A comment on the preparation of liposomes from and on the beta in equilibrium alpha acyl chain melting behaviour of rough mutant lipopolysaccharide.

We would like to comment on the investigations of Vaara, M., Plachy, W.Z. and Nikaido, H. (Vaara, M. et al. (1990) Biochim. Biophys. Acta 1024, 152-158) on the partitioning of hydrophobic probes in lipopolysaccharide bilayers. These authors reported that they did not succeed in preparing closed vesicles (liposomes) from rough mutant lipopolysaccharide. We describe the conditions under which lipopolysaccharide liposomes are formed most readily. We, furthermore, summarize data which strongly support the existence of thermotropic phase transitions of lipopolysaccharides (with transition temperatures lying in the range of 30-36 degrees C) contradictory to Vaara et al. who argue that such transitions are artefacts. Exemplary measurements of the beta in equilibrium alpha acyl chain melting for lipopolysaccharide from Escherichia coli deep rough mutant (strain F515) as compared to synthetic and natural phospholipids are presented using fluorescence spectroscopy, Fourier-transform infrared spectroscopy and differential scanning calorimetry. These results unequivocally prove the necessity to perform experiments at 37 degrees C for a determination of the outer membrane permeability under physiological conditions.

Conformational studies of synthetic lipid A analogues and partial structures by infrared spectroscopy.

Synthetic lipid A analogues and partial structures were analyzed and compared with natural hexaacyl lipid A from E. coli applying Fourier transform infrared spectroscopy. The investigations comprised (i) the measurement of the beta <=> alpha phase transition of the acyl chains via monitoring of the symmetric stretching vibration of the methylene groups, (ii) an estimation of the supramolecular aggregate structures evaluating vibrations from the interface like ester carbonyl and applying theoretical calculations (iii) a determination of the inter- and intramolecular conformations monitoring functional groups from the interface and the diglucosamine backbone (ester carbonyl, phosphate). The phase transition temperature Tc was found to be nearly a linear function of the number of acyl chains for most bisphosphoryl compounds indicating comparable packing density, whereas the deviating behaviour of some samples indicated a higher packing density. From the determination of the supramolecular aggregate structures (cubic, HII) of natural hexaacyl lipid A by X-ray small-angle diffraction, the existence of the same aggregate structures also for the synthetic hexaacyl lipid A was deduced from the nearly identical thermotropism of the ester carbonyl band. From this, a good approximation of the supramolecular structures of all synthetic samples was possible on the basis of the theory of Israelachvili. The analysis of the main phosphate band, together with that of the Tc data and former colorimetric results, allowed the establishment of a model of the intermolecular conformations of neighbouring lipid A/LPS molecules. The biological relevance of the findings is discussed in terms of the strongly varying biological activity (between high and no activity) of the samples.

Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans.

We have studied the role of lipopolysaccharide (LPS) for the insertion of LPS-free porin from Paracoccus denitrificans into planar lipid bilayers and its function therein. For this, we reconstituted the porin into different asymmetric planar lipid bilayers with or without LPS and into symmetric phospholipid bilayers. LPS-free porin added to the various bilayer systems was found to induce a step-wise increase in membrane conductance with different incorporation rates, depending on the presence of LPS in the bilayer leaflet opposite to porin addition. The incorporation rate into asymmetric LPS/phospholipid membranes from the phospholipid side was more than 10-fold higher than that observed for pure phospholipid membranes. The porin formed general diffusion pores without any salt specificity. The mean single-channel conductance did not depend on the presence of LPS and was about 4.2 nS for a subphase containing 1 M KCl in all systems tested. At certain applied transmembrane voltages, which depended on membrane composition and were approximately greater than 100 mV for the LPS/phospholipid system, single-channel closing in three steps was observed. Differences in the voltage dependence of porin-channel closing could be correlated with the surface charge of the bilayer. From the voltage-dependent gating behaviour proof for an oriented incorporation of the porin molecules, depending on the side of porin addition, and evidence for their orientation could be derived. Measurements at temperatures above and below the beta<==>alpha phase transition temperature of LPS gave evidence for the influence of membrane rigidity on the gating behaviour.

Cytoplasmic membrane of a sensitive yeast is a primary target for Cryptococcus humicola mycocidal compound (microcin).

A basidiomycetous yeast strain, Cryptococcus humicola 9-6, secretes a mycocidal compound (microcin) which is lethal for many yeasts. In this study a new protocol for microcin purification has been developed, and TLC-purity product was obtained. Using fluorescein as a pH-sensitive probe it was found that microcin treatment of Cryptococcus terreus, a model microcin-sensitive yeast, immediately caused transient alkalization followed by acidification of the cells' cytoplasm. Upon completion of this process, endogenous respiration as well as activity of unspecific esterases were inhibited, and alterations in cell wall and/or capsule started. Microcin was shown to make the cells leaky for intracellular ATP. The mycocidal effect of microcin did not depend on the cell cycle phase of Cr. terreus. Based on these observations and on electrical measurements on planar phospholipid bilayers, which indicated a microcin-induced membrane permeabilization, it is suggested that the cytoplasmic membrane of the sensitive yeast is a primary target of microcin action. The conjectured mode of microcin action involves gradual increase of the cytoplasmic membrane's unspecific permeability. Intracellular ion homeostasis changes induced by microcin are considered to be the main cause of enzyme inhibition, alterations in the outer layers of the cell envelope and, finally, division arrest.

Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis.

Two subtypes of the outer membrane porin PorA of Neisseria meningitidis, P1.6 and P1.7,16, were folded in vitro after overexpression in, and isolation from Escherichia coli. The PorA porins could be folded efficiently by quick dilution in an appropriate buffer containing the detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. Although the two PorA porins are highly homologous, they required different acidities for optimal folding, that is, a pH above the pI was needed for efficient folding. Furthermore, whereas trimers of PorA P1.7,16 were almost completely stable in 2% sodium dodecyl sulphate (SDS), those of P1.6 dissociated in the presence of SDS. The higher electrophoretic mobility of the in vitro folded porins could be explained by the stable association of the RmpM protein to the porins in vivo. This association of RmpM contributes to the stability of the porins. The P1.6 pores were moderately cation-selective and displayed a single-channel conductance of 2.8 nS in 1 M KCl. The PorA P1.6 pores, but not the PorA P1.7,16 pores, showed an unusual non-linear dependence of the single-channel conductance on the salt concentration of the subphase. We hypothesize that a cluster of three negatively charged residues in L5 of P1.6 is responsible for the higher conductance at low salt concentrations.

Physico-chemical analysis of lipid A fractions of lipopolysaccharide from Erwinia carotovora in relation to bioactivity.

Highly purified bisphosphoryl, monophosphoryl and dephosphoryl lipids A from Erwinia carotovora with different acylation patterns were characterized physico-chemically. Applying matrix assisted laser desorption/ionization mass spectrometry, the purity of the lipid A fractions was determined, and from monolayer measurements the molecular space requirement was estimated. Fourier transform infrared spectroscopy allowed the elucidation of the gel to liquid crystalline phase transition of the acyl chains as well as the determination of the tilt angle of the diglucosamine backbone with respect to the acyl chain direction applying dichroitic measurements with attenuated total reflectance. With synchrotron radiation small-angle X-ray diffraction the supramolecular aggregate structure was determined, and with fluorescence resonance energy transfer spectroscopy the lipopolysaccharide binding protein induced intercalation of lipid A into a phospholipid matrix corresponding to that of the macrophage membrane was investigated. From the results, a clear dependence of the physico-chemical parameters on the particular lipid A structure can be followed. Furthermore, these parameters correlate well with the biological activities of the various lipids A as deduced from their ability to induce biological activity (Limulus assay and cytokine induction in mononuclear cells). These results contribute to a closer interpretation of the physico-chemical prerequisites for endotoxic activity as found for enterobacterial lipid A.

Endotoxin: physical requirements for cell activation.

Lipopolysaccharide (LPS) is the eminent lipid component of the outer leaflet of the outer membrane of Gram-negative bacteria and the major initiator of innate immune response to bacterial infection. Below the critical micellar concentration (CMC), LPS is exclusively present as a monomer. Above this concentration, aggregates are formed. Increasing the concentration beyond the CMC leads to an increase in aggregate concentration, whereas the concentration of monomers remains constant or even decreases. The question how LPS activates immune cells and whether the aggregate or the monomer is the biologically active unit has been and still is controversial. To prepare clearly defined monomeric solutions, we utilized a dialysis set-up consisting of a donor and an acceptor chamber, separated by a dialysis diaphragm with a cut-off of 5 kDa, thus allowing only monomers to pass. Human mononuclear cells (MNCs) were then stimulated with equal concentrations of aggregates and monomers, respectively, of deep rough mutant LPS from Escherichia coli strain F515 (Re LPS) and TNF-alpha release was determined. In contrast to earlier and very recent work of others, we started with a preparation of aggregate-suspensions and pure monomer-solutions and show that monomers are significantly less active than aggregates in the absence and presence of serum proteins at identical concentrations. In our model, we propose that LPS aggregates are detected by membrane-associated LBP and intercalated into the cell membrane to bring LPS into close proximity to signaling proteins in the membrane, thus finally leading to cell activation. To support this model, we present data showing that LBP is indeed present in or at the cell membrane of human macrophages.

A K+ channel is involved in LPS signaling.

We previously showed a clear correlation between the molecular conformation of the lipid A moiety of endotoxin molecules and their cytokine-inducing capacity in mononuclear cells. While conically shaped lipid A moieties exhibit a high agonistic activity, a shift to a more cylindrically shaped lipid A leads to a decrease in agonistic and increase in antagonistic activity of the endotoxin molecules. Here, we show the involvement of a high-conductance Ca2+-activated potassium (MaxiK) channel in LPS signaling in macrophages. Corresponding to their biological activity, endotoxins activate a MaxiK channel as shown in outside-out patch-clamp experiments. LPS antagonists and anti-CD14 antibodies inhibit the LPS-induced activation of the channel. Blocking of the channel by specific channel blockers in macrophage cultures leads to inhibition of cytokine mRNA production. In particular, this result implies that there is no other independent transmembrane signaling pathway operative in macrophages. A shift of the molecular conformation of an a priori antagonistic lipid A from a cylindrical to a conical shape by adding the membrane-active compound chlorpromazine increases the activity of the MaxiK channel and the biological activity of the lipid A. We conclude that the activation of the MaxiK channel is a very early step in LPS-induced signaling in macrophages.

Influence of acyl chain fluidity on the lipopolysaccharide-induced activation of complement.

Lipopolysaccharides (LPSs, endotoxins) are the major amphiphilic constituents of the outer leaflet of the outer membrane of Gram-negative bacteria. They are known to activate the complement cascade to form lytic membrane pores. Here, we study the influence of the fluidity of the acyl chains of LPSs and lipid As on the formation of lytic pores. To this end, we have performed electrical measurements on asymmetric planar endotoxin/phospholipid bilayers as a reconstitution model of the outer membrane using two deep rough mutant LPSs (from Escherichia coli strains WBB01 and WBB25) and two lipid As (from E. coli WBB25 and Rhodobacter sphaeroides). The two LPSs and the two lipid As each differ in their acylation pattern which is correlated with the fluidity. The addition of human serum to the endotoxin side of the bilayers led to the formation of membrane pores, and pore formation correlated in each case with acyl chain fluidity, i.e. time required for the first lytic pore to be formed was shorter for the more fluid endotoxin. Furthermore, in the case of LPSs, the activation rate was higher for the more fluid membrane and the respective bacteria had a higher susceptibility to the growth inhibitory action of serum.

The role of membrane-bound LBP, endotoxin aggregates, and the MaxiK channel in LPS-induced cell activation.

We have previously shown in patch-clamp experiments on excised outside-out cytoplasmic membrane patches from human macrophages that the activation of a high-conductance Ca(2+)- and voltage-dependent potassium channel, the MaxiK channel, is an early step in LPS-induced transmembrane signal transduction in macrophages. MaxiK can be activated by agonistically active LPS, and activation can be completely inhibited by LPS antagonists (e.g. synthetic compound 406) and by anti-CD14 antibodies. Furthermore, by inhibiting MaxiK with the specific MaxiK blocker paxilline, we could show that activation of MaxiK is essential for LPS-induced cytokine production. As shown by RT-PCR, blockade of MaxiK by paxilline also inhibits induction of the mRNA of TNF-alpha and IL-6. This observation together with the fact that all patch-clamp experiments were done on excised outside-out patches reveal that MaxiK activation is an early step in cell activation by endotoxins. Thus, since cells lacking TLR4 on their surface can also not be activated to produce cytokines, these data allow the conclusion that TLR4 and MaxiK are both essential for activation by LPS and may form a co-operative signaling complex. We have also shown that LBP not only exists as a soluble acute-phase serum protein, but is also incorporated as a transmembrane protein (mLBP) in the cytoplasmic membrane of MNC; in this configuration, it is obviously involved in the binding of endotoxin and its transfer to the transmembrane signaling proteins finally triggering cell activation. Complexation of soluble LBP and LPS in the serum prior to binding of LPS to mLBP, in contrast, leads to neutralization of LPS. Here, we provide evidence from fluorescence resonance energy transfer spectroscopy that endotoxin aggregates are intercalated into reconstituted membranes by mLBP. In addition, cell culture assays and patch-clamp experiments demonstrate that endotoxin activates macrophages and the MaxiK channel in the aggregated, but not in the monomeric, state at similar concentrations.

Chemical structure of glycosphingolipids isolated from Sphingomonas paucimobilis.

Two novel glycosphingolipids were isolated from Sphingomonas paucimobilis and their structures were completely elucidated. The glycosyl portion of the glycosphingolipid consists of an alpha-D-Manp-[1----2)-alpha-D-Galp-(1----6)-alpha-D-GlcpN-(1 ----4)-alpha-D- GlcpA-R tetrasaccharide. The hydrophobic residue R was found to be heterogeneous with respect to the dihydrosphingosine residue. Erythro-1,3-dihydroxy-2-amino-octadecane and erythro-1,3-dihydroxy-2-amino-cis-13,14-methyleneoctadecane were identified in comparable amounts. Both dihydrosphingosine derivatives were quantitatively substituted by an (S)-2-hydroxymyristic acid in amide linkage.

Lipopolysaccharide-binding protein mediates CD14-independent intercalation of lipopolysaccharide into phospholipid membranes.

Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CD14-dependent whereas at high LPS concentrations it is CD14-independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CD14-independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide-binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14-independent cell activation at high endotoxin concentrations.

Detection of estrogen receptor alpha, carbonic anhydrase II and tartrate-resistant acid phosphatase mRNAs in putative mononuclear osteoclast precursor cells of neonatal rats by fluorescence in situ hybridization.

Increasing evidence suggests that estrogen deficiency in women promotes the expansion of populations of bone marrow cells that differentiate into osteoclasts under the influence of osteotropic hormones and local factors. A progressive cytoplasmic accumulation of osteoclastic bone resorbing enzymes, such as tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II), characterizes osteoclast differentiation. To evaluate the possibility that estrogen may have a direct effect on osteoclast precursor cells, we investigated the mRNA levels of estrogen receptor a (ERa), TRACP and CA II genes in neonatal rat bone imprints by fluorescence in situ hybridization and confocal microscopy. Morphological assessment of bone imprints has shown that the putative mononuclear osteoclast precursor cells (MOPC) display strongly basophilic cytoplasm and a low nuclear/cytoplasmic ratio, while some of these cells possess pale-staining ruffled border regions similar to those observed in osteoclasts. Both CA II and TRACP mRNAs were detected in putative MOPC as well as multinuclear osteoclasts. The gene transcripts were mainly located in the cytoplasm of these cells. To determine whether these putative MOPC possess ER mRNA, a 637 base pair antisense ER riboprobe was used. The results indicated that MOPC which show TRACP reactivity express high levels of ER gene transcripts in their cytoplasm. In contrast, only a few multinuclear osteoclasts in the bone imprints possessed ER gene transcripts. Interestingly, the levels of ER mRNA in these multinuclear osteoclasts were very low compared with those in the putative MOPC. Treatment with RNase prior to hybridization resulted in a significant loss of signal in these cells. The results of these studies suggest that estrogen may have a direct role in modulating the recruitment of osteoclast precursor cells during osteoclastogenesis.

Apolipoprotein E promotes the binding and uptake of beta-amyloid into Chinese hamster ovary cells in an isoform-specific manner.

The epsilon4 allele of apolipoprotein E gene is a major risk factor for Alzheimer's disease. However, the mechanism by which the E4 isoform of apolipoprotein E increases the risk of Alzheimer's disease is poorly understood. To determine whether the isoform-specific effects of apolipoprotein E may be mediated via clearance of bound beta-amyloid, we examined the uptake of beta-amyloid 1-40 into Chinese hamster ovary cells in the presence or absence of the apolipoprotein E isoforms E2, E3 and E4. Apolipoprotein E2 and E3 treatments were associated with higher association of beta-amyloid with cells as compared to treatment with E4. Heparin blocked the association of beta-amyloid with cells, as did an antibody to one of the apolipoprotein E receptors (the low-density lipoprotein receptor-related protein). Thus, the apolipoproteins E2 and E3, but not E4, may play important roles in the clearance of beta-amyloid from the extracellular space via the low-density lipoprotein receptor-related protein.

Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation.

Surface structures of bacteria contribute to the microbial pathogenic potential and are capable of causing local and generalized inflammatory reactions. Among these factors, endotoxin and peptidoglycan are of particular medical importance. Both toxic bacterial polymers are now recognized to interact with the same cellular receptor, the CD14 molecule, which is expressed on different types of immune cells, in particular, monocytes/macrophages. The interaction between these bacterial activators and CD14 leads to the production of endogenous mediators such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, which are ultimately responsible for phlogistic responses. The fact that CD14 recognizes not only endotoxin and peptidoglycan but also other glycosyl-based microbial polymers suggests that this cellular surface molecule represents a lectin.

Bacterial endotoxin: molecular relationships between structure and activity.

The significance of endotoxins in bacterial infection and their role as bacterial surface antigens (O antigens) have stimulated investigations into their chemical nature and the mechanisms of their biologic action during the last few decades. This article summarizes some of the recent results and emphasizes structure-activity relationships.

Development of an in vitro drug screening system for Mycobacterium leprae based on the determination of the intrabacterial sodium to potassium ratio of individual bacterial organisms.

In vitro drug effects on Mycobacterium leprae (M. leprae) in a cell-free system have been monitored by mass spectrometric determination of the ratio of the intrabacterial concentrations of the sodium and potassium ions (Na(+), K(+) ratio) of a limited number of individual bacteria per sample. From the drug-induced increase of the median values of the distributions of the Na(+), K(+) ratio, information on the concentration and time dependence of drug effects as well as on antagonistic or synergistic interactions of drugs has been obtained. Moreover, absolute values for the percentage of killed bacteria (% kill) have been derived from the distribution of the Na(+), K(+) ratios within a bacterial population. For this, the limiting value of the Na(+), K(+) ratio (up to which bacteria are viable) -which had been determined as 0.45 for cultivable bacteria - has been presumed to be valid also for M. leprae. Highest killing rates have been observed for fusidic acid and clarithromycin, followed by rifabutine, rifampin, and clofazimine. Minocycline and dapsone have shown only moderate killing effects and isoniazid and - probably due to the restricted metabolism of M. leprae in a cell-free medium - ofloxacin have been completely inactive. Strong ofloxacin effects, however, have been observed for cultivable mycobacteria and intracellular M. leprae phagocytized by a murine macrophage cell line.

Comparison of the intrabacterial Na+,K(+)-ratio and multiplication in the mouse foot pad as measures of the proportion of viable Myobacterium lepraemurium.

Drug are generally screened for activity against Mycobacterium leprae by administration to M. leprae-infected mice, and the efficacy of a chemotherapeutic regimen is assessed by inoculating mice with M. leprae recovered from the skin-biopsy specimens obtained at intervals during treatment. Both methods are expensive and time consuming. Although a number of methods has been proposed for the rapid distinction between viable and non viable M. leprae, none has found wide acceptance. Earlier work had shown that the ratios of the intrabacterial concentrations of Na(+) and K(+) (Na(+),K(+)-ratio) of individual bacterial cells, measured by means of laser microprobe mass analysis, are a sensitive indicator of the viability of cultivable organisms. Assuming that the maximal value (the "limiting value") of the Na(+),K(+)-ratio of viable cultivable organisms is valid for non-cultivable species, the degree of correspondence between intrabacterial Na(+),K(+)-ratios of M. lepraemurium after treatment in vivo with isoniazid, streptomycin and clofazimine, and the ability of these organisms to multiply in mice were examined. A linear relationship between the proportion of viable organisms, calculated from the limiting value of the Na(+),K(+)-ratio, and that calculated from ID50 was found, suggesting that, at least for M. lepraemurium, the intrabacterial Na(+),K(+)-ratio predicts the effect of drugs measured by the much more demanding technique of mouse inoculation.

Infrared spectroscopy of glycolipids.

The application of infrared spectroscopy to the physicochemical characterization of glycolipids as biologically important membrane constituents is described. In this contribution, the analysis of diacyl- and sphingosine-based (cerebroside, ganglioside) oligosaccharides and the class of lipid A-anchored lipooligo- and polysaccharides is reviewed. Furthermore, interaction of glycolipids with various agents as well as of sugars with membranes are discussed. The reviewed data prove the capacity of FTIR spectroscopy to monitor intra- and intermolecular interactions under physiologically relevant conditions refering to pH, temperature, and ion concentrations. This includes the characterization of acyl chain order and the beta<-->alpha chain melting transition, cation and drug binding to charged groups, and supramolecular organization of the glycolipid aggregates. In various examples, the biological relevance of IR data analysis are presented.

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides. Part I: monosaccharide headgroups.

A systematic structure variation of a classical amphiphile (dodecyl-beta-D-glucopyranoside) is performed, demonstrating the influence of anomeric linkage, configuration, ring size and flexibility as well as electric charges on the mesophase behaviour. In addition, we have investigated the thermotropic and lyotropic properties of some long chain alkyl glycosides with monosaccharide headgroups. The thermotropism was measured with polarizing microscopy and differential scanning calorimetry, and additionally the lyotropism with FTIR spectroscopy and X-ray diffraction.