We show here that Unc45b is a cytosolic protein in eukaryotic cells and isolates as a stable complex with Hsp90

also were shown to reach low minimum surface tensions,1 mN/m during 10 successive cycles of quasi-static or dynamic compression on the IPI 145 captive bubble surfactometer. In addition, DEPN-8+1.5% Mini-B was chemically resistant to degradation by PLA2 in vitro, while CLSE was severely degraded by this enzyme. The high surface activity, albumin inhibition resistance, and phospholipase resistance of DEPN8+1.5% Mini-B supports the continuing development of related fully-synthetic exogenous surfactants for treating inflammatory lung injuries such as ALI/ARDS. and 10 ml trifluoroacetic acid per gram of resin. The cleavage-deprotection mixture was chilled to 5uC and added to the resin, and then allowed to come to 25uC with continuous stirring over a period of 2 hrs to insure complete deprotection. The crude peptide was removed by vacuum-assisted filtration, followed by washing the resin on a medium porosity sintered glass filter with trifluoroacetic acid and then dichloromethane to 14500812 remove residual peptide. The filtrate was precipitated with ice cold tertiary butyl ether and separated by centrifugation at 20006g for 10 min. The crude peptide in the reduced state was dissolved in trifluoroethanol:10 mM HCl and freeze-dried, followed by further purification using preparative scale HPLC. The mass of final purified peptide was confirmed by MALDI TOF mass spectrometry, and peptide concentrations in physical studies were determined by UV absorbance at 280 nm. MATERIALS AND METHODS -trimethylammonium, monopropyl) ester DEPN-8 was synthesized and purified as described previously by Schwan, Notter, and co-workers. The chemical scheme for preparing DEPN-8 was based on the conversion of -1hexadecyloxy-2,3-propanediol to -2,3-bis-1-propanol by way of hydroxyl protection at the 3-position, alkylation at the 2-hydroxyl group, and deprotection. Phosphonocholine placement involved treatment of -2,3-bis-1propanol with 3-bromopropylphosphono-di-chloridic acid prepared from 3-bromopropylphosphonic acid and PCl5, followed by reaction with Me3N in CHCl3:MeOH:H2O. After concentration, the crude lipid was exposed to AmberliteH and subjected to flash chromatography with CHCl3:MeOH:H2O as the elution solvent. Final purification of DEPN-8 was through recrystallization from CHCl3/acetone, as verified by both 13C and 1H NMR spectroscopy. In the former, only peaks expected for the product were observable, and 1H NMR exhibited a lone trimethylammonium resonance. DEPN-8 also gave a single spot on thin layer chromatography using a solvent system of 30:9:25:7:25 chloroform:methanol:2-propanol:water:triethylamine. CLSE CLSE was prepared by chloroform:methanol extraction of the large aggregate fraction of lung surfactant obtained by centrifugation of saline lavage from the intact lungs of freshly-killed calves as detailed previously. Phospholipase A2 and serum albumin for inhibition studies PLA2 was suspended in 0.15 M NaCl and 1.5 mM CaCl2 and incubated with surfactants dispersed in the same solvent for 30 min at 37uC . Chemical degradation was assessed by determining phosphate levels in thin layer chromatographic bands. Albumin was combined with dispersed surfactants in 1.5M NaCl+1.5 mM CaCl2 and allowed to incubate at room temperature for 1530 min prior to activity measurements on 24847734 the pulsating bubble surfactometer. Lipid-Peptide Binding by Plasmon Resonance Binding affinities of Mini-B for DEPN-8 and DPPC were measured with a Biacore 3000 system. Mini-B films were chemically linke

Leave a Reply