Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 670
Filtrar
1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 52(1): 98-103, 2021 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-33474897

RESUMO

Objective: R6G-ddATP was used as a dideoxy fluorescence substrate to establish the single base end extension (SNaPShot)-gel fluorescence method for the rapid detection of the genotypes of three high-risk human papillomaviruses (HR-HPV) ( HPV18, HPV33 and HPV35) genotypes. Methods: HPV quality control products were used as as samples, and R6G-ddATP dideoxy fluorescence reagent was used as substrate. Firstly, HPV was amplified by using universal primers to obtain the first round of amplified products, which were purified and used as templates for subsequent SNaPShot reactions. Then, specific one-step extension primers were used to perform SNaPShot reaction to generate R6G-fluorescence-labeled DNA extension products. The product was subjected to agarose gel electrophoresis, the results of which were observed under a Gel Imager, and the HPV genotyping was done with different one-step extension primers. Each sample was tested three times and the results were compared with DNA sequencing results. Results: The preferred annealing temperature for SNaPShot reaction is 55 ℃. Three HPV genotypes were examined by R6G-ddATP/SNaPShot gel fluorescence assay under optimal conditions, and the results were consistent with DNA sequencing results. Conclusion: The R6G-ddATP/SNaPShot-gel fluorescence method for the micro-detection methods of three HR-HPV genotypes was successfully established and can be used for rapid detection of HPV genotypes.


Assuntos
Alphapapillomavirus , Papillomaviridae , Infecções por Papillomavirus , DNA Viral/genética , Nucleotídeos de Desoxiadenina , Didesoxinucleotídeos , Genótipo , Humanos , Papillomaviridae/genética , Reação em Cadeia da Polimerase
2.
Pharmacol Res Perspect ; 8(6): e00674, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33124786

RESUMO

SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected 3'-fluoro-3'-deoxythymidine triphosphate and 3'-azido-3'-deoxythymidine triphosphate, which are the active forms of two other anti-viral agents, Alovudine and AZT (an FDA-approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , /antagonistas & inibidores , Betacoronavirus/enzimologia , Carbamatos/farmacologia , Infecções por Coronavirus/virologia , Didesoxinucleotídeos/farmacologia , Combinação de Medicamentos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Pandemias , Pneumonia Viral/virologia , Sofosbuvir/farmacologia , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/farmacologia
3.
BMC Med Res Methodol ; 19(1): 216, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31775643

RESUMO

BACKGROUND: Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic benefit of ART is often limited by delayed drug-associated toxicity. Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of ART regimens. NRTIs compete with endogenous deoxyribonucleotide triphosphates (dNTPs) in incorporation into elongating DNA chain resulting in their cytotoxic or antiviral effect. Thus, the efficacy of NRTIs could be affected by direct competition with endogenous dNTPs and/or feedback inhibition of their metabolic enzymes. In this paper, we assessed whether the levels of ribonucleotides (RN) and dNTP pool sizes can be used as biomarkers in distinguishing between HIV-infected patients with ART-induced mitochondrial toxicity and HIV-infected patients without toxicity. METHODS: We used data collected through a case-control study from 50 subjects. Cases were defined as HIV-infected individuals with clinical and/or laboratory evidence of mitochondrial toxicity. Each case was age, gender, and race matched with an HIV-positive without evidence of toxicity. We used a range of machine learning procedures to distinguish between patients with and without toxicity. Using resampling methods like Monte Carlo k-fold cross validation, we compared the accuracy of several machine learning algorithms applied to our data. We used the algorithm with highest classification accuracy rate in evaluating the diagnostic performance of 12 RN and 14 dNTP pool sizes as biomarkers of mitochondrial toxicity. RESULTS: We used eight classification algorithms to assess the diagnostic performance of RN and dNTP pool sizes distinguishing HIV patients with and without NRTI-associated mitochondrial toxicity. The algorithms resulted in cross-validated classification rates of 0.65-0.76 for dNTP and 0.72-0.83 for RN, following reduction of the dimensionality of the input data. The reduction of input variables improved the classification performance of the algorithms, with the most pronounced improvement for RN. Complex tree-based methods worked the best for both the deoxyribose dataset (Random Forest) and the ribose dataset (Classification Tree and AdaBoost), but it is worth noting that simple methods such as Linear Discriminant Analysis and Logistic Regression were very competitive in terms of classification performance. CONCLUSIONS: Our finding of changes in RN and dNTP pools in participants with mitochondrial toxicity validates the importance of dNTP pools in mitochondrial function. Hence, levels of RN and dNTP pools can be used as biomarkers of ART-induced mitochondrial toxicity.


Assuntos
Antirretrovirais/efeitos adversos , Desoxirribonucleotídeos/metabolismo , Didesoxinucleotídeos/metabolismo , Infecções por HIV/tratamento farmacológico , Aprendizado de Máquina , Ribonucleotídeos/metabolismo , Algoritmos , Biomarcadores/metabolismo , Estudos de Casos e Controles , Infecções por HIV/diagnóstico , Humanos
4.
Methods Mol Biol ; 2054: 243-261, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482460

RESUMO

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Sondas de DNA/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Genômica/métodos , Hibridização in Situ Fluorescente/métodos , Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Sondas de DNA/genética , DNA Bacteriano/genética , Nucleotídeos de Desoxiuracil/química , Didesoxinucleotídeos/química , Digoxigenina/análogos & derivados , Digoxigenina/química , Fluoresceínas/química , Corantes Fluorescentes/química , Secções Congeladas , Genômica/instrumentação , Humanos , Hibridização in Situ Fluorescente/instrumentação , Rodaminas/química , Coloração e Rotulagem/instrumentação , Coloração e Rotulagem/métodos , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/química
5.
Cell Metab ; 29(4): 871-885.e5, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30853213

RESUMO

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.


Assuntos
Inflamação/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sirtuínas/metabolismo , Fatores Etários , Animais , Didesoxinucleotídeos/administração & dosagem , Didesoxinucleotídeos/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas de Ligação a RNA/antagonistas & inibidores , Sirtuínas/deficiência , Estavudina/administração & dosagem , Estavudina/farmacologia , Nucleotídeos de Timina/administração & dosagem , Nucleotídeos de Timina/farmacologia , Zidovudina/administração & dosagem , Zidovudina/análogos & derivados , Zidovudina/farmacologia
6.
Elife ; 82019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30681409

RESUMO

Transcription by RNA polymerase II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the promoter. In vitro, some GTFs are essential for transcription, whereas others are not required under certain conditions. PICs are stable in the absence of nucleotide triphosphates, and subsets of GTFs can form partial PICs. By depleting individual GTFs in yeast cells, we show that all GTFs are essential for TBP binding and transcription, suggesting that partial PICs do not exist at appreciable levels in vivo. Depletion of FACT, a histone chaperone that travels with elongating Pol II, strongly reduces PIC formation and transcription. In contrast, TBP-associated factors (TAFs) contribute to transcription of most genes, but TAF-independent transcription occurs at substantial levels, preferentially at promoters containing TATA elements. PICs are absent in cells deprived of uracil, and presumably UTP, suggesting that transcriptionally inactive PICs are removed from promoters in vivo.


Assuntos
RNA Polimerase II/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Genéricos de Transcrição/genética , Transcrição Genética , Proteínas de Ligação a DNA/genética , Didesoxinucleotídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIID/genética
7.
J Microbiol Biotechnol ; 29(3): 367-372, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30661323

RESUMO

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.


Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Genes Bacterianos/genética , Fosfotransferases/genética , Sequência de Aminoácidos , Aminoglicosídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nucleotídeos de Desoxiguanina/biossíntese , Nucleotídeos de Desoxiguanina/genética , Didesoxinucleotídeos/biossíntese , Didesoxinucleotídeos/genética , Gentamicinas/biossíntese , Micromonospora/genética , Micromonospora/metabolismo , Alinhamento de Sequência , Streptomyces/genética , Streptomyces/metabolismo
8.
Nat Commun ; 9(1): 3872, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250201

RESUMO

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.


Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Inibidores Enzimáticos/farmacologia , Hidroxibenzoatos/farmacologia , Neoplasias/terapia , Fosfofrutoquinase-2/antagonistas & inibidores , Sulfonas/farmacologia , Antineoplásicos/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia/métodos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Didesoxinucleotídeos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidroxibenzoatos/uso terapêutico , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Radiação Ionizante , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos da radiação , Sulfonas/uso terapêutico
9.
Anal Bioanal Chem ; 410(21): 5245-5253, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29947896

RESUMO

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal-organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delivery applications and gave fundamental data on their size distribution and stability. Graphical abstract ᅟ.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Complexos de Coordenação/química , Portadores de Fármacos/química , Estruturas Metalorgânicas/química , Nanopartículas/química , Zidovudina/administração & dosagem , Fármacos Anti-HIV/química , Antimetabólitos/administração & dosagem , Antimetabólitos/química , Didesoxinucleotídeos/administração & dosagem , Didesoxinucleotídeos/química , Difusão Dinâmica da Luz , Fracionamento por Campo e Fluxo , Modelos Moleculares , Tamanho da Partícula , Refratometria , Espectrofotometria Ultravioleta , Nucleotídeos de Timina/administração & dosagem , Nucleotídeos de Timina/química , Zidovudina/análogos & derivados , Zidovudina/química
10.
Bioorg Med Chem Lett ; 28(7): 1248-1251, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29506959

RESUMO

A conjugate of triphosphorylated 2',3'-dideoxyuridine (ddU) with SiO2 nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO2 nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO2 nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183 ±â€¯57 µg/mL, which corresponds to 22 ±â€¯7 µM of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO2 nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Didesoxinucleotídeos/farmacologia , Nanopartículas/química , Dióxido de Silício/farmacologia , Nucleotídeos de Uracila/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Didesoxinucleotídeos/síntese química , Didesoxinucleotídeos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Estrutura Molecular , Dióxido de Silício/química , Relação Estrutura-Atividade , Nucleotídeos de Uracila/síntese química , Nucleotídeos de Uracila/química
11.
Nat Microbiol ; 2(11): 1513-1522, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28871089

RESUMO

In this study, we report that the tetraspanin CD81 enhances human immunodeficiency virus (HIV)-1 reverse transcription in HIV-1-infected cells. This is enabled by the direct interaction of CD81 with the deoxynucleoside triphosphate phosphohydrolase SAMHD1. This interaction prevents endosomal accumulation and favours the proteasome-dependent degradation of SAMHD1. Consequently, CD81 depletion results in SAMHD1 increased expression, decreasing the availability of deoxynucleoside triphosphates (dNTP) and thus HIV-1 reverse transcription. Conversely, CD81 overexpression, but not the expression of a CD81 carboxy (C)-terminal deletion mutant, increases cellular dNTP content and HIV-1 reverse transcription. Our results demonstrate that the interaction of CD81 with SAMHD1 controls the metabolic rate of HIV-1 replication by tuning the availability of building blocks for reverse transcription, namely dNTPs. Together with its role in HIV-1 entry and budding into host cells, the data herein indicate that HIV-1 uses CD81 as a rheostat that controls different stages of the infection.


Assuntos
Didesoxinucleotídeos/metabolismo , HIV-1/genética , Transcrição Reversa , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Tetraspanina 28/metabolismo , Replicação do DNA , HIV-1/fisiologia , Células HeLa , Humanos , Macrófagos/virologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Tetraspanina 28/genética , Replicação Viral
12.
RNA ; 23(10): 1582-1591, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28698239

RESUMO

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.


Assuntos
DNA Nucleotidilexotransferase/metabolismo , Hibridização in Situ Fluorescente/métodos , Sondas RNA/química , Animais , Biotina , Didesoxinucleotídeos/química , Didesoxinucleotídeos/metabolismo , Drosophila melanogaster/genética , Feminino , Corantes Fluorescentes/química , Sondas de Oligonucleotídeos/química , Oligonucleotídeos/química , Ovário/fisiologia , Sondas RNA/metabolismo , Nucleotídeos de Uracila/química , Nucleotídeos de Uracila/metabolismo
13.
J Biol Chem ; 292(34): 14016-14025, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28684413

RESUMO

Retrovirus integration into the host genome relies on several host enzymes, potentially including DNA polymerase ß (Pol ß). However, whether human Pol ß is essential for lentivirus replication in human cells is unclear. Here, we abolished DNA polymerase ß (Pol ß) expression by targeting its DNA polymerase domain with CRISPR/Cas9 in human monocytic THP-1 cells to investigate the role of Pol ß in HIV-1 transduction in both dividing and nondividing macrophage stages of THP-1 cells. Pol ß-knock-out was confirmed by enhanced sensitivity to methyl methanesulfonate-induced DNA damage. Of note, nuclear extracts from Pol ß-knock-out THP-1 cells prepared from both dividing and nondividing stages displayed significantly reduced capability to repair the gapped HIV-1 integration intermediate DNA substrate in a biochemical simulation. However, nuclear extract from both dividing and nondividing stages of the Pol ß-KO cells had detectable gap repair activity, suggesting that other host DNA polymerases also repair gapped HIV-1 DNA, particularly in dividing cells. Next, when we compared transduction using HIV-1 and simian immunodeficiency virus in control and Pol ß-KO cells, the loss of the Pol ß expression did not affect transduction efficiency of these lentiviruses in both dividing and nondividing stages. Finally, the gap repair assay indicated that limited cellular dNTP pools, but not Pol ß expression, are a primary factor for HIV-1 DNA gap repair, particularly in nondividing cells. These data support the idea that Pol ß polymerase activity is dispensable for HIV-1 infection in both dividing and nondividing stages of human cells targeted by the virus.


Assuntos
DNA Polimerase beta/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Macrófagos/virologia , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , DNA Polimerase beta/antagonistas & inibidores , DNA Polimerase beta/química , DNA Polimerase beta/genética , Reparo do DNA , Didesoxinucleotídeos/metabolismo , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Domínios e Motivos de Interação entre Proteínas , RNA/metabolismo , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Integração Viral
14.
DNA Repair (Amst) ; 49: 51-59, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27989484

RESUMO

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+ and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).


Assuntos
Coenzimas/metabolismo , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Enzimas Multifuncionais/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Antivirais/metabolismo , Antivirais/uso terapêutico , Arabinofuranosilcitosina Trifosfato/metabolismo , Arabinofuranosilcitosina Trifosfato/uso terapêutico , Cátions Bivalentes/metabolismo , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/metabolismo , Citidina Trifosfato/uso terapêutico , Desoxirribonucleotídeos/metabolismo , Didesoxinucleotídeos/metabolismo , Didesoxinucleotídeos/uso terapêutico , Emtricitabina/análogos & derivados , Emtricitabina/metabolismo , Emtricitabina/uso terapêutico , Humanos , Cinética , Lamivudina/análogos & derivados , Lamivudina/metabolismo , Lamivudina/uso terapêutico
15.
Biochemistry ; 56(1): 33-46, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27936595

RESUMO

Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg2+, whereas the free Mg2+ concentration in cells is much lower. Artificially high Mg2+ concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg2+. At low concentrations of Mg2+, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg2+, whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg2+ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg2+ concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on kcat/Km) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg2+ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC50 values) more effective at low than at high Mg2+ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg2+-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg2+ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg2+ in vitro conditions. These results also emphasize differences in Mg2+ sensitivity between the translocation inhibitor EFdATP and other NRTIs.


Assuntos
Didesoxinucleotídeos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Magnésio/farmacologia , Nucleosídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Nucleotídeos de Desoxicitosina/farmacologia , Nucleotídeos de Desoxiguanina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Eletroforese em Gel de Poliacrilamida , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Humanos , Cinética , Mutação , Nucleotídeos de Timina/farmacologia , Zalcitabina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/farmacologia
16.
PLoS One ; 11(11): e0165772, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27812166

RESUMO

BACKGROUND: Pediatric uptake and outcomes in antiretroviral treatment (ART) programmes have lagged behind adult programmes. We describe outcomes from a population-based pediatric ART cohort in rural southern Malawi. METHODS: Data were analyzed on children who initiated ART from October/2003 -September/2011. Demographics and diagnoses were described and survival analyses conducted to assess the impact of age, presenting features at enrolment, and drug selection. RESULTS: The cohort consisted of 2203 children <15 years of age. Age at entry was <1 year for 219 (10%), 1-1.9 years for 343 (16%), 2-4.9 years for 584 (27%), and 5-15 years for 1057 (48%) patients. Initial clinical diagnoses of tuberculosis and wasting were documented for 409 (19%) and 523 (24%) patients, respectively. Median follow-up time was 1.5 years (range 0-8 years), with 3900 patient-years of follow-up. Over the period of observation, 134 patients (6%) died, 1324 (60%) remained in the cohort, 345 (16%) transferred out, and 387 (18%) defaulted. Infants <1 year of age accounted for 19% of deaths, with a 2.7-fold adjusted mortality hazard ratio relative to 5-15 year olds; median time to death was also shorter for infants (60 days) than older children (108 days). Survival analysis demonstrated younger age at ART initiation, more advanced HIV stage, and presence of tuberculosis to each be associated with shorter survival time. Among children <5 years, severe wasting (weight-for-height z-score

Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV , Adolescente , Alquinos , Benzoxazinas/uso terapêutico , Contagem de Linfócito CD4 , Criança , Pré-Escolar , Estudos de Coortes , Ciclopropanos , Didesoxinucleotídeos/uso terapêutico , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Infecções por HIV/mortalidade , Síndrome de Emaciação por Infecção pelo HIV/mortalidade , Humanos , Lactente , Lamivudina/uso terapêutico , Malaui/epidemiologia , Masculino , Modelos de Riscos Proporcionais , Estudos Retrospectivos , População Rural , Estavudina/análogos & derivados , Estavudina/uso terapêutico , Análise de Sobrevida , Tuberculose Pulmonar/mortalidade , Zidovudina/uso terapêutico
17.
PLoS One ; 11(7): e0153201, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27380276

RESUMO

MicroRNAs (miRNAs) are important regulators of gene translation and have been suggested as potent biomarkers in various disease states. In this study, we established an efficient method for simultaneous determination of multiple miRNA levels, employing the previously developed SPC-SBE (solid phase capture-single base extension) approach and MALDI-TOF mass spectrometry (MS). In this approach, we first perform reverse transcription of miRNAs extracted using stem-loop primers. Then the cDNA is co-amplified with competitors, synthetic oligonucleotides whose sequences precisely match cDNA except for one base, and the amplicons serve as templates for a multiplexed SBE reaction. Extension products are isolated using SPC and quantitatively analyzed with MALDI-TOF MS to determine multiple miRNA levels. Here we demonstrated concurrent analysis of four miRNA levels utilizing the approach. Furthermore, we showed the presented method significantly facilitated MS analysis of peak area ratio owing to SPC. The SPC process allowed effective removal of irrelevant reaction components prior to MS and promoted MS sample purification. Data obtained in this study was verified with RT-qPCR and agreement was shown on one order of magnitude scale, suggesting the SPC-SBE and MS approach has strong potential as a viable tool for high throughput miRNA analysis.


Assuntos
Biotinilação , Didesoxinucleotídeos/genética , MicroRNAs/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Células A549 , Animais , Calibragem , DNA Complementar/química , DNA Complementar/genética , Didesoxinucleotídeos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
18.
Nucleic Acids Res ; 44(5): 2310-22, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26850643

RESUMO

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.


Assuntos
Farmacorresistência Viral Múltipla/genética , Inibidores Enzimáticos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Clonagem Molecular , Didesoxinucleotídeos/química , Didesoxinucleotídeos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease H do Vírus da Imunodeficiência Humana/genética , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/química , Zidovudina/farmacologia
19.
J Acquir Immune Defic Syndr ; 72(3): 246-53, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-26859826

RESUMO

OBJECTIVES: Zidovudine (AZT) is mainly used to prevent mother-to-child HIV-1 transmission (PMTCT). Despite serious concerns on AZT-associated toxicity, there is little information on pharmacokinetics of intracellular AZT metabolites in infants. METHODS: We conducted a prospective study in 31 HIV-uninfected infants who received AZT for PMTCT. Blood samples were obtained from 14 infants on postdelivery days (PDD) 1, 7, 14, and 28 and from 17 infants at 0 and 4 hours after dosing on PDD-1. Plasma AZT concentrations (pAZT) and intracellular concentrations of AZT-monophosphate (icAZT-MP), diphosphate (icAZT-DP), and triphosphate (icAZT-TP) were determined. RESULTS: Plasma AZT and icAZT-MP concentrations were 2713 nmol/L and 79 fmol/10 cells in PDD-1, but decreased to 1437 nmol/L and 31 fmol/10 cells by PDD-28 (P = 0.02 and P = 0.07 for all PDDs, respectively), whereas those of icAZT-DP and icAZT-TP remained low throughout the sampling period (P = 0.29 and P = 0.61 for all PDDs, respectively) There were no differences in icAZT-TP between infants of the 2 mg/kg 4 times a day dose and 4 mg/kg twice daily dose (P = 0.25), whereas pAZT and icAZT-MP levels were higher in the latter (P < 0.01 and <0.01, respectively). The pAZT and icAZT-MP significantly increased from 0 to 4 hours after dosing (P < 0.001 and <0.001, respectively), whereas icAZT-DP, icAZT-TP levels were not changed (P = 0.41 and 0.33, respectively). CONCLUSIONS: The level of icAZT-TP did not change with age, time, or a single dose despite the wide range of pAZT concentration. A safer dosage needs to be determined because high pAZT levels do not parallel those of icAZT-TP.


Assuntos
Fármacos Anti-HIV/farmacocinética , Didesoxinucleotídeos/farmacocinética , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , HIV-1/efeitos dos fármacos , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Complicações Infecciosas na Gravidez/tratamento farmacológico , Nucleotídeos de Timina/farmacocinética , Zidovudina/análogos & derivados , Zidovudina/farmacocinética , Adulto , Fármacos Anti-HIV/sangue , Cromatografia Líquida de Alta Pressão/métodos , Didesoxinucleotídeos/sangue , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Recém-Nascido , Estudos Longitudinais , Masculino , Mães , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Estudos Prospectivos , Nucleotídeos de Timina/sangue , Resultado do Tratamento , Adulto Jovem , Zidovudina/sangue
20.
Assay Drug Dev Technol ; 13(10): 628-37, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26690766

RESUMO

The Plasmodium falciparum telomerase reverse transcriptase (PfTERT) is a ribonucleoprotein that assists the maintenance of the telomeric ends of chromosomes by reverse transcription of its own RNA subunit. It represents an attractive therapeutic target for eradication of the plasmodial parasite at the asexual liver stage. Automated modeling using MUSTER and knowledge-based techniques were used to obtain a three-dimensional model of the active site of reverse transcriptase domain of PfTERT, which is responsible for catalyzing the addition of incoming dNTPs to the growing DNA strand in presence of divalent magnesium ions. Further, the ternary complex of the active site of PfTERT bound to a DNA-RNA duplex was also modeled using Haddock server and represents the functional form of the enzyme. Initially, established nucleoside analog inhibitors of PfTERT, AZTTP, and ddGTP were docked in the modeled binding site of the PfTERT ternary complex using AutoDock v4.2. Subsequently, docking studies were carried out with 14 approved nucleoside analog inhibitors. Docking studies predicted that floxuridine, gemcitabine, stavudine, and vidarabine have high affinity for the PfTERT ternary complex. Further analysis on the basis of known side effects led us to propose repositioning of vidarabine as a suitable drug candidate for inhibition of PfTERT.


Assuntos
Antimaláricos/farmacologia , Reposicionamento de Medicamentos/métodos , Nucleosídeos/farmacologia , Plasmodium falciparum/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Telomerase/antagonistas & inibidores , Sequência de Aminoácidos , Antimetabólitos/farmacologia , Nucleotídeos de Desoxiguanina/antagonistas & inibidores , Nucleotídeos de Desoxiguanina/genética , Didesoxinucleotídeos/antagonistas & inibidores , Didesoxinucleotídeos/genética , Humanos , Magnésio/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Estrutura Terciária de Proteína , DNA Polimerase Dirigida por RNA/genética , Telomerase/genética , Nucleotídeos de Timina/antagonistas & inibidores , Nucleotídeos de Timina/genética , Vidarabina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/antagonistas & inibidores
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...