug-drug interactions and the lack of any activity against other viruses are the main shortcomings of letermovir.


Assuntos
Acetatos/farmacologia , Antibioticoprofilaxia , Antivirais/farmacologia , Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Quinazolinas/farmacologia , Acetatos/efeitos adversos , Acetatos/uso terapêutico , Adulto , Antivirais/efeitos adversos , Antivirais/uso terapêutico , Citomegalovirus/patogenicidade , Avaliação de Medicamentos , Interações de Medicamentos , Farmacorresistência Viral , Endodesoxirribonucleases/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Quinazolinas/efeitos adversos , Quinazolinas/uso terapêutico , Transplantados , Resultado do Tratamento
4.
Future Med Chem ; 11(2): 137-154, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30648904

RESUMO

Acyclic nucleoside phosphonates represent a well-defined class of clinically used nucleoside analogs. All acyclic nucleoside phosphonates need intracellular phosphorylation before they can bind viral DNA polymerases. Recently, a novel class of alpha-carboxynucleoside phosphonates have been designed to mimic the natural 2'-deoxynucleotide 5'-triphosphate substrates of DNA polymerases. They contain a carboxyl group in the phosphonate moiety linked to the nucleobase through a cyclic or acyclic bridge. Alpha-carboxynucleoside phosphonates act as viral DNA polymerase inhibitors without any prior requirement of metabolic conversion. Selective inhibitory activity against retroviral reverse transcriptase and herpesvirus DNA polymerases have been demonstrated. These compounds have a unique mechanism of inhibition of viral DNA polymerases, and provide possibilities for further modifications to optimize and fine tune their antiviral DNA polymerase spectrum.


Assuntos
Antivirais/química , Antivirais/farmacologia , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleosídeos/análogos & derivados , Nucleosídeos/farmacologia , Organofosfonatos/química , Organofosfonatos/farmacologia , Animais , DNA Polimerase Dirigida por DNA , Descoberta de Drogas , Exodesoxirribonucleases/antagonistas & inibidores , Herpes Simples/tratamento farmacológico , Humanos , Modelos Moleculares , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Proteínas Virais/antagonistas & inibidores , Viroses/tratamento farmacológico , Vírus/efeitos dos fármacos , Vírus/enzimologia
5.
Viruses ; 11(1)2018 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-30583530

RESUMO

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.


Assuntos
Genoma Viral , Prófagos/imunologia , Streptococcus thermophilus/imunologia , Streptococcus thermophilus/virologia , Integração Viral , Genoma Bacteriano/genética , Lipoproteínas/genética , Mitomicina/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA não Traduzido/genética , Streptococcus thermophilus/efeitos dos fármacos
6.
World J Gastroenterol ; 24(34): 3834-3848, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-30228778

RESUMO

Colorectal cancer (CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo- and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibody-drug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Imunoconjugados/farmacologia , Evasão Tumoral/efeitos dos fármacos , Alfa-Amanitina/farmacologia , Alfa-Amanitina/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Receptores Coestimuladores e Inibidores de Linfócitos T/antagonistas & inibidores , Receptores Coestimuladores e Inibidores de Linfócitos T/imunologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Imunoconjugados/uso terapêutico , Imunoterapia/métodos , Instabilidade de Microssatélites/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/uso terapêutico , RNA Polimerase II/antagonistas & inibidores , Resultado do Tratamento , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/genética
7.
PLoS Pathog ; 14(5): e1007070, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29813138

RESUMO

Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results in demethylated DNA, topological changes, removal of proteins and recruitment of proteins to promoters. One or more of these effects of DNA replication may facilitate late gene transcription. Using 5-azacytidine to promote demethylation of DNA, we demonstrate that late gene transcription cannot be rescued by DNA demethylation. Late gene transcription precedes significant increases in DNA copy number, indicating that increased template numbers also do not contribute to the linkage between replication and late gene transcription. By using serial, timed blockade of DNA replication and measurement of late gene mRNA accumulation, we demonstrate that late gene transcription requires ongoing DNA replication. Consistent with these findings, blocking DNA replication led to dissolution of DNA replication complexes which also contain RNA polymerase II and BGLF4, an EBV protein required for transcription of several late genes. These data indicate that ongoing DNA replication maintains integrity of a replication-transcription complex which is required for recruitment and retention of factors necessary for late gene transcription.


Assuntos
Replicação do DNA/fisiologia , Gammaherpesvirinae/genética , Transcrição Genética/fisiologia , Replicação Viral/fisiologia , Azacitidina/farmacologia , Desmetilação do DNA , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Gammaherpesvirinae/fisiologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Precoces , Cinética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ácido Fosfonoacéticos/farmacologia , Regiões Promotoras Genéticas/genética
8.
Molecules ; 23(1)2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29320423

RESUMO

In this study, novel N'-(3-cyclohexyl/phenyl-4-(substituted phenyl)thiazole-2(3H)-ylidene)-2-[(5,6,7,8-tetrahydronaphthalen-2-yl)oxy]acetohydrazide (4a-4k) derivatives were synthesized and their anticancer potency were evaluated on human breast adenocarcinoma cell line (MCF-7), human lung carcinoma cell line (A549) and mouse embryoblast cell line (NIH/3T3) using the MTT method, DNA synthesis inhibition and flow cytometric analysis. Compound 4e bearing 4-methoxyphenyl moiety exhibited the highest antitumor efficiency against MCF-7 cell line with higher DNA synthesis inhibition and apoptotic cell percentages (ealy+late apoptotic cell). On the other hand, compounds 4f, 4g, and 4h bearing 4-bromo, 4-chloro and 4-florophenyl moieties, respectively caused excellent apoptosis levels against A549 cell line when treated with lower concentration even than cisplatin. Anticholinesterase activity of the compounds were also tested, compound 4h showed 49.92% inhibition of acetylcholinesterase (AChE).


Assuntos
Antineoplásicos/química , Hidrazinas/química , Triazóis/química , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose , Sobrevivência Celular , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Cisplatino/química , Cisplatino/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Hidrazinas/farmacologia , Células MCF-7 , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Camundongos , Estrutura Molecular , Células NIH 3T3 , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Relação Estrutura-Atividade , Triazóis/farmacologia
9.
J Cell Physiol ; 233(3): 2170-2182, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28407293

RESUMO

Natural products have gained a wide popularity as chemopreventive and anti-cancer agents owing to their multi-mechanistic mode of action, availability and synergism with several conventional chemotherapeutic agents. Crocetin is a carotenoid compound isolated from the stigma of Crocus sativus L. (saffron). Crocetin has shown promising effects as an anti-tumor agent in animal models and cell culture systems. Crocetin retards the growth of cancer cells via inhibiting nucleic acid synthesis, enhancing anti-oxidative system, and inducing apoptosis and differentiation pathways. The present review outlines natural sources of crocetin, and its pharmacokinetic and pharmacological properties relevant to the prevention and treatment of cancer. Also, we discuss molecular targets underlying the putative anti-tumor effects of crocetin.


Assuntos
Anticarcinógenos/farmacologia , Antineoplásicos/farmacologia , Carotenoides/farmacologia , Quimioprevenção/métodos , Neoplasias/tratamento farmacológico , Inibidores da Síntese de Ácido Nucleico/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Crocus/química , Humanos , Neoplasias/prevenção & controle
10.
Respir Med ; 133: 6-11, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29173450

RESUMO

INTRODUCTION: The discrepancy rates of drug susceptibility testing (DST) results between solid and liquid media have been reported to range from 2.4 to 7.4% for isoniazid. Most isolate with isoniazid DST discrepancies between solid and liquid media test as susceptible on solid medium and resistant in liquid medium, however, the optimal management of patients with discordant testing is unknown. This study was conducted to evaluate the effect of treatment regimen on treatment outcomes when patients with rifampicin-susceptible pulmonary tuberculosis have isoniazid resistance (INH-R) in liquid medium but isoniazid susceptibility (INH-S) on solid medium. METHOD: This study was retrospectively conducted by reviewing patient medical records on the liquid compared to solid culture based phenotypic testing at Samsung Medical Center between January 2009 and December 2015. The study population which have INH-R in liquid medium and INH-S on solid medium was divided into two groups: group A (n = 30), which included patients treated for INH-S tuberculosis by discontinuing pyrazinamide (and ethambutol), and group B (n = 56), which included patients treated for INH-R tuberculosis by continuing pyrazinamide and/or adding fluoroquinolone. Unfavorable outcomes included treatment failure and relapse. RESULTS: There were no statistically significant differences between the two groups including demographic data, comorbidities, radiologic data, and treatment duration. However, baseline smear positive rates were more frequent in group A (19/30, 63.3%) than in group B (22/56, 39.3%; P = 0.033). Only three patients had unfavorable outcomes; one was bacteriologically proven treatment failure and the other two were clinically judged as unfavorable outcomes. All of them were in the group A (3/30, 10%); no unfavorable outcomes occurred in the group B (0/56, 0%; P = 0.040). CONCLUSIONS: Unfavorable outcomes were less frequent in the group B than in the group A, indicating that treatment regimen modification according to DST results on liquid medium could improve treatment outcomes in patients with rifampicin-susceptible pulmonary tuberculosis. Further studies are required to confirm these findings to overcome the small number of unfavorable outcomes.


Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Pulmonar/tratamento farmacológico , Idoso , Antituberculosos/farmacologia , Quimioterapia Combinada/métodos , Etambutol/farmacologia , Feminino , Fluoroquinolonas/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Síntese de Ácido Nucleico/farmacologia , Pirazinamida/farmacologia , República da Coreia/epidemiologia , Estudos Retrospectivos , Rifampina/farmacologia , Falha de Tratamento , Resultado do Tratamento , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
11.
Nucleic Acids Res ; 45(18): 10783-10799, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28985404

RESUMO

In Chlamydomonas reinhardtii, regulation of chloroplast gene expression is mainly post-transcriptional. It requires nucleus-encoded trans-acting protein factors for maturation/stabilization (M factors) or translation (T factors) of specific target mRNAs. We used long- and small-RNA sequencing to generate a detailed map of the transcriptome. Clusters of sRNAs marked the 5' end of all mature mRNAs. Their absence in M-factor mutants reflects the protection of transcript 5' end by the cognate factor. Enzymatic removal of 5'-triphosphates allowed identifying those cosRNA that mark a transcription start site. We detected another class of sRNAs derived from low abundance transcripts, antisense to mRNAs. The formation of antisense sRNAs required the presence of the complementary mRNA and was stimulated when translation was inhibited by chloramphenicol or lincomycin. We propose that they derive from degradation of double-stranded RNAs generated by pairing of antisense and sense transcripts, a process normally hindered by the traveling of the ribosomes. In addition, chloramphenicol treatment, by freezing ribosomes on the mRNA, caused the accumulation of 32-34 nt ribosome-protected fragments. Using this 'in vivo ribosome footprinting', we identified the function and molecular target of two candidate trans-acting factors.


Assuntos
Chlamydomonas reinhardtii/genética , RNA de Cloroplastos/metabolismo , Pequeno RNA não Traduzido/metabolismo , Transcriptoma , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Perfilação da Expressão Gênica , Inibidores da Síntese de Ácido Nucleico/farmacologia , Processos Fototróficos , Proteínas de Plantas/metabolismo , Biossíntese de Proteínas , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Análise de Sequência de RNA , Transcrição Genética/efeitos dos fármacos
12.
J Neurosci ; 37(40): 9675-9685, 2017 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-28887385

RESUMO

Reactivated memories can be modified during reconsolidation, making this process a potential therapeutic target for posttraumatic stress disorder (PTSD), a mental illness characterized by the recurring avoidance of situations that evoke trauma-related fears. However, avoidance memory reconsolidation depends on a set of still loosely defined boundary conditions, limiting the translational value of basic research. In particular, the involvement of the hippocampus in fear-motivated avoidance memory reconsolidation remains controversial. Combining behavioral and electrophysiological analyses in male Wistar rats, we found that previous learning of relevant nonaversive information is essential to elicit the participation of the hippocampus in avoidance memory reconsolidation, which is associated with an increase in theta- and gamma-oscillation power and cross-frequency coupling in dorsal CA1 during reactivation of the avoidance response. Our results indicate that the hippocampus is involved in memory reconsolidation only when reactivation results in contradictory representations regarding the consequences of avoidance and suggest that robust nesting of hippocampal theta-gamma rhythms at the time of retrieval is a specific reconsolidation marker.SIGNIFICANCE STATEMENT Posttraumatic stress disorder (PTSD) is characterized by maladaptive avoidance responses to stimuli or behaviors that represent or bear resemblance to some aspect of a traumatic experience. Disruption of reconsolidation, the process by which reactivated memories become susceptible to modifications, is a promising approach for treating PTSD patients. However, much of what is known about fear-motivated avoidance memory reconsolidation derives from studies based on fear conditioning instead of avoidance-learning paradigms. Using a step-down inhibitory avoidance task in rats, we found that the hippocampus is involved in memory reconsolidation only when the animals acquired the avoidance response in an environment that they had previously learned as safe and showed that increased theta- and gamma-oscillation coupling during reactivation is an electrophysiological signature of this process.


Assuntos
Aprendizagem da Esquiva/fisiologia , Hipocampo/fisiologia , Consolidação da Memória/fisiologia , Alfa-Amanitina/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Ondas Encefálicas/efeitos dos fármacos , Ondas Encefálicas/fisiologia , Hipocampo/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Aprendizagem/fisiologia , Masculino , Consolidação da Memória/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ratos , Ratos Wistar
13.
Peptides ; 97: 1-7, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28917652

RESUMO

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.


Assuntos
Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Hormônio do Crescimento/genética , Hormônios Peptídicos/metabolismo , Hipófise/metabolismo , Tilápia/fisiologia , Animais , Dactinomicina/farmacologia , Masculino , Inibidores da Síntese de Ácido Nucleico/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Hipófise/citologia , Cultura Primária de Células , RNA Mensageiro/antagonistas & inibidores , Tilápia/genética
15.
G3 (Bethesda) ; 7(10): 3269-3279, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28818866

RESUMO

To ensure genomic integrity, living organisms have evolved diverse molecular processes for sensing and repairing damaged DNA. If improperly repaired, DNA damage can give rise to different types of mutations, an important class of which are genomic structural variants (SVs). In spite of their importance for phenotypic variation and genome evolution, potential contributors to SV formation in Saccharomyces cerevisiae (budding yeast), a highly tractable model organism, are not fully recognized. Here, we developed and applied a genome-wide assay to identify yeast gene knockout mutants associated with de novo deletion formation, in particular single-strand annealing (SSA)-mediated deletion formation, in a systematic manner. In addition to genes previously linked to genome instability, our approach implicates novel genes involved in chromatin remodeling and meiosis in affecting the rate of SSA-mediated deletion formation in the presence or absence of stress conditions induced by DNA-damaging agents. We closely examined two candidate genes, the chromatin remodeling gene IOC4 and the meiosis-related gene MSH4, which when knocked-out resulted in gene expression alterations affecting genes involved in cell division and chromosome organization, as well as DNA repair and recombination, respectively. Our high-throughput approach facilitates the systematic identification of processes linked to the formation of a major class of genetic variation.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Camptotecina/farmacologia , Dano ao DNA , Reparo do DNA , DNA Fúngico/genética , DNA de Cadeia Simples , Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Instabilidade Genômica , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Topoisomerase I/farmacologia , Inibidores da Topoisomerase II/farmacologia
16.
J Biol Chem ; 292(39): 16003-16013, 2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28827310

RESUMO

Germline stem cells are essential in the generation of both male and female gametes. In mammals, the male testis produces sperm throughout the entire lifetime, facilitated by testicular germline stem cells. Oocyte renewal ceases in postnatal or adult life in mammalian females, suggesting that germline stem cells are absent from the mammalian ovary. However, studies in mice, rats, and humans have recently provided evidence for ovarian female germline stem cells (FGSCs). A better understanding of the role of FGSCs in ovaries could help improve fertility treatments. Here, we developed a rapid and efficient method for isolating FGSCs from ovaries of neonatal mice. Notably, our FGSC isolation method could efficiently isolate on average 15 cell "strings" per ovary from mice at 1-3 days postpartum. FGSCs isolated from neonatal mice displayed the string-forming cell configuration at mitosis (i.e. a "stringing" FGSC (sFGSC) phenotype) and a disperse phenotype in postnatal mice. We also found that sFGSCs undergo vigorous mitosis especially at 1-3 days postpartum. After cell division, the sFGSC membranes tended to be connected to form sFGSCs. Moreover, F-actin filaments exhibited a cell-cortex distribution in sFGSCs, and E-cadherin converged in cell-cell connection regions, resulting in the string-forming morphology. Our new method provides a platform for isolating FGSCs from the neonatal ovary, and our findings indicate that FGCSs exhibit string-forming features in neonatal mice. The sFGSCs represent a valuable resource for analysis of ovary function and an in vitro model for future clinical use to address ovarian dysfunction.


Assuntos
Células-Tronco de Oogônios/citologia , Ovário/citologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Varredura , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oogênese/efeitos dos fármacos , Células-Tronco de Oogônios/efeitos dos fármacos , Células-Tronco de Oogônios/metabolismo , Células-Tronco de Oogônios/ultraestrutura , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Ovário/ultraestrutura , Interferência de RNA
17.
Biosci Rep ; 37(4)2017 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-28679648

RESUMO

The present study investigated the effects of microRNA-374 (miR-374) on human squamous cell carcinoma (SCC) cell proliferation, migration, invasion, and apoptosis through P53 signaling pathway by targeting growth arrest and DNA-damage-inducible protein 45 α (Gadd45a). Skin samples were collected from patients with skin SCC and normal skin samples. Expression of miR-374, Gadd45a, P53, P73, P16, c-myc, bcl-2, Bax, caspase-3, and caspase-9 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. A431 and SCL-1 cells were divided into blank, negative control (NC), miR-374 mimics, miR374 inhibitors, siRNA-Gadd45a, and miR-374 inhibitors + siRNA-Gadd45a groups. Their proliferation, migration, invasion, cell cycle, and apoptosis were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, scratch test, Transwell assay, and flow cytometry. SCC skin tissues exhibited decreased expression of miR-374, P73, P16, Bax caspase-3 and caspase-9, and increased levels of Gadd45a, P53, c-myc, and Bcl-2 compared with the normal skin tissues. The miR-374 inhibitors group exhibited decreased expression of miR-374, P73, P16, Bax caspase-3 and caspase-9, and increased expression of Gadd45a, P53, c-myc, and Bcl-2, enhanced cell proliferation, migration, and invasion, and reduced apoptosis compared with the blank and NC groups; the miR-374 mimics group followed opposite trends. Compared with the blank and NC groups, the miR-374 inhibitors + siRNA-Gadd45a group showed decreased miR-374 level; the siRNA-Gadd45a group showed elevated levels of P73, P16, Bax, caspase-3 and caspase-9, decreased levels of Gadd45a, P53, c-myc, and Bcl-2, reduced cell proliferation, migration, and invasion, and accelerated apoptosis. miR-374 induces apoptosis and inhibits proliferation, migration, and invasion of SCC cells through P53 signaling pathway by down-regulating Gadd45a.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Nucleares/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteína Supressora de Tumor p53/genética
18.
J Biol Chem ; 292(37): 15489-15500, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28743741

RESUMO

DNA replication greatly enhances expression of the herpes simplex virus 1 (HSV-1) γ2 late genes by still unknown mechanisms. Here, we demonstrate that 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK9, suppresses expression of γ2 late genes with an IC50 of 5 µm, which is at least 10 times lower than the IC50 value required for inhibition of expression of early genes. The effect of DRB could not be explained by inhibition of DNA replication per se or loading of RNA polymerase II to late promoters and subsequent reduction of transcription. Instead, DRB reduces accumulation of γ2 late mRNA in the cytoplasm. In addition, we show that siRNA-mediated knockdown of the transcription factor SPT5, but not NELF-E, also gives rise to a specific inhibition of HSV-1 late gene expression. Finally, addition of DRB reduces co-immunoprecipitation of ICP27 using an anti-SPT5 antibody. Our results suggest that efficient expression of replication-dependent γ2 late genes is, at least in part, regulated by CDK9 dependent co- and/or post-transcriptional events involving SPT5 and ICP27.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Replicação do DNA , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Replicação Viral , Substituição de Aminoácidos , Antivirais/farmacologia , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Replicação do DNA/efeitos dos fármacos , Diclororribofuranosilbenzimidazol/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/genética , Imunoprecipitação , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
19.
Exp Mol Med ; 49(7): e357, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28729638

RESUMO

Our study aims to investigate the roles that microRNA-214 (miR-214) plays in the epithelial mesenchymal transition (EMT) process and the development of interstitial cystitis (IC) in postmenopausal women by targeting Mitofusin 2 (Mfn2). IC bladder tissues and adjacent normal bladder tissues were collected from postmenopausal women. Immunohistochemistry (IHC) staining was conducted. The target relationship between miR-214 and Mfn2 was determined by a dual luciferase reporter gene assay. Adipose-derived mesenchymal stem cells (ADMSCs) were extracted from postmenopausal rats and assigned to the blank, mimics, miR-214 inhibitors, mimics negative control (NC), inhibitors NC, Mfn2 siRNA, miR-214 inhibitors and Mfn2 siRNA groups. Exosomes secreted by transfected ADMSCs were instilled into the bladders of postmenopausal rats. The expression of miR-214 and Mfn2 mRNA and EMT markers was assessed by qRT-PCR and western blotting. It was confirmed that Mfn2 was the target gene of miR-214 in IC. Compared with the normal bladder tissues, miR-214 decreased, but Mfn2 increased in IC bladder tissues. Compared with the blank group, the expression of miR-214 and the expression levels of N-cadherin, Fibronectin, Twist1, Snail and Vimentin mRNA and protein increased, whereas the expression levels of Mfn2, E-cadherin and ZO-1 mRNA and protein decreased in the miR-214 mimics and Mfn2 groups. The expression of MiR-214 and the expression levels of N-cadherin, Fibronectin, Twist1, Snail and Vimentin mRNA and protein decreased, whereas the expression levels of Mfn2, E-cadherin and ZO-1 mRNA and protein increased in the miR-214 inhibitors group. Our findings indicate that the inhibition of miR-214 promotes the EMT process and contributes to bladder wall fibrosis by up-regulating Mfn2, thus leading to the occurrence of IC in postmenopausal women.


Assuntos
Cistite Intersticial/metabolismo , Transição Epitelial-Mesenquimal , GTP Fosfo-Hidrolases/metabolismo , MicroRNAs/fisiologia , Proteínas Mitocondriais/metabolismo , Pós-Menopausa/metabolismo , Bexiga Urinária/patologia , Tecido Adiposo/citologia , Animais , Biomarcadores/análise , Cistite Intersticial/patologia , Modelos Animais de Doenças , Exossomos/metabolismo , Feminino , Fibrose , GTP Fosfo-Hidrolases/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/antagonistas & inibidores , Proteínas Mitocondriais/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima
20.
Learn Mem ; 24(8): 375-380, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28716957

RESUMO

Activity-regulated cytoskeleton-associated protein (Arc) supports fear memory through synaptic plasticity events requiring actin cytoskeleton rearrangements. We have previously shown that reducing hippocampal Arc levels through antisense knockdown leads to the premature extinction of contextual fear. Here we show that the AMPA receptor antagonist CNQX elevates hippocampal Arc levels during extinction and blocks extinction that can be rescued by reducing Arc. Increasing Arc levels with CNQX also overcomes the actin-destabilizing properties of cytochalasin D and promotes extinction. Therefore, extinction is dependent on AMPA-mediated reductions of Arc via a mechanism consistent with a role for Arc in stabilizing the actin cytoskeleton to constrain extinction.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Extinção Psicológica/fisiologia , Medo/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Citocalasina D/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Extinção Psicológica/efeitos dos fármacos , Medo/efeitos dos fármacos , Reação de Congelamento Cataléptica/efeitos dos fármacos , Reação de Congelamento Cataléptica/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Testes Neuropsicológicos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ratos , Receptores de AMPA/antagonistas & inibidores
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