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1.
RNA Biol ; 8(3): 478-87, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21508682

RESUMO

Global mapping of rRNA structure by traditional methods is prohibitive in terms of time, labor and expense. High throughput selective 2' hydroxyl acylation analyzed by primer extension (hSHAPE) bypasses these problems by using fluorescently labeled primers to perform primer extension reactions, the products of which can be separated by capillary electrophoresis, thus enabling long read lengths in a cost effective manner. The data so generated is analyzed in a quantitative fashion using SHAPEFinder. This approach was used to map the flexibility of nearly the entire sequences of the 3 largest rRNAs from intact, empty yeast ribosomes. Mapping of these data onto near-atomic resolution yeast ribosome structures revealed the binding sites of known trans-acting factors, as well as previously unknown highly flexible regions of yeast rRNA. Refinement of this technology will enable nucleotide-specific mapping of changes in rRNA structure depending on the status of tRNA occupancy, the presence or absence of other trans-acting factors, due to mutations of intrinsic ribosome components or extrinsic factors affecting ribosome biogenesis, or in the presence of translational inhibitors.


Assuntos
RNA Fúngico/química , RNA Ribossômico/química , Ribossomos/química , Saccharomyces cerevisiae/genética , Sequência de Bases , Sítios de Ligação , Microscopia Crioeletrônica , Primers do DNA/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae/metabolismo
2.
RNA Biol ; 7(3): 354-60, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20404492

RESUMO

One of the major challenges facing researchers working with eukaryotic ribosomes lies in their lability relative to their eubacterial and archael counterparts. In particular, lysis of cells and purification of eukaryotic ribosomes by conventional differential ultracentrifugation methods exposes them for long periods of time to a wide range of co-purifying proteases and nucleases, negatively impacting their structural integrity and functionality. A chromatographic method using a cysteine charged Sulfolink resin was adapted to address these problems. This fast and simple method significantly reduces co-purifying proteolytic and nucleolytic activities, producing good yields of highly biochemically active yeast ribosomes with fewer nicks in their rRNAs. In particular, the chromatographic purification protocol significantly improved the quality of ribosomes isolated from mutant cells. This method is likely applicable to mammalian ribosomes as well. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.


Assuntos
RNA Ribossômico/isolamento & purificação , Ribossomos/química , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Leveduras/química , Sítios de Ligação , Fracionamento Celular/métodos , Cromatografia/métodos , Contaminação de Medicamentos , Ensaios Enzimáticos , Modelos Biológicos , Conformação de Ácido Nucleico , Peptídeo Hidrolases/metabolismo , Conformação Proteica , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Ribonucleases/metabolismo , Ribossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Leveduras/citologia
3.
Antioxid Redox Signal ; 19(17): 2141-56, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-23621620

RESUMO

SIGNIFICANCE: Ewan Cameron reported that ascorbate, given orally and intravenously at doses of up to 10 g/day, was effective in the treatment of cancer. Double-blind placebo-controlled clinical trials showed no survival advantage when the same doses of ascorbate were given orally, leading the medical and scientific communities to dismiss the use of ascorbate as a potential cancer treatment. However, the route of administration results in major differences in ascorbate bioavailability. Tissue and plasma concentrations are tightly controlled in response to oral administration, but this can be bypassed by intravenous administration. These data provide a plausible scientific rationale for the absence of a response to orally administered ascorbate in the Mayo clinic trials and indicate the need to reassess ascorbate as a cancer therapeutic. RECENT ADVANCES: High dose ascorbate is selectively cytotoxic to cancer cell lines through the generation of extracellular hydrogen peroxide (H2O2). Murine xenograft models confirm a growth inhibitory effect of pharmacological concentrations. The safety of intravenous ascorbate has been verified in encouraging pilot clinical studies. CRITICAL ISSUES: Neither the selective toxicity of pharmacologic ascorbate against cancer cells nor the mechanism of H2O2-mediated cytotoxicity is fully understood. Despite promising preclinical data, the question of clinical efficacy remains. FUTURE DIRECTIONS: A full delineation of mechanism is of interest because it may indicate susceptible cancer types. Effects of pharmacologic ascorbate used in combination with standard treatments need to be defined. Most importantly, the clinical efficacy of ascorbate needs to be reassessed using proper dosing, route of administration, and controls.


Assuntos
Antineoplásicos/administração & dosagem , Ácido Ascórbico/administração & dosagem , Neoplasias/tratamento farmacológico , Administração Oral , Animais , Antineoplásicos/farmacocinética , Ácido Ascórbico/farmacocinética , Disponibilidade Biológica , Morte Celular/efeitos dos fármacos , Interações Medicamentosas , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
4.
J Vis Exp ; (56)2011 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-22042245

RESUMO

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.


Assuntos
Cromatografia em Gel/métodos , Ribossomos/química , Leveduras/ultraestrutura , Cisteína/química , Reagentes de Sulfidrila/química , Leveduras/química
5.
Toxicon ; 55(8): 1475-83, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20211195

RESUMO

The preproricin gene encodes ricin, the highly toxic, type II ribosome-inactivating protein of castor bean (Ricinus communis L.). As a generalist plant defense gene, preproricin is expected to exhibit population-level variation consistent with the neutral equilibrium model and to comprise few functionally different alleles. We first test the hypothesis that the preproricin gene family should comprise six to eight members by searching the publicly available draft genome sequence of R. communis and analyzing its ricin-like loci. We then test the neutral equilibrium expectation for the preproricin gene by characterizing its allelic variation among 25 geographically diverse castor bean plants. We confirm the presence of six ricin-like loci that share with the preproricin gene 62.9-96.3% nucleotide identity and intact A-chains. DNA sequence variation among the preproricin haplotypes significantly rejects tests of the neutral equilibrium model. Replacement mutations preserve the 12 amino acids known to affect catalytic and electrostatic interactions of the native protein toxin, which suggests functional divergence among alleles has been minimal. Nucleotide polymorphism is maintained by purifying selection (omega < 0.3) yet includes an excess of rare silent mutations greater than predicted by the neutral equilibrium model. Development of robust detection methods for ricin contamination must account for the presence of these other ricin-like molecules and should leverage the specificity provided by the numerous single nucleotide polymorphisms in the preproricin gene.


Assuntos
Genética Populacional , Precursores de Proteínas/genética , Ricina/genética , Ricinus communis/fisiologia , Toxinas Biológicas/genética , DNA de Plantas/análise , Evolução Molecular , Frequência do Gene , Genômica , Polimorfismo de Nucleotídeo Único , Análise de Sequência de Proteína
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