Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study investigated the potential of intravesical instillation for localized reduction of NGF (nerve growth factor) expression in the urinary bladder. Overexpression of NGF has been linked to the pathogenesis of interstitial cystitis (IC). A minimum free energy algorithm was used to predict suitable regions in mRNA of rat betaNGF, which can be targeted for an antisense approach. The candidate antisense oligos were evaluated for their ability to reduce NGF expression in vitro by cotransfecting HEK293 cells with NGF cDNA. A single oligonucleotide ODN sequence was chosen for testing in an acute cystitis model in rat induced by cyclophosphamide. Overexpression of NGF is known to mediate inflammation of bladder in this model. For improved stability, antisense ODN was replaced with antisense peptide nucleic acid (PNA) and its penetration into bladder was facilitated by tethering TAT peptide sequence. Rat bladders were instilled with either antisense or its scrambled control prior to cystitis induction. Cystometrograms performed on rats under urethane anaesthesia exhibited bladder contraction frequency that was significantly decreased in the antisense treated rats than rats treated with the control. NGF immunoreactivity was also decreased in the urothelium of the antisense treated bladders. Our findings demonstrate the feasibility of using TAT-PNA conjugates for intravesical antisense therapy.
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PMID:Intravesical antisense therapy for cystitis using TAT-peptide nucleic acid conjugates. 1688 33

Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT-mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)-TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP-TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum-containing medium; (2) monocultures grown in serum-free medium; (3) cocultures with neurons in serum-free medium. The efficiency of GFP-TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP-TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT-mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT-mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell-type and phenotype-dependence of TAT-mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs.
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PMID:TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression. 1944 55