Hermann J. Roth

Loading of preformed liposomes with high trapping efficiency by detergent-induced formation of transient membrane holes

Abstract Transient membrane holes induced by sodium cholate (Schubert et al., 1986, Biochemistry 25, 5263–5269) were used to encapsulate molecules at least up to M r = 70,000 into preformed liposomes. High trapping efficiencies (TE) close to 50% were achieved with concentrated suspensions of liposomes independent of their lamellarity, which was evaluated by cryomicroscopy of cryofixated suspensions. Another suitable hole-forming detergent is n -octyl β- d -glucopyranoside. Maximum trapping efficiency was measured within a narrow concentration range of added detergent below the onset of membrane solubilization. Using pure egg yolk lecithin (egg PC) or egg PC/bovine brain phosphatidylserine (7:3 mol/mol), trapping efficiency compared with the ratio of the volume inside the liposomes to the total aqueous volume in the samples. Therefore, it is probable that solutes that have been added to the external liposomal space diffuse into the liposomes, when the membranes are opened for several minutes. TE is slightly reduced by 30 mol% cholesterol in the membrane or if highly concentrated protein solutions (insulin or hemoglobin) or molecules with elongated form (polyuridylic acid) are to be encapsulated. An especially low TE into DPPC liposomes at 25°C is indicative of suppressed hole formation in the gel phase of the membrane. The residual amount of detergent after rechromatography of the loaded liposomes and liposome alterations are negligible. Detergent-induced liposome loading (DILL) is a convenient method for entrapping small molecules, larger carbohydrates, proteins, and nucleic acids into preformed liposomes with membranes in the liquid crystal phase. In addition, the ratio of calculated to found trapping efficiency of inulin is a suitable value for the mean lamellarity of vesicles up to a diameter of 200 nm.

Surface and bulk interactions of organic molecules with calixarene layers

Modified calixarenes can be used as model compounds to study molecular recognition since their molecular cavities reversibly incorporate small organic molecules. This effect has been used in chemical sensors with bulk and surface acoustic wave devices, coated with thin calixarene layers. These devices sensitively convert the mass changes during molecule/calixarene interactions into electronic signals. Thin films of modified calixarenes were prepared with various side groups and various sizes by Knudsen sublimation under well-defined, ultra-high-vacuum conditions. The interaction with perchloroethylene, chloroform, benzene, and toluene at constant temperatures, T, and partial pressures, pi, was studied systematically for different film thicknesses, d, by means of mass changes Δm=f(T,pi,d). From the thickness dependence of values, Δmeq, obtained under thermodynamic equilibrium conditions, surface and bulk effects during molecule calixarene interactions were separated. Pronounced excesses of surface concentrations of organic molecules have been found. Activation energies for molecular desorption from surface sites and for diffusion to subsurface sites were determined from mass spectroscopic results of thermal desorption behaviour. Experimental data of adsorption and desorption energies were confirmed by theoretical force field calculations.

Supramolecular complexes based on calixarenes: force field calculations and applications for chemical sensors

Abstract The inclusion of organic molecules to different calixarenes was investigated both experimentally and theoretically. Experimental data were obtained for various calixarenes of different thicknesses interacting with perchloroethylene, chloroform, benzene and toluene at different partial pressures and temperatures. The amount of included molecules was monitored by frequency responses of mass-sensitive bulk acoustic wave devices. To estimate ‘key-lock’ binding energies, the activation energy of desorption was determined experimentally from thermodesorption spectroscopy. The host/guest interaction energies were calculated theoretically with the TRIPOS force field approach which, in contrast to current quantum chemical approaches (MOPAC/AM1, MOPAC/PM3), gave satisfactory distance-dependent energy minima. Experimentally observed changes in the binding energies for the inclusion of C 2 Cl 4 , CHCl 3 , CH 3 C 5 H 6 and C 6 H 6 molecules in calixarenes agree surprisingly well with results with theoretical calculations.

A new synthesis of thiophosphoric acid esters with a C-S-P bond

Abstract Various S-substituted thiosuccinimides 1a-d and thiophthalimides 2a-d were found to react with trialkylphosphites according to a Michaelis-Arbuzov type mechanism. This provides an efficient way to prepare thiophosphoric acid esters, particularly thiophospholipids, with a C-S-P bond.

New series of lipoxins isolated from human eosinophils

Granulocytes from human eosinophilic donors were incubated with arachidonic acid or 15‐hydroxyeicosatetraenoic acid (15‐HETE) and stimulated with the ionophore A23187. The eicosanoids were extracted with reversed‐phase cartridges and subjected to RP‐HPLC analysis. When extracts from eosinophil‐enriched populations were analysed and compared with extracts from human neutrophils, three additional peaks were detected which coeluted with 15‐hydroxy‐Δ13‐trans‐15H derivatives of leukotriene C4, D4 and E4 in different HPLC systems. The recorded absorbance spectra of the eluted compounds and the standards were identical and showed a maximum at 307 nm which is characteristic for a conjugated tetraene system with a bathochromic shift by the sulfur moiety in α‐position to the tetraene system. The compound which coeluted with the 15‐hydroxy‐LTC4 standard was treated with γ‐glutamyltransferase and converted to the corresponding leukotriene D4 derivative. The results indicate that interaction between the 5‐ and 15‐lipoxygenase pathways leads to the formation of a new series of arachidonic acid metabolites in human eosinophils. Since the biosynthetic route is similar to that of lipoxin A4 and lipoxin B4, we suggest the trivial names lipoxin C4, D4 and E4.

New class of 5-lipoxygenase inhibitors: correlation between inhibition of LTB4 production and chemiluminescence of human polymorphonuclear granulocytes

The effect of several series of compounds on the biosynthesis of leukotriene B4 in human polymorphonuclear granulocytes (PMNL) was measured after stimulation of the cells by the Ca2+ ionophore A23187. Phenylhydrazone derivatives and some pyrrole derivatives strongly inhibited 5-lipoxygenase activity at 10 µM. In contrast, 4-aminopyrrolopyrimidines and 4-oxopyrrolopyrimidmes did not exhibit any effect. The compounds examined for inhibition of 5-lipoxygenase were also tested in a chemiluminescence assay for potential effects on the emission of photons as a measure of activated oxygen species generated by the stimulated granulocytes. There was a good correlation between lipoxygenase inhibition and suppression of chemiluminescence; however, some derivatives with no inhibitory activity against lipoxygenase still reduced chemiluminescence, which suggests that an alternative inhibitory mechanism of chemiluminescence must be present. Cyclooxygenase inhibitors such as aspirin did not suppress chemiluminescence of granulocytes. It is, therefore, unlikely that cyclooxygenase is required in the chemiluminescence reaction. In contrast, LTB4 seems to play an important role in the zymosan-induced chemiluminescence of PMNL.

Lipophilic disulfide prodrugs — syntheses and disulfide bond cleavage

Abstract Various drugs bearing a thiol or thione function were coupled with activated lipophilic thiol derivatives to afford unsymmetrical disulfides. Synthetic methods were developed to introduce one or two long alkyl chains into drug molecules in order to obtain highly lipophilic prodrugs. These might be suitable to form bilayers or for integration into liposomes. Disulfide bond cleavage was assessed by preincubation of the 6-MP prodrug 2 in serum followed by a bioassay. The preincubation period did not increase the inhibitory potency of the prodrug on lymphocyte proliferation as compared to the parent drug. Thus disulfide bond cleavage of prodrug 2 is assumed to be an active cellular process.

Determination of picogram amounts of lipoxin A4 and lipoxin B4 by high-performance liquid chromatography with electrochemical detection

A new method for the determination of lipoxins with electrochemical detection after high-performance liquid chromatography is described. The half-wave potentials of lipoxin A4 and lipoxin B4 at a glassy carbon electrode and a mobile phase of methanol-water (65:35 v/v) and 1 mM trifluoroacetic acid was found to be +1.14 V versus an Ag/AgCl reference electrode. The use of trifluoroacetic acid instead of sulphuric acid and lithium perchlorate led to a background current of 6-8 nA at +1.20 V. The detection limits for both lipoxins, based on a signal-to-noise ratio of 3:1 were found to be 5-10 pg (15-30 fmol). The new method was applied to an extract of human polymorphonuclear granulocytes, preincubated with 15-hydroxyeicosatetraenoic acid and stimulated with Ca2+ ionophore A23187.

L-ascorbic acid modulates 5-lipoxygenase activity in human polymorphonuclear leukocytes.

Leukotriene B4 (LTB4), a metabolite of the arachidonic acid pathway mediated by 5-lipoxygenase, is released by stimulated polymorphonuclear neutrophils (PMNL) and has been postulated to be an important mediator of the inflammatory response. Extracellular L-ascorbate at concentrations in the range of 0.5-5.0 mM effectively inhibits the biosynthesis of LTB4 and 5-hydroxyeicosatetraenoic acid (5-HETE) stimulated by the calcium ionophore A 23187. The ionophore-activated LTB4 production is reduced after incubation of PMNL with opsonized zymosan as a phagocytic stimulus. Extracellular L-ascorbate at concentrations above 0.1 mM reverses the zymosan-induced deactivation of 5-lipoxygenase, resulting in significantly higher LTB4 and 5-HETE yields. The inhibitory effect of zymosan preincubation on LTB4 production is independent of calcium or free arachidonic acid in the incubation medium. Interaction of L-ascorbate with the catabolism of LTB4 was excluded by measuring the trihydroxy metabolites which were unchanged. Furthermore, the formation of glutathione derivatives of leukotriene A4 can be excluded due to the lack of glutathione transferase activity in PMNL. In order to link the intracellular function of L-ascorbate with the serum level, the ascorbate uptake has been studied in more detail. The L-ascorbate transport into PMNL is stereospecific and can best be described by kinetics consisting of a saturation part, Km and Vmax being 39 microM and 0.28 nmol/10(8) cells.min, respectively, plus passive diffusion, the diffusion coefficient P being 1.75 microliter/10(8) cells.min. Furthermore, the uptake is inhibited by the isomers D-ascorbic acid and D-erythorbic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of picomole amounts of lipoxins C4, D4 and E4 by high-performance liquid chromatography with electrochemical detection

A method for the sensitive determination of the sulphopeptide lipoxins (LXs) C4, D4 and E4 by high-performance liquid chromatography with subsequent electrochemical detection is described. The best results were obtained when the analysis was carried out with the solvent system methanol-water-trifluoroacetic acid (66:34:0.008, v/v/v). The acquired half-wave potentials were different for all investigated compounds: +1.18 V for LXC4 +1.3 V for LXD4 and +1.25 V for LXE4. The detection limits of LXC4, LXD4 and LXE4, based on a signal-to-noise ratio of 3:1, were found to be 200-700 fmol. Although sulphopeptide lipoxins possess a high molar absorptivity, electrochemical detection still is three times more sensitive than ultraviolet detection.