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“ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present study reports on the characterization of a P-glycoprotein (Pgp), TgABC.B1, in the protozoan parasite Toxoplasma gondii. The protein encoded by the TgABC.B1 gene displays the typical (TMD-NBD)(2) Structural organization of the “full” ABC transporter and shows significant identity
and similarity with two apicomplexan Pgps; Pgh1 in Plasmodium falciparum and CpABC3 in Cryptosporidium parvum. The TgABC.B1 gene is a single copy gene transcribed into a full-length mRNA of 4.3 kb and expressed as a protein of approximately 150 kDa. which AMN-107 cellular localization revealed a membrane-associated labelling in tachyzoites. The TgABC.B1 gene is constitutively expressed in the three major T gondii genotypes but demonstrated a higher expression in virulent type 1, at both transcriptional and translational levels. Further characterization of this Pgp-like protein will increase our knowledge
of the membrane transport system in this parasite and could result in the identification of a new therapeutic target in Toxoplasma. (C) 2008 Elsevier B.V. All rights reserved.”
“Background CYP2C19 is a polymorphic enzyme that plays a pivotal role in the metabolism of 10% of clinically used drugs worldwide. The CYP2C19*3 allele is characterized by a premature stop codon that leads to a truncated CBL0137 cost nonfunctional protein and consequently a poor metabolizer phenotype. Aminoglycoside antibiotics have been shown to induce readthrough of premature stop codons and partial restoration of protein function. We investigated the ability of the aminoglycosides gentamicin and G418 to induce readthrough of CYP2C19*3 premature stop codon in human cells.\n\nMethods A CYP2C19*3 expression model in HeLa cells was used in all experiments. CYP2C19-EGFP expression was assessed by flow cytometry, immunoblotting, and fluorescence microscopy, whereas CYP2C19 enzymatic activity was
quantified by hydroxylation of 3-cyano-7-ethoxycoumarin.\n\nResults G418 and gentamicin promoted readthrough of the CYP2C19*3 premature stop codon in a concentration-dependent manner. Flow cytometry revealed a maximum 23% protein restoration with the highest aminoglycoside concentrations tested, namely 300 mg/ml G418 and 1000 mg/ml gentamicin. At these Vorinostat nmr concentrations, G418 was more effective than gentamicin in restoring CYP2C19 expression in immunoblotting and fluorescence microscopy assays, as well as in restoring CYP2C19 enzymatic activity.\n\nConclusion This is the first demonstration of readthrough of a stop codon in a pharmacogenetic target of clinical relevance, namely CYP2C19*3. The experimental models may be adapted to explore readthrough of stop codons in other genes of pharmacogenetic interest. Pharmacogenetics and Genomics 21: 694-700 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.