RESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508 (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic-reticulum-associated degradation. This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements was expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady-state levels of the mRNA varied by as much as 30-fold. Experiments support that this apparent inconsistency is attributed to nonsense mediated decay independent of exon junction complex. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high-expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped endoplasmic-reticulum-associated degradation resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that codon usage has an effect on mRNA levels and protein expression, for CFTR, and likely on chaperone-assisted folding pathway, for F508del CFTR.
Assuntos
Membrana Celular/metabolismo , Códon/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fases de Leitura Aberta/genética , Deleção de Sequência , Northern Blotting , Western Blotting , Cloretos/metabolismo , Imunofluorescência , Células HEK293 , Humanos , Mutação/genética , Dobramento de Proteína , Transporte Proteico , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: To investigate the feasibility of electrically stimulating the lateral rectus muscle to recover its physiologic abduction ability in cases of complete sixth cranial (abducens) nerve palsy. METHODS: In the feline lateral rectus muscle model, the effects of a charge-balanced, biphasic, current-controlled stimulus on the movement of the eye were investigated while stimulation frequency, amplitude, and pulse duration was varied. Eye deflection was measured with a force transducer. Denervated conditions were simulated by injection of botulinum toxin A. RESULTS: Three chemically denervated and 4 control lateral rectus muscles were analyzed. In control lateral rectus muscles, the minimum fusion frequency was approximately 170 Hz, and the maximum evoked abduction was 27 degrees. The minimum fusion frequency was unchanged after 4 weeks of chemical denervation. Stimulation of chemically denervated lateral rectus muscle resulted in 17 degrees of abduction. For both innervated and chemically denervated lateral rectus muscle, frequencies greater than 175 Hz yielded very little increase in abduction. Modulating amplitude produced noticeable movement throughout the tested range (0.2 to 9 mA). CONCLUSIONS: Results from the feline lateral rectus muscle showed that electrical stimulation is a feasible approach to evoke a contraction from a denervated lateral rectus muscle. The degree of denervation of the feline lateral rectus muscle was indeterminate. Varying the stimulation amplitude allowed greater eye movement. It is very likely that both frequency and amplitude must be modulated for finer control of static eye position.
Assuntos
Doenças do Nervo Abducente/terapia , Terapia por Estimulação Elétrica/instrumentação , Terapia por Estimulação Elétrica/métodos , Músculos Oculomotores/fisiologia , Estrabismo/terapia , Animais , Toxinas Botulínicas Tipo A/farmacologia , Gatos , Denervação , Modelos Animais de Doenças , Eletrodos , Estudos de Viabilidade , Neurotoxinas/farmacologia , TransdutoresRESUMO
The mineralogical and elemental compositions of the martian soil are indicators of chemical and physical weathering processes. Using data from the Mars Exploration Rovers, we show that bright dust deposits on opposite sides of the planet are part of a global unit and not dominated by the composition of local rocks. Dark soil deposits at both sites have similar basaltic mineralogies, and could reflect either a global component or the general similarity in the compositions of the rocks from which they were derived. Increased levels of bromine are consistent with mobilization of soluble salts by thin films of liquid water, but the presence of olivine in analysed soil samples indicates that the extent of aqueous alteration of soils has been limited. Nickel abundances are enhanced at the immediate surface and indicate that the upper few millimetres of soil could contain up to one per cent meteoritic material.
