SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

The electrostatic properties of charged bilayers and the bilayer component of biological membranes are often described theoretically by assuming the charge is smeared uniformly over the surface. This is one of the fundamental assumptions in the Gouy-Chapman-Stern (GCS) theory. However, the average distance between the charged phospholipids in a typical biological membrane is 2-3 nm, which is 2-3 times the Debye length in a 0.1 M salt solution. Existing discreteness-of-charge theories predict significant deviations from the GCS theory for the adsorption of ions to such membranes. We considered the predictions of the simplest discreteness-of-charge theory [Nelson, A. P., & McQuarrie, D. A. (1975) J. Theor. Biol. 55, 13-27], in which the charges are assumed to be fixed in a square lattice and the potential is described by the linearized Poisson-Boltzmann relation. This theory predicts deviations that are larger for counterions than for co-ions and much larger for divalent than for monovalent counterions. We tested these predictions by measuring the adsorption of a fluorescent monovalent anion and a paramagnetic divalent cation to both positive and negative membranes, which we demonstrated experimentally had the same average surface potential. All our experimental results with probes, including those obtained on membranes in the gel rather than in the liquid-crystalline state, agreed with the predictions of the GCS theory rather than with the discreteness-of-charge theory. A simple calculation indicates that the agreement between the experimental results and the predictions of the GCS theory could be due to the finite size of the lipids.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.

Although the Gouy-Chapman-Stern theory of the aqueous diffuse double layer describes well the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes, it does not describe adequately the zeta potential of biological membranes: the zeta potential of an erythrocyte is about half the value predicted from the theory by using the known density of negatively charged sialic acid residues. To investigate the factors responsible for this low electrophoretic mobility, we formed membranes from mixtures of the zwitterionic lipid phosphatidylcholine, PC, and the glycolipid galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl) -galactosylglucosylceramide, GM1. This glycolipid differs from phospholipids in two respects. First, the negative charge on GM1 is located about 1 nm from the surface, which tends to increase the electrophoretic mobility of vesicles. Second, the head group of GM1 contains five sugar groups that exert a hydrodynamic drag, which tends to decrease the mobility of the vesicles. In a decimolar monovalent salt solution, where the Debye length is about 1 nm, the electrophoretic mobility of the PC-GM1 vesicles is about half the mobility of PC-phosphatidylserine or PC-phosphatidylglycerol vesicles of equivalent composition. In addition, conductance measurements with planar bilayer membranes as well as 31P nuclear magnetic resonance and fluorescence measurements with sonicated vesicles indicate that the potential at the surface of PC-GM1 membranes is about half the value measured for PC-phosphatidylserine membranes in a 0.1 M monovalent salt solution.

We formed vesicles from mixtures of egg phosphatidylcholine (PC) and the gangliosides GM1, GD1a, or GT1 to model the electrokinetic properties of biological membranes. The electrophoretic mobilities of the vesicles are similar in NaCl, CsCl, and TMACl solutions, suggesting that monovalent cations do not bind significantly to these gangliosides. If we assume the sialic acid groups on the gangliosides are located some distance from the surface of the vesicle and the sugar moieties exert hydrodynamic drag, we can describe the mobility data in 1, 10, and 100 mM monovalent salt solutions with a combination of the Navier-Stokes and nonlinear Poisson-Boltzmann equations. The values we assume for the thickness of the ganglioside head group and the location of the charge affect the theoretical predictions markedly, but the Stokes radius of each sugar and the location of the hydrodynamic shear plane do not. We obtain a reasonable fit to the mobility data by assuming that all ganglioside head groups project 2.5 nm from the bilayer and all fixed charges are in a plane 1 nm from the bilayer surface. We tested the latter assumption by estimating the surface potentials of PC/ganglioside bilayers using four techniques: we made 31P nuclear magnetic resonance, fluorescence, electron spin resonance, and conductance measurements. The results are qualitatively consistent with our assumption.