RESUMO
Fibroblast growth factor 2 (basic FGF or FGF2) has been shown to affect growth and differentiation in some tissues and to be required for cardiac hypertrophy in vivo. FGF2 has been shown in vitro to signal through the mitogen-activated protein kinase (MAPK) to affect cell survival and growth. To ascertain the role of FGF2 in cardiac hypertrophy, wildtype, Fgf2 knockout, non-transgenic, and FGF2 transgenic mice were treated with isoproterenol or saline via subcutaneous mini-osmotic pump implants to induce a hypertrophic response to ß-adrenergic stimulation. Fgf2 knockout hearts are protected from isoproterenol-induced cardiac hypertrophy; whereas, FGF2 transgenic hearts show exacerbated cardiac hypertrophy as assessed by heart weight-to-body weight ratios and myocyte cross-sectional area. Echocardiography reveals significantly decreased fractional shortening in isoproterenol-treated FGF2 transgenic mice but not in Fgf2 knockout mice suggesting that FGF2 mediates the maladaptive cardiac dysfunction seen in cardiac hypertrophy induced by isoproterenol. Western blot analysis also reveals alterations in MAPK signaling in Fgf2 knockout and FGF2 transgenic hearts subjected to isoproterenol treatment, suggesting that this cascade mediates FGF2's pro-hypertrophic effect. Pharmacologic inhibition of extracellular signal-regulated kinase (ERK) signaling results in an attenuated hypertrophic response in isoproterenol-treated FGF2 transgenic mice, but this response is not seen with p38 mitogen-activated protein kinase (p38) pathway inhibition, suggesting that FGF2 activation of ERK but not p38 is necessary for FGF2's role in the mediation of cardiac hypertrophy.
RESUMO
A cleft-[2]rotaxane (CR2+2-) was derivatized with carboxylic acids to enhance the intracellular delivery of a highly cationic or anionic pentapeptide. CR2+2- delivers the fluorescein (Fl) tagged peptide Fl-KKALR to a greater amount than Fl-QEAVD, and at a higher concentration, a greater amount than Fl-AVWAL. The level of delivery is largely temperature and ATP independent, suggesting that the Fl-peptide.CR2+2- complexes pass through the cellular membrane without requiring active cell-mediated processes. This study shows that selective delivery of peptides is possible by using a suitably derivatized host-rotaxane as the transporter.
Assuntos
Ácidos Carboxílicos/química , Peptídeos/química , Rotaxanos/química , Cátions , FluorescênciaRESUMO
A host-[2]rotaxane was constructed by converting a diaminophenylcalix[4]arene into a [2]rotaxane using the DCC-rotaxane method (Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486). N-Ac-Arg groups were attached to the dibenzo-24-crown-8 ring of the rotaxane to provide a convergent functional group. To demonstrate the advantage provided by the rotaxane architecture for recognition of guests that contain a variety of functional groups, association constants (K(A)) for N-Ac-Trp, indole, N-Ac-Gly, fluorescein, 1-(dimethylamino)-5-naphthalenesulfonate, and pyrene bound to the [2]rotaxane were determined by performing (1)H NMR and fluorescence spectroscopic experiments. The host-[2]rotaxane had the highest affinity for fluorescein with a K(A) = 4.6 x 10(6) M(-)(1) in a 98/2 buffer (1 mM phosphate, pH 7)/DMSO solution. A comparison of K(A) values demonstrates that both the aromatic pocket and ring of the host-[2]rotaxane contribute binding free energy for complexation. Association constants were also derived for the same guests bound to the diaminophenylcalix[4]arene and to a diphenylcalix[4]arene that contained arginine residues displayed in a nonconvergent fashion. The host-[2]rotaxane provides higher affinity and specificity for most guests than the host with divergent N-Ac-Arg groups of the one that only has an aromatic pocket. For example, the K(A) for the complex of the host-[2]rotaxane and fluorescein in the DMSO/water mixture is more than 2 orders of magnitude greater than association constants derived for the other hosts.
RESUMO
Host-[2]rotaxanes, containing a diarginine-derivatized dibenzo-24-crown-8 (DB24C8) ether as the ring and a cyclophane pocket or an aromatic cleft as one blocking group, are cell transport agents. These hosts strongly associate with a variety of amino acids, dipeptides, and fluorophores in water (1 mM phosphate buffer, pH 7.0), DMSO, and a 75/25 (v/v) buffer to DMSO solution. All peptidic guests in all solvent systems have association constants (K(A)'s) in the range of 1 x 10(4) to 5 x 10(4) M(-)(1), whereas the K(A) range for the fluorophores is 1 x 10(4) to 9 x 10(5) M(-)(1). Association constants for the cyclophane itself, cyclophane 3, are smaller. These values are in the 1 x 10(3) to 5 x 10(3) M(-)(1) range, which shows that the rotaxane architecture is advantageous for guest binding. Cyclophane-[2]rotaxane 1 efficiently transports fluorescein and a fluorescein-protein kinase C (PKC) inhibitor into eukaryotic COS-7 cells, including the nucleus. Interestingly, cleft-[2]rotaxane 2 does not transport fluorescein as efficiently, even though the results from the fluorescence assays show that both [2]rotaxanes bind fluorescein with the same ability.