RESUMO
Telomerase overexpression has been associated directly with cancer, and the enzyme itself is recognized within the scientific community as a cancer biomarker. BIDEA's biosensing strip (BBS) is an innovative technology capable of detecting the presence of telomerase activity (TA) using electrochemical impedance spectroscopy (EIS). This BBS is an interdigital gold (GID) electrode array similar in size and handling to a portable glucose sensor. For the detection of the biomarker, BBS was modified by the immobilization of a telomere-like single strand DNA (ssDNA) on its surface. The sensor was exposed to telomerase-positive extract from commercially available cancer cells, and the EIS spectra were measured. Telomerase recognizes the sequence of this immobilized ssDNA probe on the BBS, and the reverse transcription process that occurs in cancer cells is replicated, resulting in the ssDNA probe elongation. This surface process caused by the presence of TA generates changes in the capacitive process on the electrode array microchip surface, which is followed by EIS as the sensing tool and correlated with the presence of cancer cells. The telomerases' total cell extraction protocol results demonstrate significant changes in the charge-transfer resistance (R ct) change rate after exposure to telomerase-positive extract with a detection limit of 2.94 × 104 cells/mL. Finally, a preliminary study with a small set of "blind" uterine biopsy samples suggests the feasibility of using the changes in the R ct magnitude change rate (Δ(ΔR ct/R cti)/Δt) to distinguish positive from negative endometrial adenocarcinoma samples by the presence or absence of TA.
RESUMO
Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells.
Assuntos
Regiões 3' não Traduzidas , Proteínas ELAV/fisiologia , Interleucina-3/genética , Interleucina-3/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteína Semelhante a ELAV 1 , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Células Jurkat , Ativação Linfocitária , Proteínas de Ligação a Poli(A)/fisiologia , Antígeno-1 Intracelular de Células T , Transformação GenéticaRESUMO
The enzyme telomerase is present in about 85% of human cancers which makes it not only a good target for cancer treatment but also an excellent marker for cancer detection. Using a single stranded DNA probe specific for telomerase binding and reverse transcription tethered to an interdigital gold electrode array surface, the chromosome protection provided by the telomerase was replicated and followed by Electrochemical Impedance Spectroscopy as an unlabeled biosensor. Using this system designed in-house, easy and affordable, impedance measurements were taken while incubating at 37 °C and promoting the probe elongation. This resulted in up to 14-fold increase in the charge transfer resistance when testing a telomerase-positive nuclear extract from Jurkat cells compared to the heat-inactivated telomerase-negative nuclear extract. The electron transfer process at the Au electrodes was studied before the elongation, at different times after the elongation, and after desorption of non-specific binding.
RESUMO
One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.
Assuntos
RNA Helicases/química , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Degradação do RNAm Mediada por Códon sem Sentido , Terminação Traducional da Cadeia Peptídica , Fosforilação , Alinhamento de Sequência , Tirosina/metabolismoRESUMO
The siRNA transfection efficiency of nanoparticles (NPs), composed of a superparamagnetic iron oxide core modified with polycationic polymers (poly(hexamethylene biguanide) or branched polyethyleneimine), were studied in CHO-K1 and HeLa cell lines. Both NPs demonstrated to be good siRNA transfection vehicles, but unmodified branched polyethyleneimine (25 kD) was superior on both cell lines. However, application of an external magnetic field during transfection (magnetofection) increased the efficiency of the superparamagnetic NPs. Furthermore, our results reveal that these NPs are less toxic towards CHO-K1 cell lines than the unmodified polycationic-branched polyethyleneimine (PEI). In general, the external magnetic field did not alter the cell's viability nor it disrupted the cell membranes, except for the poly(hexamethylene biguanide)-modified NP, where it was observed that in CHO-K1 cells application of the external magnetic field promoted membrane damage. This paper presents new polycationic superparamagnetic NPs as promising transfection vehicles for siRNA and demonstrates the advantages of magnetofection.
RESUMO
Heterogeneous nuclear ribonucleoproteins are multifunctional proteins that bind to newly synthesized mRNAs in the nucleus and participate in many subsequent steps of gene expression. A well-studied Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein that has several nuclear functions is Npl3p. Here, we provide evidence that Npl3p also has a cytoplasmic role: it functions in translation termination fidelity. Yeast harboring the npl3-95 mutant allele have an impaired ability to translate lacZ, enhanced sensitivity to cycloheximide and paromomycin, and increased ability to read through translation termination codons. Most of these defects are enhanced in yeast that also lack Upf1p, an RNA surveillance factor crucial for translation termination. We show that the npl3-95 mutant allele encodes a form of Npl3p that is part of high molecular-weight complexes that cofractionate with the poly(A)-binding protein Pab1p. Together, these results lead us to propose a model in which Npl3p engenders translational fidelity by promoting the remodeling of mRNPs during translation termination.