ABSTRACT
Attempts have been made to treat nonsense-associated genetic disorders by chemical agents and hence an improved mechanistic insight into the decoding of readthrough signals is essential for the identification and characterisation of factors for the treatment of these disorders. To identify either novel compounds or genes that modulate translation readthrough, we have employed dual reporter-based high-throughput screens that use enzymatic and fluorescence activities and screened bioactive National Institute of Neurological Disease Syndrome (NINDS) compounds (n = 1000) and siRNA (n = 288) libraries. Whilst siRNAs targeting kinases such as CSNK1G3 and NME3 negatively regulate readthrough, neither the bioactive NINDS compounds nor PTC124 promote readthrough. Of note, PTC124 has previously been shown to promote readthrough. Furthermore, the impacts of G418 on the components of eukaryotic selenocysteine incorporation machinery have also been investigated. The selenocysteine machinery decodes the stop codon UGA specifying selenocysteine in natural selenoprotein genes. We have found that the eukaryotic SelC gene promotes the selenocysteine insertion sequence (SECIS)-mediated readthrough but inhibits the readthrough activity induced by G418. We have previously reported that SECIS-mediated readthrough at UGA codons follows a non-processive mechanism. Here, we show that G418-mediated promotion of readthrough also occurs through a non-processive mechanism which competes with translation termination. Based on our observations, we suggest that proteins generated through a non-processive mechanism may be therapeutically beneficial for the resolution of nonsense-associated genetic disorders.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Small Molecule Libraries/pharmacology , Aminoglycosides/metabolism , Base Sequence , Casein Kinase Ialpha/metabolism , Codon, Nonsense , Codon, Terminator , Humans , NM23 Nucleoside Diphosphate Kinases/metabolism , Nucleic Acid Conformation , Oxadiazoles/pharmacology , Peptide Chain Termination, Translational , Protein Biosynthesis , Protein Synthesis Inhibitors , RNA, Messenger/genetics , RNA, Small Interfering/geneticsABSTRACT
Resting neutrophils generate NO, while activation leads to the production of reactive oxygen and nitrogen species. Nowadays cardiovascular pathological conditions such as hypertension, cardiac ischemia, reperfusion and heart failure are associated with inflammation. This project explores the respiratory burst potential and NO generation status in the neutrophils, plasma, aorta, and kidneys from normotensive Wistar and spontaneously hypertensive rats (SHR). Total and protein associated nitrite content was quantitated using Griess reagent following cadmium reduction and mercuric chloride treatment respectively. NO and superoxide generation evaluated by Flowcytometry and peroxynitrite by spectrofluorimetric method. Expression of NOS isoforms was analyzed by RT-PCR. NO generation from SHR neutrophils was significantly augmented in comparison to normotensive counterparts. Neutrophils activated in response to arachidonic acid, PMA, fMLP or E. coli generated more superoxide radicals among SHR, and consequentially peroxynitrite. Expression of iNOS was significantly more in the SHR neutrophils, while that of nNOS remained unaffected. Results suggest that NO generated in SHR is utilized in scavenging superoxide radicals thereby limiting its bioavailability. Thus induction of NOS in neutrophils combined with augmented oxidative stress might influence its association with endothelium and contribute to inflammatory responses under hypertensive condition.
