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2.
Endocrinology ; 161(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31742329

RESUMO

Many neural sex differences are differences in the number of neurons of a particular phenotype. For example, male rodents have more calbindin-expressing neurons in the medial preoptic area (mPOA) and bed nucleus of the stria terminalis (BNST), and females have more neurons expressing estrogen receptor alpha (ERα) and kisspeptin in the ventromedial nucleus of the hypothalamus (VMH) and the anteroventral periventricular nucleus (AVPV), respectively. These sex differences depend on neonatal exposure to testosterone, but the underlying molecular mechanisms are unknown. DNA methylation is important for cell phenotype differentiation throughout the developing organism. We hypothesized that testosterone causes sex differences in neurochemical phenotype via changes in DNA methylation, and tested this by inhibiting DNA methylation neonatally in male and female mice, and in females given a masculinizing dose of testosterone. Neonatal testosterone treatment masculinized calbindin, ERα and kisspeptin cell number of females at weaning. Inhibiting DNA methylation with zebularine increased calbindin cell number only in control females, thus eliminating sex differences in calbindin in the mPOA and BNST. Zebularine also reduced the sex difference in ERα cell number in the VMH, in this case by increasing ERα neuron number in males and testosterone-treated females. In contrast, the neonatal inhibition of DNA methylation had no effect on kisspeptin cell number. We conclude that testosterone normally increases the number of calbindin cells and reduces ERα cells in males through orchestrated changes in DNA methylation, contributing to, or causing, the sex differences in both cell types.


Assuntos
Encéfalo/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Diferenciação Sexual/efeitos dos fármacos , Testosterona/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/metabolismo , Química Encefálica/efeitos dos fármacos , Calbindinas/metabolismo , Citidina/administração & dosagem , Citidina/análogos & derivados , Citidina/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Kisspeptinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Diferenciação Sexual/fisiologia , Fatores Sexuais , Testosterona/administração & dosagem
3.
Anal Bioanal Chem ; 411(27): 7221-7231, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31583449

RESUMO

DNA methylation is a typical epigenetic phenomenon. Numerous methods for detecting global DNA methylation levels have been developed, among which LC-MS/MS has emerged as an excellent method from the viewpoint of sensitivity, reproducibility, and cost. However, LC-MS/MS methods have limitations due to a lack of the stability and the standardization required for a laboratory assay. The present study aimed to establish a robust assay that guarantees highly accurate measurements of global DNA methylation levels. There are at least three facets of the developed method. The first is discovery of the solvent conditions to minimize sodium adducts. The second is improvement of separation of nucleosides by LC using the columns that had not been used in previous similar studies. The third is success in reduction of the uncertainty of the measurement results, which was achieved by the calibration using the ratio of mdC but not the absolute amount in the presence of internal standards. These facets represent the advantage over methods reported previously. Our developed method enables quantification of DNA methylation with a short time length (8 min) for one analysis as well as with the high reproducibility of measurements that is represented by the inter-day CV% being less than 5%. In addition, data obtained from measuring global DNA methylation levels in cultured cell lines, with or without pharmacological demethylation, support its use for biomedical research. This assay is expected to allow us to conduct initial screening of epigenetic alterations or aberration in a variety of cells.


Assuntos
Metilação de DNA , DNA/química , Espectrometria de Massas em Tandem/métodos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/economia , Cromatografia Líquida de Alta Pressão/métodos , Citidina/análogos & derivados , Citidina/análise , Citidina/genética , DNA/genética , Humanos , Espectrometria de Massas em Tandem/economia , Fatores de Tempo
4.
Org Biomol Chem ; 17(43): 9435-9441, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31603457

RESUMO

To restrict pathogens, in a normal human cell, APOBEC3 enzymes mutate cytosine to uracil in foreign single-stranded DNAs. However, in cancer cells, APOBEC3B (one of seven APOBEC3 enzymes) has been identified as the primary source of genetic mutations. As such, APOBEC3B promotes evolution and progression of cancers and leads to development of drug resistance in multiple cancers. As APOBEC3B is a non-essential protein, its inhibition can be used to suppress emergence of drug resistance in existing anti-cancer therapies. Because of the vital role of APOBEC3 enzymes in innate immunity, selective inhibitors targeting only APOBEC3B are required. Here, we use the discriminative properties of wild-type APOBEC3A, APOBEC3B and APOBEC3G to deaminate different cytosines in the CCC-recognition motif in order to best place the cytidine analogue 2'-deoxyzebularine (dZ) in the CCC-motif. Using several APOBEC3 variants that mimic deamination patterns of wild-type enzymes, we demonstrate that selective inhibition of APOBEC3B in preference to other APOBEC3 constructs is feasible for the dZCC motif. This work is an important step towards development of in vivo tools to inhibit APOBEC3 enzymes in living cells by using short, chemically modified oligonucleotides.


Assuntos
Citidina Desaminase/antagonistas & inibidores , Citidina/análogos & derivados , DNA de Cadeia Simples/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas/antagonistas & inibidores , Linhagem Celular , Citidina/química , Citidina/farmacologia , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Proteínas/metabolismo
5.
Anal Chim Acta ; 1081: 103-111, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446947

RESUMO

Both DNA cytosine methylation (5-methyl-2'-deoxycytidine, m5dC) and RNA cytosine methylation (5-methylcytidine, m5rC) are important epigenetic marks that play regulatory roles in diverse biological processes. m5dC and m5rC can be further oxidized by the ten-eleven translocation (TET) proteins to form 5-hydroxymethyl-2'-deoxycytidine (hm5dC) and 5-hydroxymethylcytidine (hm5rC), respectively. 2'-O-methyl-5-hydroxymethylcytidine (hm5rCm) was recently also identified as a second oxidative metabolite of m5rC in RNA. Previous studies showed that the dysregulation of cytidine modifications in both DNA and RNA are closely related to a variety of human diseases. These cytidine modifications are generally excreted from cell into urine. If these cytidine modifications exhibit specific features related to certain diseases, determination of the cytidine modifications in urine could be utilized as non-invasive diagnostic of diseases. Here, we established a solid-phase extraction in combination with liquid chromatography-mass spectrometry (LC-MS/MS) analysis for simultaneous detection of these cytidine modifications in human urine samples. The developed method enabled the distinct detection of these cytidine modifications. We reported, for the first time, the presence of hm5rCm in human urine. Furthermore, we found that compared to the healthy controls, the contents of hm5dC, hm5rC, and hm5rCm showed significant increases in urine samples of cancer patients, including lymphoma patients, gastric cancer patients, and esophageal cancer patients. This study indicates that the urinary hydroxylmethylation modifications of hm5dC, hm5rC, and hm5rCm may serve as potential indicator of cancers.


Assuntos
Cromatografia Líquida/métodos , Citidina/análogos & derivados , Citidina/urina , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA/química , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA/química
6.
Anticancer Res ; 39(7): 3609-3614, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262886

RESUMO

BACKGROUND/AIM: The novel cytidine analog RX-3117, which is activated by uridine-cytidine kinase 2 (UCK2), shows encouraging activity in pancreatic and bladder cancer Phase IIa studies. In this study we highlight the potential role of UCK2 as a biomarker for selecting patients for RX-3117 treatment. PATIENTS AND METHODS: The online genomics analysis and visualization platform, R2, developed by the Oncogenomics department at the AMC (Amsterdam, The Netherlands) was used for in silico UCK2-mRNA correlation with overall survival of pancreatic cancer patients, while UCK2 protein expression was evaluated by immunohistochemistry on pancreatic tumor formalin-fixed-paraffin-embedded sections from independent pancreatic cancer patients. mRNA expression was also determined for SUIT-2, PANC-1 and PDAC-3. Lastly, the drug sensitivity to RX-3117 was investigated using the Sulforhodamine-B cytotoxicity assay. RESULTS: The in silico data showed that a high UCK2-mRNA expression was correlated with a shorter overall survival in pancreatic cancer patients. Moreover, UCK2 protein expression was high in 21/25 patients, showing a significantly shorter mean. Overall Survival (8.4 versus 34.3 months, p=0.045). Sensitivity to RX-3117 varied between 0.6 and 11 µM. CONCLUSION: Pancreatic cancer cells are sensitive to pharmacologically achievable RX-3117 concentrations and UCK2 might be exploited as a biomarker for patient treatment selection.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Citidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Uridina Quinase/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Idoso , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Citidina/farmacologia , Feminino , Humanos , Masculino , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , RNA Mensageiro/metabolismo , Uridina Quinase/genética
7.
EBioMedicine ; 46: 317-329, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31303499

RESUMO

BACKGROUND: Most studies on regenerative medicine focus on cell-based therapies and transplantations. Small-molecule therapeutics, though proved effective in different medical conditions, have not been extensively investigated in regenerative research. It is known that healing potential decreases with development and developmental changes are driven by epigenetic mechanisms, which suggests epigenetic repression of regenerative capacity. METHODS: We applied zebularine, a nucleoside inhibitor of DNA methyltransferases, to stimulate the regenerative response in a model of ear pinna injury in mice. FINDINGS: We observed the regeneration of complex tissue that was manifested as improved ear hole repair in mice that received intraperitoneal injections of zebularine. Six weeks after injury, the mean hole area decreased by 83.2 ±â€¯9.4% in zebularine-treated and by 43.6 ±â€¯15.4% in control mice (p < 10-30). Combined delivery of zebularine and retinoic acid potentiated and accelerated this effect, resulting in complete ear hole closure within three weeks after injury. We found a decrease in DNA methylation and transcriptional activation of neurodevelopmental and pluripotency genes in the regenerating tissues. INTERPRETATION: This study is the first to demonstrate an effective induction of complex tissue regeneration in adult mammals using zebularine. We showed that the synergistic action of an epigenetic drug (zebularine) and a transcriptional activator (retinoic acid) could be effectively utilized to induce the regenerative response, thus delineating a novel pharmacological strategy for regeneration. The strategy was effective in the model of ear pinna regeneration in mice, but zebularine acts on different cell types, therefore, a similar approach can be tested in other tissues and organs.


Assuntos
Citidina/análogos & derivados , Epigênese Genética/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/genética , Animais , Biomarcadores , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG , Citidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Pavilhão Auricular/efeitos dos fármacos , Pavilhão Auricular/lesões , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Medicina Regenerativa , Tretinoína/farmacologia
8.
ACS Chem Biol ; 14(7): 1403-1409, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31243956

RESUMO

Post-transcriptional modifications to messenger RNAs (mRNAs) have the potential to alter the biological function of this important class of biomolecules. The study of mRNA modifications is a rapidly emerging field, and the full complement of chemical modifications in mRNAs is not yet established. We sought to identify and quantify the modifications present in yeast mRNAs using an ultra-high performance liquid chromatography tandem mass spectrometry method to detect 40 nucleoside variations in parallel. We observe six modified nucleosides with high confidence in highly purified mRNA samples (N7-methylguanosine, N6-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, N4-acetylcytidine, and 5-formylcytidine) and identify the yeast protein responsible for N4-acetylcytidine incorporation in mRNAs (Rra1). In addition, we find that mRNA modification levels change in response to heat shock, glucose starvation, and/or oxidative stress. This work expands the repertoire of potential chemical modifications in mRNAs and highlights the value of integrating mass spectrometry tools in the mRNA modification discovery and characterization pipeline.


Assuntos
Nucleosídeos/análise , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina/análogos & derivados , Adenosina/análise , Adenosina/metabolismo , Citidina/análogos & derivados , Citidina/análise , Citidina/metabolismo , Glucose/metabolismo , Guanosina/análogos & derivados , Guanosina/análise , Guanosina/metabolismo , Resposta ao Choque Térmico , Nucleosídeos/metabolismo , Estresse Oxidativo , RNA Fúngico/química , RNA Mensageiro/química , Saccharomyces cerevisiae/química
9.
ACS Chem Biol ; 14(7): 1418-1425, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31188562

RESUMO

RNA contains diverse modifications that exert important influences in a variety of cellular processes. So far more than 150 modifications have been identified in various RNA species, mainly in rRNA and tRNA. Recent research advances in RNA modifications have been sparked by the discovery of dynamic and reversible modifications in mRNA. Moving beyond the abundant tRNA and rRNA to mRNA is opening new directions in understanding RNA modification-mediated regulation of gene expression. Recently, it was reported that N3-methylcytidine (m3C) existed in mRNA of mammalian cells, and methyltransferase-like 8 (METTL8) was identified to be the writer enzyme of m3C. However, little is known about the eraser enzyme of m3C in mRNA. In the current study, we found that the AlkB homologue 1 (ALKBH1) was capable of demethylating m3C in mRNA of mammalian cells in vitro. Overexpression and knockdown of ALKBH1 in cultured human cells can induce decrease and increase of the level of m3C in mRNA, respectively, revealing the eraser enzyme property of ALKBH1 on m3C in mRNA. In addition, we observed significant decrease of the level of m3C in mRNA in hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues, which could be attributed to the increased expression of ALKBH1 as well as the decreased expression of METTL8 in HCC tissues. These results indicated that m3C in mRNA may play certain roles in tumorigenesis. Our study shed light on understanding the demethylation of m3C in mRNA.


Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Citidina/análogos & derivados , RNA Mensageiro/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Citidina/metabolismo , Desmetilação , Células HEK293 , Humanos , Neoplasias Hepáticas/metabolismo , Mamíferos
10.
Methods Enzymol ; 621: 31-51, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31128786

RESUMO

Posttranscriptional modifications of RNA represent an emerging class of regulatory elements in human biology. Improved methods for studying how these elements are controlled and where they occur has the potential to transform our understanding of gene expression in development and disease. Here we describe a chemical method for nucleotide resolution sequencing of N4-acetylcytidine (ac4C), a highly conserved modified nucleobase whose formation is catalyzed by the essential cytidine acetyltransferase enzyme NAT10. This approach enables the sensitive, PCR-amplifiable detection of individual ac4C sites from nanograms of unfractionated cellular RNA. The sensitive and quantitative nature of this assay provides a powerful tool to understand how cytidine acetylation is targeted, profile RNA acetyltransferase dynamics, and validate the sites and stoichiometry of ac4C in novel RNA species.


Assuntos
Citidina/análogos & derivados , RNA/química , Análise de Sequência de RNA/métodos , Acetilação , Animais , Linhagem Celular , Citidina/análise , Citidina/genética , Citidina/metabolismo , Humanos , Acetiltransferases N-Terminal/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA
11.
Pestic Biochem Physiol ; 156: 116-122, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31027570

RESUMO

Cucumber mosaic virus (CMV) is a plant virus with one of the largest host ranges, the widest distribution, and economic importance, and ningnanmycin (NNM) is a commercial antiviral agent. Studies have shown that NNM induces and promotes pathogenesis-related proteins in tobacco mosaic virus-inoculated tobacco. In the present study, the defense enzymes and the biochemical factors of CMV-inoculated tobacco treated with NNM were measured. The biochemical factors of CMV-inoculated tobacco leaves treated with NNM were analyzed. Results showed that the phenylalanine ammonia-lyase, peroxidase, polypheuoloxidase, and superoxide in the CMV-inoculated tobacco leaves treated with NNM were higher than those in non-treated tobacco leaves. Furthermore, NNM activated the oxidation-reduction process, metabolic process, and oxidoreductase activity in the CMV-infected tobacco.


Assuntos
Cucumovirus/patogenicidade , Citidina/análogos & derivados , Tabaco/metabolismo , Tabaco/virologia , Citidina/farmacologia , Oxirredução , Peroxidase , Fenilalanina Amônia-Liase/metabolismo , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Superóxidos/metabolismo
12.
Neurobiol Learn Mem ; 161: 72-82, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30930287

RESUMO

Beta-adrenergic receptor (b-AR) activation by noradrenaline (NA) enhances memory formation and long-term potentiation (LTP), a form of synaptic plasticity characterized by an activity-dependent increase in synaptic strength. LTP is believed to be a cellular mechanism for contextual learning and memory. In the mammalian hippocampus, LTP can be observed at multiple synaptic pathways after strong stimulation of a single synaptic pathway. This heterosynaptic LTP is believed to involve synaptic tagging of active synapses and capture of plasticity-related proteins that enable heterosynaptic transfer of persistent potentiation. These processes may permit distinct neural pathways to associate information transmitted by separate, but convergent, synaptic inputs. We had previously shown that transcription and epigenetic modifications were necessary for stabilization of homosynaptic LTP. However, it is unclear whether transfer of LTP to a second, heterosynaptic pathway involves b-ARs signalling to the nucleus. Using electrophysiologic recordings in area CA1 of murine hippocampal slices, we show here that pharmacologically inhibiting b-AR activation, transcription, DNA methyltransferase or histone acetyltransferase activation, prevents stabilization of heterosynaptic LTP. Our data suggest that noradrenergic stabilization of heterosynaptic ("tagged") LTP requires not only transcription, but specifically, DNA methylation and histone acetylation. NA promotes stable heterosynaptic plasticity through engagement of nuclear processes that may contribute to prompt consolidation of short-term memories into resilient long-term memories under conditions when the brain's noradrenergic system is recruited.


Assuntos
Região CA1 Hipocampal/fisiologia , Epigênese Genética/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciação de Longa Duração/fisiologia , Norepinefrina/fisiologia , Receptores Adrenérgicos beta/fisiologia , Transdução de Sinais/fisiologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Citidina/análogos & derivados , Citidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Histona Acetiltransferases/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Propranolol/farmacologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
13.
Expert Opin Investig Drugs ; 28(4): 311-322, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30879349

RESUMO

INTRODUCTION: RX-3117 is an oral, small molecule cytidine analog anticancer agent with an improved pharmacological profile relative to gemcitabine and other nucleoside analogs. The agent has excellent activity against various cancer cell lines and xenografts including gemcitabine-resistant variants and it has excellent oral bioavailability; it is not a substrate for the degradation enzyme cytidine deaminase. RX-3117 is being evaluated at a daily oral schedule of 700 mg (5 days/week for 3 weeks) which results in plasma levels in the micromolar range that have been shown to be cytotoxic to cancer cells. It has shown clinical activity in refractory bladder cancer and pancreatic cancer. Areas covered: The review provides an overview of the relevant market and describes the mechanism of action, main pharmacokinetic/pharmacodynamic features and clinical development of this investigational small molecule. Expert opinion: RX-3117 is selectively activated by uridine-cytidine kinase 2 (UCK2), which is expressed only in tumors and has a dual mechanism of action: DNA damage and inhibition of DNA methyltransferase 1 (DNMT1). Because of its tumor selective activation, novel mechanism of action, excellent oral bioavailability and candidate biomarkers for patient selection, RX-3117 has the potential to replace gemcitabine in the treatment of a spectrum of cancer types.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Citidina/análogos & derivados , Neoplasias/tratamento farmacológico , Administração Oral , Antimetabólitos Antineoplásicos/farmacocinética , Antimetabólitos Antineoplásicos/farmacologia , Disponibilidade Biológica , Citidina/farmacocinética , Citidina/farmacologia , Citidina/uso terapêutico , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Humanos , Neoplasias/patologia , Seleção de Pacientes
14.
Adv Mater ; 31(17): e1806957, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30856290

RESUMO

Patients with advanced melanoma that is of low tumor-associated antigen (TAA) expression often respond poorly to PD-1/PD-L1 blockade therapy. Epigenetic modulators, such as hypomethylation agents (HMAs), can enhance the antitumor immune response by inducing TAA expression. Here, a dual bioresponsive gel depot that can respond to the acidic pH and reactive oxygen species (ROS) within the tumor microenvironment (TME) for codelivery of anti-PD1 antibody (aPD1) and Zebularine (Zeb), an HMA, is engineered. aPD1 is first loaded into pH-sensitive calcium carbonate nanoparticles (CaCO3 NPs), which are then encapsulated in the ROS-responsive hydrogel together with Zeb (Zeb-aPD1-NPs-Gel). It is demonstrated that this combination therapy increases the immunogenicity of cancer cells, and also plays roles in reversing immunosuppressive TME, which contributes to inhibiting the tumor growth and prolonging the survival time of B16F10-melanoma-bearing mice.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/síntese química , Citidina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Imunossupressores/química , Melanoma/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Carbonato de Cálcio/química , Linhagem Celular Tumoral , Citidina/farmacologia , Liberação Controlada de Fármacos , Quimioterapia Combinada/métodos , Humanos , Concentração de Íons de Hidrogênio , Imunossupressores/farmacologia , Imunoterapia/métodos , Nanopartículas/química , Receptor de Morte Celular Programada 1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/efeitos dos fármacos
15.
Curr Protoc Nucleic Acid Chem ; 76(1): e75, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30725523

RESUMO

The detailed synthetic protocols for the preparation of phosphoramidite reagents compatible with standard, automated oligonucleotide synthesis for the 2'-deoxy- and ribo-6-phenylpyrrolocyitidine are reported. Each protocol starts with the parent nucleoside and prepares the 5'-O-dimethoxytrityl-N4 -benzoyl-5-iodocytosine derivative for the nucleobase modification chemistry. The key step is the direct formation of 6-phenylpyrrolocytosine aglycon via a sequential, one-pot Pd-catalyzed Sonogashira-type cross- coupling followed by a 5-endo-dig cyclization. Subsequent standard transformations provide the deoxy- and 2'-O-tert-butyldimethysilyl protected ribo- nucleoside phosphoramidite reagents. © 2019 by John Wiley & Sons, Inc.


Assuntos
Citidina/análogos & derivados , Corantes Fluorescentes/química , Nucleosídeos/química , Pirróis/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Citidina/química , Compostos Organofosforados/química , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray
16.
Chem Commun (Camb) ; 55(16): 2328-2331, 2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30723849

RESUMO

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), two of the best-studied DNA modifications, play crucial roles in normal development and disease in mammals. Although 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) have also been identified in RNA, their distribution and biological function in RNA remain largely unexplored, due to the lack of suitable sequencing methods. Here, we report a base-resolution sequencing method for hm5C in RNA. We applied the selective oxidation of hm5C to trihydroxylated-thymine (thT) mediated by peroxotungstate. thT was subsequently converted to T during cDNA synthesis using a thermostable group II intron reverse transcriptase (TGIRT). Base-resolution analysis of the hm5C sites in RNA was performed using Sanger sequencing. Furthermore, in combination with the TET enzyme oxidation of m5C to hm5C in RNA, we expand the use of peroxotungstate oxidation to detect m5C in RNA at base-resolution. By using this method, we confirmed three known m5C sites in human tRNA, demonstrating the applicability of our method in analyzing real RNA samples.


Assuntos
Citidina/análogos & derivados , RNA/genética , RNA/metabolismo , Análise de Sequência de RNA/métodos , Compostos de Tungstênio/química , Compostos de Tungstênio/metabolismo , Sequência de Bases , Citidina/metabolismo
17.
Electrophoresis ; 40(11): 1535-1539, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30767246

RESUMO

Methylating substances alter DNA by forming N3-methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), we synthesized the N3mT-3'-phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N'-nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5-hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5-methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE-LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.


Assuntos
Eletroforese Capilar/métodos , Neoplasias/patologia , Nitrosaminas/farmacologia , Timidina/análogos & derivados , Células A549 , Citidina/análogos & derivados , Citidina/análise , Fluorescência , Humanos , Neoplasias/química , Timidina/análise
18.
Nucleic Acids Res ; 47(5): 2487-2505, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30759234

RESUMO

TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs (C/D scaRNAs; i.e. scaRNA2, 7, 9 and 28) through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2'-O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.


Assuntos
Esclerose Amiotrófica Lateral/genética , Corpos Enovelados/genética , Proteínas de Ligação a DNA/genética , Proteínas/genética , Esclerose Amiotrófica Lateral/patologia , Nucléolo Celular/genética , Corpos Enovelados/metabolismo , Citidina/análogos & derivados , Citidina/genética , Células HeLa , Humanos , Chaperonas Moleculares , RNA Guia/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Telomerase
19.
J Agric Food Chem ; 67(7): 1795-1806, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30681853

RESUMO

Plant viral diseases cause tremendous decreases in crop yield and quality. Natural products have always been a valuable source for lead discovery in medicinal and agricultural chemistry. A series of pimprinine alkaloids and their derivatives were prepared and identified by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). The antiviral activities of these alkaloids against tobacco mosaic virus (TMV) were systematically investigated for the first time. Most of the compounds exhibited higher antiviral activities than ribavirin. Compounds 5l, 9h, and 10h, which had similar or higher antiviral activities than ningnanmycin (perhaps the most widely used antiviral agent at present), emerged as new antiviral pilot compounds. This systematic structure-activity-relationship research lays the foundation for simplifying the structure of these alkaloids. The ring-open products, acylhydrazones 9a-9u, were also found to possess good antiviral activities. Moreover, all the synthesized compounds displayed broad-spectrum fungicidal activities. This study provides important information for the research and development of pimprinine alkaloids as novel antiviral agents.


Assuntos
Alcaloides/farmacologia , Antivirais , Oxazóis/farmacologia , Alcaloides/química , Citidina/análogos & derivados , Citidina/farmacologia , Estrutura Molecular , Oxazóis/química , Doenças das Plantas/virologia , Vírus de Plantas/efeitos dos fármacos , Ribavirina/farmacologia , Relação Estrutura-Atividade , Vírus do Mosaico do Tabaco/efeitos dos fármacos
20.
Invest New Drugs ; 37(3): 415-423, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30019100

RESUMO

We performed an in-vitro study testing the chemosensitivity of peritoneal cancer cell lines (SW620, HCT116, MKN45, 23,132/87, OAW42) to various cytostatic drug regimens. A duplex drug, characterized by reversible linking of the antimetabolites 2'-deoxy-5-fluorouridine (5-FdU) and 3'-C-ethynylcytidine (ECyd), was compared to oxaliplatin or to cisplatin plus doxorubicin. The experiments were designed to reflect the conditions of intraperitoneal chemotherapy. CASY® (Cell Analysis System) technology was used to compare the impact of incubation temperature/duration and drug concentration on the viability of the cancer cell lines versus normal human dermal fibroblasts. Two incubation scenarios were explored: (i) hyperthermic intraperitoneal chemotherapy (HIPEC) with 1 h of incubation at 42 °C, and (ii) pressurized intraperitoneal aerosol chemotherapy (PIPAC) with several successive incubations at 37 °C. Under HIPEC conditions, oxaliplatin induced a potent temperature-dependent growth inhibition of colon cancer cells not seen with the duplex drug. Under PIPAC conditions, the duplex drug achieved the same growth inhibition at a fraction of the dose level required with oxaliplatin. Gastric and ovarian cancer cells were more sensitive to cisplatin plus doxorubicin than to the duplex drug under PIPAC conditions. The duplex drug suggests itself, notably in cases of platinum resistance, as an alternative or addition to intraperitoneal chemotherapies when platinum-based PIPAC technology is used. Using it with HIPEC technology is not recommended. Higher doses of the duplex drug will enhance growth inhibition, albeit at the cost of a severely reduced difference in chemosensitivity between tumor and normal cells. Our findings provide orientation for PIPAC-based personalized intraperitoneal chemotherapy.


Assuntos
Proliferação de Células/efeitos dos fármacos , Citidina/análogos & derivados , Citostáticos/farmacologia , Desoxiuridina/análogos & derivados , Hipertermia Induzida , Neoplasias Peritoneais/tratamento farmacológico , Adulto , Idoso , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Citidina/farmacologia , Desoxiuridina/farmacologia , Doxorrubicina/farmacologia , Feminino , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Oxaliplatina/farmacologia , Neoplasias Peritoneais/patologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas
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