The peptides comprising the sequence of HIV-1 Tat protein (positions 48-60), Antennapedia (positions 43-58), and HIV-1 Rev protein (positions 34-50) are known to be cell-permeating. In this study, we examined how the distribution of Fab fragments in rats is affected by conjugation with these peptides. Fab fragment was iodinated by a chloramine-T method and then chemically conjugated with cell-permeating peptide. The complex of 125I-Fab and cell-permeating peptide was administered to male rats intravenously at a dose of 1 mg/kg, and whole-body autoradiography was performed at 4 and 24 h after administration. The patterns of distribution of 125I-Fab exhibited remarkable variation depending on the cell-permeating peptide used. In particular, at 4 h, high concentrations of radioactivity were observed in the spleen, adrenal gland, renal medulla, and liver with Rev peptide-Fab complex, in the liver and spleen with Tat peptide-Fab complex, and in the spleen, adrenal gland, and liver with Antennapedia peptide-Fab complex. Even at 24 h, high concentrations of radioactivity were still observed in the spleen and renal medulla of rat with Rev peptide-Fab complex, and in the spleen and renal cortex of rat with Antennapedia peptide-Fab complex. These findings demonstrate that the patterns of distribution of peptide-125I-Fab complexes can be modulated by selection of cell-penetrating peptides. Moreover, the patterns of retention of peptide-125I-Fab complexes in internal organs also differed at 24 h after administration. These findings provide valuable information for the development of novel antibody pharmaceuticals and therapeutic systems.
Successful intracellular delivery of various bioactive molecules has been reported using cell‐permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio‐iodinated Fab fragment with CPPs (CPP‐125I‐Fab) derived from HIV‐1 TAT, HIV‐1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine–0.15M NaCl, pH 3.0) was thus employed in this study to remove cell‐surface adsorbed CPP‐125I‐Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP‐125I‐Fab conjugates. The kinetics of internalization of REV‐125I‐Fab conjugate was rapid, and a considerable fraction of REV‐125I‐Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/ macropinocytosis inhibitors, in the order REV‐125I‐Fab ≥ TAT‐125I‐Fab ≥ ANP‐125I‐Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate‐buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell‐surface‐adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 98–107, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Near therapeutic concentrations of cibenzoline and disopyramide inhibit KATP channel activities in both heart and pancreatic beta cells. This may be causally related to the fasting hypoglycaemia which is sometimes reported in patients receiving the drugs. These antiarrhythmic agents may also modulate myocardial electrical properties during hypoxia or ischaemia.
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