Hansjörg Eibl

Structure and biochemistry of mouse hepatic gap junctions.

A new method for the isolation of gap junctions from mouse liver is described. Particular attention has been directed to minimising the effects of proteolysis during isolation. The purified membrane fragments retain the typical morphological features found in junctions of the intact liver. The junctions show two major polypeptides upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The apparent molecular weights are 26,000 for the more abundant species and 21,000 for the minor component. Preliminary protein chemical characterisation by fingerprint analysis suggests that the two polypeptides are structurally related. While an in vivo origin of the 21,000 molecular weight species cannot be excluded, the sensitivity of the junction proteins to proteolytic degradation in vitro suggests that the 21,000 molecular weight molecule may be a breakdown product of the major component. Image reconstruction methods applied to micrographs of negatively stained isolated junctions show that the membrane contains a close-packed hexagonal lattice of components having marked 6-fold symmetry. It is suggested that these represent hexamers of the 26,000 molecular weight protein. Lipid analysis performed on gap junctions isolated by different procedures shows that the lipid composition is strongly affected by the detergents employed during the isolation. A large amount of phopholipid, but not cholesterol, can be extracted from the structure without affecting its gross morphology. This result suggests that cholesterol is tightly bound to the junction protein and may play a role in determining the structure of the gap junction.

Hexadecylphosphocholine: oral treatment of visceral leishmaniasis in mice.

Hexadecylphosphocholine (He-PC), a novel phospholipid derivative, was tested against Leishmania donovani and Leishmania infantum, the causative agents of visceral leishmaniasis. In vitro, promastigotes were highly susceptible to He-PC; the 50% inhibitory concentrations were between 0.89 and 2.25 micrograms/ml for the different leishmanial strains. In vivo, a marked antileishmanial activity in infected BALB/c mice could be demonstrated after oral administration of He-PC. Whereas parasite suppression and killing in the liver were comparable after 5 days of treatment with He-PC (10 or 20 mg/kg of body weight per day administered orally) and sodium stibogluconate (120 mg of pentavalent antimonal agent per kg/day administered subcutaneously), a superior reduction in the parasite load in the spleen and bone marrow was observed after oral treatment with He-PC. After a 4-week treatment period, parasite suppression in the spleen was better than that observed with standard sodium stibogluconate therapy by a factor of more than 600.

Synthesis of glycerophospholipids

Recent advances in the synthesis of phosphatidic acids, phosphatidylethanolamines and phosphatidylcholines are described. Methods for the synthesis of some alkylacyl and alk-1-enylacyl analogues of the common diacylglycerophospholipids are also discussed. In addition, synthetic routes are described, that lead to unusual phospholipids such as compounds containing the polar group at position 2 of the glycerol moiety, glycerophospholipids containing alkanolamines of different chain lengths, and glycolphospholipids. All of the common glycerophospholipids can be prepared without the use of protecting groups. Major advances in phospholipid synthesis involve the application of novel phosphorylating agents and the use of cyclic intermediates. Although phosphatidylserines and phosphatidylthreonines as well as phosphatidylglycerols and cardiolipins can be prepared by chemical synthesis, further systematic studies are required to work out procedures that lead to these compounds in high yields.

The influence of charge on phosphatidic acid bilayer membranes

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, 1,2-dihexadecyl-sn-glycerol-3-phosphoric acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of the phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; 1,2-dimyristoyl-sn-glycerol-3-phosphoric acid, 1,3-dimyristoylglycerol-2-phosphoric acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoric acid. The phase transition temperatures (Tt) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90 degrees light scattering. The Tt values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states. The Tt vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (pK1). The regions between the first and the second pK of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the Tt vs. pH diagram beyond pK2 becomes very steep. This is the result of reduced hydrocarbon interaction energy, which was demonstrated by differential scanning calorimetry (Blume, A. and Eibl, H., unpublished data).

HEXADECYLPHOSPHOCHOLINE - A NEW AND SELECTIVE ANTITUMOR DRUG

Interdisciplinary cooperation between basic and clinical research has resulted in the discovery and development of alkylphosphocholines, a new class of substances for the treatment of breast cancer. In contrast to most antitumor substances, the alkylphosphocholines do not attack the cell nucleus, but the cell membrane. This report presents a systematic study which, for the first time, provides a correlation between their chemical structure, antitumor efficacy and selectivity. Through an understanding of the metabolism of tumor growth inhibiting (ether)-lysolecithins, the minimal structural requirements for the antineoplastic efficacy of these substances have been obtained. This knowledge was used to identify molecular structures which are more effective and less toxic for the organism. The active principle derived from a study of (ether)-lysolecithins active as antitumor agents represents a new class of compounds: the alkylphosphocholines. As reported here, hexadecylphosphocholine is the most promising candidate of this group of compounds. It has an extremely selective action against chemically induced, autochthonous rat mammary carcinomas. No loss of activity was observed when comparing oral and intravenous administration. Particularly striking (and favorable for long-term therapy) is the fact that immunosuppression and hematotoxicity were not found at drug concentrations which lead to complete tumor remissions. Results obtained from animal experiments have been confirmed by preliminary clinical investigations.

X-ray analysis and calorimetry on phosphatidylcholine model membranes. The influence of length and position of acyl chains upon structure and phase behaviour

The effect of variation of the acyl chain composition of phosphatidylcholines upon thermal behaviour of multilamellar liposomes was evaluated by calorimetry and X-ray studies. A total of thirteen different phosphatidylcholines were examined. They differed from each other in the length as well as in the position of the acyl chains in the glycerol backbone. The experimental results show that the hitherto accepted phase scheme for phosphatidylcholine-water systems is incomplete and has to be extended to include the behaviour of samples that have been stored for long times at low temperatures. The X-ray results show that the structure of the new low-temperature phase is not in agreement with the hexagonal packing of the acyl chains. To explain the X-ray results, a two-dimensional orthorhombic unit cell has to be assumed in order to fit all the observed reflexes in the wide-angle region.

BCR‐ABL influences the antileukaemic efficacy of alkylphosphocholines

We have compared the antileukaemic efficacy of a series of new i.v. injectable alkylphosphocholines (APC) with their clinically used congeners miltefosine and perifosine. The test system consisted of four leukaemic cell lines carrying the bcr‐abl rearrangement (K‐562, LAMA‐84, CML‐T1 and BV‐173) and two other leukaemic cell lines (HL‐60 and SKW‐3) without this genetic alteration. The prototype of i.v. injectable APC, erucylphosphocholine, was more active against BCR‐ABL‐positive cell lines than the two reference APC. It induced programmed cell death in HL‐60 and SKW‐3 cells after exposure for 24 h, and in bcr‐abl expressing cells after a prolonged incubation period (48 h). LAMA‐84 cells responded to i.v. injectable APC with increased conversion to an adherent, fibroblast‐like phenotype. Experiments with a cell‐free system showed that the target structures of APC are localized within the cytoplasmic compartment. Blockade of ceramide synthase by fumonisin B1 was insufficient to prevent oligonucleosomal DNA fragmentation. Using RT‐PCR we confirmed that K‐562 and LAMA‐84 cells carry the b3a2 fusion type, and CML‐T1 and BV‐173 the b2a2 variant. BV‐173 cells had the lowest level of bcr‐abl mRNA which correlated with their increased sensitivity. Transfection of K‐562 cells with antisense oligonucleotides directed against bcr‐abl caused a specific suppression of K‐562 clonogenicity. Our data indicated that i.v. injectable alkylphosphocholines are potent inducers of apoptosis and display increased antileukaemic efficacy against BCR‐ABL‐positive blasts as compared with miltefosine and perifosine. The expression of BCR‐ABL cannot prevent apoptosis but delays erucylphosphocholine‐induced programmed cell death. Transfection with bcr‐abl directed antisense oligonucleotides reduces the clonogenicity of K‐562 cells.

[53] Preparation of phospholipids and their analogs by phospholipase D

Publisher Summary This chapter discusses the preparation of phospholipids and their analogs by phospholipase D. Egg phosphatidylcholine was extracted from egg yolk and purified on silica gel 60, using solvent mixtures of chloroform-methanol-ammonia in ratios of 200:15:1 to start with and 65: 15:1 for the elution of egg phosphatidylcholine. The pure lipid dissolved in chloroform-methanol, 9:1 (by volume), was stored at 0° under nitrogen. The transfer of the phosphatidyl residue from phosphatidylcholine to primary alcohols or ethanolamines was detected by TLC in the solvent system chloroform-methanol-ammonia 65:30:3. The hydrolysis of phosphatidylethanolamines was performed in the presence of Triton X-100, which also facilitated the formation of clear emulsions and thus allowed complete hydrolysis of the substrates. It is found that no difference in the extent and rate of transphosphatidylation was observed when smaller samples were used in the experiments with respectively reduced amounts of enzyme and primary alkanol and volumes of the buffer–ether mixture.

Conformation and packing properties of membrane lipids: The crystal structure of sodium dimyristoylphosphatidylglycerol

The conformation and molecular packing of sodium 1,2-dimyristoyl-sn-glycero-phospho-rac-glycerol (DMPG) have been determined by single crystal analysis (R = 0.098). The lipid crystallizes in the monoclinic spacegroup P2(1) with the unit cell dimensions a = 10.4, b = 8.5, c = 45.5 A and beta = 95.2 degrees. There are two independent molecules (A and B) in the asymmetric unit which with respect to configuration and conformation of their glycerol headgroup are mirror images. The molecules pack tail to tail in a bilayer structure. The phosphoglycerol headgroups have a layer-parallel orientation giving the molecules an L-shape. At the bilayer surface the (-) phosphoglycerol groups are arranged in rows which are separated by rows of (+) sodium ions. Laterally the polar groups interact by an extensive network of hydrogen, ionic and coordination bonds. The packing cross-section per molecule is 44.0 A2. The hydrocarbon chains are tilted (29 degrees) and have opposite inclination in the two bilayer halves. In the chain matrix the chain planes are arranged according to a so far unknown hybride packing mode which combines the features of T parallel and O perpendicular subcells. The two fatty acid substituted glycerol oxygens have mutually a - synclinal rather than the more common + synclinal conformation. The conformation of the diacylglycerol part of molecule A and B is distinguished by an axial displacement of the two hydrocarbon chains by four methylene units. This results in a reorientation of the glycerol back bone and a change in the conformation and stacking of the hydrocarbon chains. In molecule A the beta-chain is straight and the gamma-chain is bent while in molecule B the chain conformation is reversed.

Cytotoxic Activities of Alkylphosphocholines against Clinical Isolates of Acanthamoeba spp.

ABSTRACT Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.