RESUMO
Aptamers are single-stranded DNA or RNA molecules which bind with high specificity to the molecular target for which they have been selected. Through a number of unnatural modifications, the diversity of interactions available for this class of molecules can be greatly expanded, potentially leading to better binding properties. Herein we describe a method to prepare an initial chemically modified DNA library for SELEX. It comprises the synthesis of a modified nucleotide in the CuAAC reaction and its purification by an adapted HPLC method. Subsequent stage is the incorporation of the prepared modified nucleotide into a DNA library in a primer extension reaction and a quick and easy method of its purification using an electroelution device. This allows the recovery of the resulting chemically modified ssDNA library with high efficiency to kick off the aptamer selection process.
Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples , Biblioteca Gênica , RNA , Técnica de Seleção de Aptâmeros/métodosRESUMO
Changes in the functional state of mitochondria have profound effects on other cellular compartments. Genome-wide expression analysis of Arabidopsisrps10 mutants with an RNAi-silenced expression of mitoribosomal S10 protein has revealed extensive transcriptional reprogramming. A meta-analysis comparing expression datasets of 25 mitochondrial perturbations showed a high similarity of the aox1a:rpoTmp mutant, which is defective in the alternative oxidase (AOX1a) and dual-targeted mitochondrial and plastid RNA polymerase (RPOTmp), to rps10. Both rps10 and aox1a:rpoTmp showed a significantly decreased electron flux through both the cytochrome and the alternative respiratory pathways, and a markedly decreased the expression of nuclear-encoded components of the chloroplast transcription machinery. In line with this, a decreased level of plastid transcripts was observed in rps10 and aox1a:rpoTmp, which was reflected in a reduced rate of chloroplast transcription. Chemical treatment of wild-type seedlings with respiratory inhibitors showed that only simultaneous and direct inhibition of complex IV and AOX activity decreased the level of plastid transcripts. Taken together, both chemical and genetic studies show that the limitation of the activity of two mitochondrial terminal oxidases, complex IV and AOX, negatively impacts chloroplast transcription. Salicylic acid and oxygen are discussed as putative mediators of the signalling pathway between mitochondria, nucleus and chloroplasts. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Transcrição Gênica , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismoRESUMO
The activity of a cell-surface ecto-adenosine deaminase (eADA) is markedly increased in the endothelial activation and vascular inflammation leading to decreased adenosine concentration and alterations in adenosine signalling. Depending on the specific pathway activated, extracellular purines mediate host cell response or regulate growth and cytotoxicity on tumour cells. The aim of this study was to test the effects of adenosine deaminase inhibition by 2'deoxycoformycin (dCF) on the breast cancer development. dCF treatment decreased a tumour growth and a final tumour mass in female BALB/c mice injected orthotopically with 4T1 cancer cells. dCF also counteracted cancer-induced endothelial dysfunction in orthotopic and intravenous 4T1 mouse breast cancer models. In turn, this low dCF dose had a minor effect on immune stimulation exerted by 4T1 cell implantation. In vitro studies revealed that dCF suppressed migration and invasion of 4T1 cells via A2a and A3 adenosine receptor activation as well as 4T1 cell adhesion and transmigration through the endothelial cell layer via A2a receptor stimulation. Similar effects of dCF were observed in human breast cancer cells. Moreover, dCF improved a barrier function of endothelial cells decreasing its permeability. This study highlights beneficial effects of adenosine deaminase inhibition on breast cancer development. The inhibition of adenosine deaminase activity by dCF reduced tumour size that was closely related to the decreased aggressiveness of tumour cells by adenosine receptor-dependent mechanisms and endothelial protection.
