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The combination of grazing-incidence X-ray scattering experiments and Monte Carlo simulation unravels the physics of bacterial survival against cationic antimicrobial peptides (protamine). As a realistic model of bacterial outer membranes, an insoluble monolayer of lipopolysaccharide from Salmonella enterica sv. Minnesota Ra (LPS Ra) is spread on buffered subphase. In the presence of Ca 2+ , vertical electron density profiles reconstructed from X-ray scattering imply the “collapse” of saccharide chains, suggesting that Ca 2+ bridges the negatively charged saccharide units. Under this condition, the LPS monolayer remains intact even after injection of protamine near the minimum inhibitory concentration. This can theoretically be accounted in terms of the formation of an electrostatic energy barrier that prevents the approach of protamine to the hydrophobic region. In contrast, as predicted from in vivo experiments, the intrusion of protamine in the absence of Ca 2+ results in the complete destruction of the layered structure of LPS Ra monolayers.

Lipopolysaccharide (LPS), which constitutes the outermost layer of Gram-negative bacterial cells as a typical component essential for their life, induces the first line defense system of innate immunity of higher animals. To understand the basic mode of interaction between bacterial LPS and phospholipid cell membranes, distribution patterns were studied by various physical methods of deep rough mutant LPS (ReLPS) of Escherichia coli incorporated in phospholipid bilayers as simple models of cell membranes. Solid-state 31P-NMR spectroscopic analysis suggested that a substantial part of ReLPS is incorporated into 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid bilayers when multilamellar vesicles were prepared from mixtures of these. In egg L-α-phosphatidylcholine (egg-PC)-rich membranes, ReLPS undergoes micellization. In phosphatidylethanolamine-rich membranes, however, micellization was not observed. We studied by microscopic techniques the location of ReLPS in membranes of ReLPS/egg-PC (1:10 M/M) and ReLPS/egg-PC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (1:9:1 M/M/M). The influence of ReLPS on the physicochemical properties of the membranes was studied as well. Microscopic images of both giant unilamellar vesicles and supported planar lipid bilayers showed that LPS was uniformly incorporated in the egg-PC lipid bilayers. In the egg-PC/POPG (9:1M/M) lipid bilayers, however, ReLPS is only partially incorporated and becomes a part of the membrane in a form of aggregates (or as mixed aggregates with the lipids) on the bilayer surface. The lipid lateral diffusion coefficient measurements at various molar ratios of ReLPS/egg-PC/POPG indicated that the incorporated ReLPS reduces the diffusion coefficients of the phospholipids in the membrane. The retardation of diffusion became more significant with increasing POPG concentrations in the membrane at high ReLPS/phospholipid ratios. This work demonstrated that the phospholipid composition has critical influence on the distribution of added ReLPS in the respective lipid membranes and also on the morphology and physicochemical property of the resulting membranes. A putative major factor causing these phenomena is reasoned to be the miscibility between ReLPS and individual phospholipid compositions.

Macrophages have a pivotal function in innate immunity against bacterial infections. They are present in all body compartments and able to detect invading microorganisms with high sensitivity. LPS (endotoxin) of Gram-negative bacteria is among the most potent stimuli for macrophages and initiates a wide panel of cellular activation responses. The release of mediators such as TNF-α and ILs is essential for the initiation of a proinflammatory antibacterial response. Here, we show that blockade of the large-conductance Ca2+-activated potassium channel MaxiK (BK) inhibited cytokine production from LPS-stimulated macrophages at the transcriptional level. This inhibitory effect of channel blockade was specific to stimulation with LPS and affected neither stimulation of macrophages with the cytokine TNF-α nor LPS-induced activation of cells that do not express MaxiK. Investigation of the upstream intracellular signaling pathways induced by LPS revealed that the blockade of MaxiK selectively inhibited signaling pathways leading to the activation of the transcription factor NF-κB and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor IκB-α is critically involved in the observed inhibition of NF-κB translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-κB and subsequent cytokine production is not restricted to in vitro-generated monocyte-derived macrophages but also can be observed in primary cells. Thus, MaxiK appears to be a central molecule in the NF-κB-dependent inflammatory response of macrophages to bacterial LPS.

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.

Defensins represent a major component of innate host defense against bacteria, fungi, and enveloped viruses. One potent defensin found, e.g., in epithelia, is the polycationic human beta-defensin-3 (hBD3). We investigated the role of the lipid matrix composition, and in particular the presence of negatively charged lipopolysaccharides (LPS) from sensitive (Escherichia coli, Salmonella enterica serovar Minnesota) or resistant (Proteus mirabilis) Gram-negative bacteria or of the zwitterionic phospholipids of human cells, in determining the action of polycationic hBD3 on the different membranes, and related to their biological activity. The main focus was directed on data derived from electrical measurements on a reconstitution system of the OM as a planar asymmetric bilayer composed on one side of LPS and on the other of a phospholipid mixture. Our results demonstrate that the antimicrobial activity and the absence of cytotoxicity can be explained by the lipid-specificity of the peptide. A clear correlation between these aspects of the biological activity of hBD3 and its interaction with lipid matrices could be found. In particular, hBD3 could only induce lesions in those membranes resembling the lipid composition of the OM of sensitive bacterial strains. The permeation through the membrane is a decisive first step for the biological activity of many antimicrobial peptides. Therefore, we propose that the lipid-specificity of hBD3 as well as some other membrane-active antimicrobial peptides is important for their activity against bacteria or mammalian cells.

Lipopolysaccharides (LPS; endotoxin) activate immunocompetent cells of the host via a transmembrane signaling process. In this study, we investigated the function of the LPS-binding protein (LBP) in this process. The cytoplasmic membrane of the cells was mimicked by lipid liposomes adsorbed on mica, and the lateral organization of LBP in these membranes and its interaction with LPS aggregates were characterized by atomic force microscopy. Using cantilever tips functionalized with anti-LBP antibodies, single LBP molecules were localized in the membrane at low concentrations. At higher concentrations, LBP formed clusters of several molecules and caused cross-linking of lipid bilayers. The addition of LPS to LBP-containing liposomes led to the formation of LPS domains in the membranes, which could be inhibited by anti-LBP antibodies. Thus, LBP mediates the fusion of lipid membranes and LPS aggregates.

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-α 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.

Lipopolysaccharides (LPSs) play a dual role as target and as effector molecules. The knowledge of the LPS-induced activation of human immune cells is increasing; however, surprisingly, much less effort seems to be directed towards the understanding of the mechanisms leading to the killing of the bacterial organisms, which eventually results in the release of LPS from the bacterial surface into the blood circulation. We demonstrate mechanisms of interaction of peptides of the innate immune system ( e.g. defensins and cathelicidins) as well as of externally administered antibiotics ( e.g. Polymyxin B) with Gram-negative bacteria. The main focus is directed on data derived from electrical measurements on a reconstitution system of the outer membrane as an asymmetric bilayer composed on one side of LPS and on the other of phospholipids. All these antimicrobial peptides (AMPs) are membrane-active and induce the permeabilization of the reconstituted membranes by the formation of lesions. We found that differences in the activity of the AMPs against various sensitive and resistant Gram-negative bacteria can be explained solely by variations in the chemical structure of LPS, e.g. in the composition of the sugar head group. A reduction of the net negative charge of LPS is responsible for a reduced interaction with the polycationic AMPs and thus for resistance. A most important side effect of positively charged AMPs is the neutralization of the negatively charged LPS released from the bacterial surface as a consequence of AMP-induced killing.

The biophysical properties of a series of glycosyl dialkyl- and diacyl-glycerols bearing unsaturated or chiral methyl branched chains in the tail, and di- and trisaccharide carbohydrate headgroups are described. Thermotropism was investigated by polarising microscopy, the lyotropism was investigated by small angle X-ray diffraction and by the contact preparation method, and the gel to liquid crystalline phase transition by FT-IR-spectroscopy. The compounds displayed thermotropic Smectic A (SmA), cubic and columnar phases, whereas in the lyotropic phase diagram lamellar, hexagonal and cubic phases are found. The introduction of unsaturated or methyl branched chains leads to liquid crystallinity at ambient temperature. The difference between the 1,3-oleyl-glycerol maltoside and the corresponding 1,2-oleoyl-glycerol maltoside is small.

The inhibition of LPS-induced cell activation by specific antagonists is a long-known phenomenon; however, the underlying mechanisms are still poorly understood. It is commonly accepted that the membrane-bound receptors mCD14 and TLR4 are involved in the activation of mononuclear cells by LPS and that activation may be enhanced by soluble LPS-binding protein (LBP). Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity, whereas lipid A with four fatty acids (precursor IVa, synthetic compound 406) is endotoxically inactive, but expresses antagonistic activity against active LPS. Seeking to unravel basic molecular principles underlying antagonism, we investigated phospholipids with structural similarity to compound 406 with respect to their antagonistic activity. The tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-α release when added to the cells before stimulation or as a CL/LPS mixture. Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production. Using Abs against LBP, we could show that the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids. In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, we could show that preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation. In summary, we show that LBP is a target for the inhibitory function of phospholipids.

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the ‘endotoxic principle’ of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular (‘endotoxic’) conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-d-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.

Symmetric and asymmetric planar lipid bilayers prepared according to the Montal-Mueller method are a powerful tool to characterize peptide-membrane interactions. Several electrical properties of lipid bilayers such as membrane current, membrane capacitance, and the inner membrane potential differences and their changes can be deduced. The time-resolved determination of peptide-induced changes in membrane capacitance and inner membrane potential difference are of high importance for the characterization of peptide-membrane interactions. Intercalation and accumulation of peptides lead to changes in membrane capacitance, and membrane interaction of charged peptides induces changes in the charge distribution within the membrane and with that to changes in the membrane potential profile. In this study, we establish time-resolved measurements of the capacitance minimization potential DeltaPsi on various asymmetric planar lipid bilayers using the inner field compensation method. The results are compared to the respective ones of inner membrane potential differences DeltaPhi determined from ion carrier transport measurements. Finally, the time courses of membrane capacitances and of DeltaPsi have been used to characterize the interaction of cathelicidins with reconstituted lipid matrices of various Gram-negative bacteria.

Amoebapores from protozoan parasite Entamoeba histolytica and NK-lysin of porcine cytotoxic lymphocytes belong to the same family of saposin-like proteins. In addition to the structural similarity, amoebapores and NK-lysin are both highly effective against prokaryotic and eukaryotic target cells in that they permeabilize the target cell membranes. Here, we have investigated in detail the protein/lipid interaction for the three isoforms of amoebapore and NK-lysin. Results obtained from electrical measurements on planar bilayer membranes, including reconstitution models of the lipid matrix of the outer membrane of Escherichia coli and phospholipid membranes, fluorescence energy transfer spectroscopy with liposomes, and monolayer measurements on a Langmuir trough, provided information on lipid preferences, pH dependences, and membrane interaction mechanisms. The three amoebapores led to the formation of transient pores with similar characteristics in conductance, sublevels, and lifetime for the different isoforms. The conductance of the pores was dependent on the polarity of the applied clamp voltage, and the distribution of the sublevels was affected by the value of the clamp voltage. The size of the pores and distribution of conductance sublevels differed between symmetric phospholipid and asymmetric lipopolysaccharide/phospholipid bilayers. Notably, NK-lysin caused the formation of well-defined pores, which were lipid- and voltage-dependent, and their characteristics differed from those induced by amoebapores; e.g., the protein concentration necessary to induce pore formation was 20 times higher. The biophysical data give important information on the mode of action of these small effector proteins, which may further lead to a better understanding of peptide-membrane interactions in general.

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 Ca2+ - 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-α 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.

Lipopolysaccharides (LPSs) play a dual role as inflammation-inducing and as membrane-forming molecules. The former role attracts significantly more attention from scientists, possibly because it is more closely related to sepsis and septic shock. This review aims to focus the reader's attention to the other role, the function of LPS as the major constituent of the outer layer of the outer membrane of Gram-negative bacteria, in particular those of enterobacterial strains. In this function, LPS is a necessary component of the cell envelope and guarantees survival of the bacterial organism. At the same time, it represents the first target for attacking molecules which may either be synthesized by the host's innate or adaptive immune system or administered to the human body. The interaction of these molecules with the outer membrane may not only directly cause the death of the bacterial organism, but may also lead to the release of LPS into the circulation. Here, we review membrane model systems and their application for the study of molecular mechanisms of interaction of peptides such as those of the human complement system, the bactericidal/permeability-increasing protein (BPI), cationic antibacterial peptide 18 kDa (CAP18) as an example of cathelicidins, defensins, and polymyxin B (PMB). Emphasis is on electrical measurements with a reconstitution system of the lipid matrix of the outer membrane which was established in the authors' laboratory as a planar asymmetric bilayer with one leaflet being composed solely of LPS and the other of the natural phospholipid mixture. The main conclusion, which can be drawn from these investigations, is that LPS and in general its negative charges are the dominant determinants for specific peptide—membrane interactions. However, the detailed mechanisms of interaction, which finally lead to bacterial killing, may involve further steps and differ for different antibacterial peptides.

Escherichia coli low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C-terminal amphiphilic α-helices. Nonetheless, the membrane interactive mechanisms utilized by the C-terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment-based analyses have predicted that, in contrast to the PBP4 and PBP6b C-termini, those of PBP5 and PBP6 are candidates to form oblique orientated α-helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C-terminal regions, P4 and P5, respectively, both possessed the ability to adopt α-helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal α-helices, the PBP5 and PBP6 C-terminal α-helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.

The activity of m-calpain, a heterodimeric, Ca2+-dependent cysteine protease appears to be modulated by membrane interactions involving oblique-orientated α-helix formation by a segment, GTAMRILGGVI, in the protein's smaller subunit. Here, graphical and hydrophobic moment-based analyses predicted that this segment may form an α-helix with strong structural resemblance to the influenza virus peptide, HA2, a known oblique-orientated α-helix former. Fourier transform infrared spectroscopy showed that a peptide homologue of the GTAMRILGGVI segment, VP1, adopted low levels of α-helical structure (≈ 20%) in the presence of zwitterionic lipid and induced a minor decrease (3 °C) in the gel to liquid-crystalline phase transition temperature, TC, of the hydrocarbon chains of zwitterionic membranes, suggesting interaction with the lipid headgroup region. In contrast, VP1 adopted high levels of α-helical structure (65%) in the presence of anionic lipid, induced a large increase (10 °C) in the TC of anionic membranes, and showed high levels of anionic lipid monolayer penetration (ΔSP = 5.5 mN·m−1), suggesting deep levels of membrane penetration. VP1 showed strong haemolytic ability (LD50 = 1.45 mm), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid monolayers, was greatly reduced. In combination, our results suggest that m-calpain domain V may penetrate membranes via the adoption of an oblique-orientated α-helix and electrostatic interactions. We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this α-helix.

Synthetic triacyl glucosamine monosaccharide lipid A part structures corresponding to the non-reducing moiety of enterobacterial lipid A with an acyloxyacyl chain linked to position 3 of the glucosamine and an unbranched chain linked to position 2 (group 1) and vice versa (group 2) were analyzed biophysically: Fourier-transform infrared spectroscopy was performed to characterize the gel-to-liquid crystalline phase transition, the phosphate band contour, and the orientation of the glucosamine with respect to the membrane surface. Small-angle x-ray diffraction was applied for the elucidation of the supramolecular aggregate structure and, with that, of the molecular shape. With fluorescence resonance energy transfer the lipopolysaccharide-binding protein (LBP)-mediated intercalation of the lipid A partial structures into phospholipid liposomes was monitored. The physical data clearly exhibit a classification of the synthetic compounds into two groups: group 1 compounds have sharp phase transitions, indicating dense acyl chain packing and an inclination of the glucosamine backbone with respect to the membrane surface of 30 degrees with the phosphate buried in the membrane. Group 2 compounds have a very broad phase transition, indicating poorly packed acyl chains, and an inclination of -30 degrees with the phosphate group sticking outward. For the first group unilamellar phases are observed superimposed by a non-lamellar structure, and for the second one only multilamellar aggregate structures. The cytokine-inducing capacity in human mononuclear cells is relatively high for the first group and low or absent for the second group. Based on these data a model of the intra and intermolecular conformations is proposed which also extends the concept of "endotoxic conformation."

It is reported on the interaction of bacterial lipopolysaccharide (LPS, endo-toxin), having different sugar chain length covalently bound to its hydrophobic moiety lipid A, with the polycationic antibiotics polymyxin B (PMB) and PMB-nonapeptide (PMBN). The binding enthalpies and the lipid:peptide binding stoichiometries were determined by isothermal titration calorimetry (ITC). For LPS with a long sugar chain (S: smooth form LPS from Yersinia enterocolitica), the titration curves exhibit a strong exo-therm which can be interpreted to result from the electrostatic interaction of the negative charges of the LPS with the positive charges of the peptides. In contrast, the titration curves of LPS with a short sugar chain (LPS Re from Escherichia coli) and of free lipid A yield complex patterns of endo- and exo-therms, which result from the superposition of electrostatic binding, fluidization of the acyl chains and a transition between different three-dimensional aggregate structures of the endo-toxins due to peptide binding. Infrared spectroscopy indicates that the fluidizing effect of the polymyxins is similar for both types of LPS and for lipid A. Small-angle X-ray diffraction reveals, however, that the Re-type LPS and lipid A are converted from a cubic into a multilamellar structure, whereas, the S-form LPS transforms from a unilamellar into a multilamellar structure with a small number of lamellae. The presented data allow a better understanding of the interaction of peptides with endo-toxin molecules.

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.

Antimicrobial peptides with alpha-helical structures and positive net charges are in the focus of interest with regard to the development of new antibiotic agents, in particular against Gram-negative bacteria. Interaction between seven polycationic alpha-helical CAP18-derived peptides and different types of artificial membranes composed of phosphatidylcholine or lipopolysaccharide of the Gram-negative bacterium Escherichia coli were investigated using different biophysical techniques. Results obtained from fluorescence energy transfer spectroscopy with liposomes, monolayer measurements on a Langmuir trough, and electrophysiological measurements on planar reconstituted asymmetric bilayer membranes including the lipid matrix of the outer membrane of E. coli were correlated, and these data were, furthermore, correlated with structural parameters of the peptides (net charge, alpha-helical content, hydrophobic moment, and hydrophobicity). All peptides induced current fluctuations in planar membranes due to the formation of transient lesions above a peptide- and lipid-specific minimal clamp voltage. Antibacterial activity was exhibited only by those peptides that induced lesion formation in the reconstituted outer membrane at clamp voltages below the transmembrane potential of the natural membrane. Thus, we propose that the physicochemical properties of both the peptides as well as of the target membranes are important for antibacterial activity.

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.

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.

LPS (endotoxins) activate cells of the human immune system, among which are monocytes and macrophages, to produce endogenous mediators. These regulate the immune response, but may also cause severe harm leading to septic shock. The activation of monocytes/macrophages by LPS is mediated by a membrane-bound LPS receptor, mCD14. As mCD14 lacks a transmembrane domain, a further protein is required for the signal transducing step to the cell interior. Here we show, using excised outside-out membrane patches, that activation of a high-conductance Ca2+- and voltage-dependent potassium channel is an early step in the transmembrane signal transduction in macrophages. The channel is activated by endotoxically active LPS in a dose-dependent manner. Channel activation can be completely inhibited by LPS antagonists and by anti-CD14 Abs. Activation of the channel is essential for LPS-induced cytokine production as shown by its inhibition by selective K+ channel blockers.

We have investigated the thermotropic and lyotropic properties of some long chain alkyl glycosides with disaccharide headgroups. The thermotropism was measured with polarising microscopy and additionally the lyotropism with the contact preparation method, Fourier-transform Infrared (FTIR) spectroscopy, X-ray diffraction and small angle neutron scattering. A broad thermotropic as well as lyotropic polymorphism was found. The compounds displayed thermotropic S(A) (lamellar) and cubic phases, and the investigation of the lyotropic phase behaviour led to the observation of inverted bicontinuous cubic V(II) phases, lamellar L(alpha) phases, normal bicontinuous cubic V(I) phases, normal columnar H(I) phases, normal discontinuous cubic I(I) phases and lyotropic cholesteric phases. The phases are discussed with respect to the chemical structures that have been varied systematically to derive structure-property relationships.

The triacylated lipid A partial structure OM-174 was characterized in detail using a variety of physical and biological techniques. OM-174 aggregates adopt the micellar HI structure. The temperature (Tc) of the gel to liquid-crystalline phase transition of the hydrocarbon chains is 0 degrees C, from which high fluidity of the acyl chains at 37 degrees C can be deduced. The molecular area of a single OM-174 molecule at a surface pressure of 30 mN x m-1 is 0.78 +/- 0.04 nm2. Conformational analyses, using IR spectroscopy, of the behavior of the various functional groups of OM-174 as compared with hexa-acyl lipid A suggest altered hydration of the phosphate charges and unusually strong hydration of the ester groups, which is probably related to the high accessibility of these groups to water in the micellar aggregate structure. OM-174 was shown to intercalate into a phospholipid membrane corresponding to the macrophage membrane within seconds in the presence, and within minutes to hours in the absence, of LPS-binding protein. In the Limulus amebocyte lysate assay, the triacyl lipid A is more than 105-fold less active than hexa-acyl lipid A, but only 10-fold less active in inducing IL-6 in human mononuclear cells, and equally active in inducing NO production in murine macrophages. These findings are used to explain the mechanism of the lipid A-induced cell activation.

Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man. The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the ‘endotoxic principle’ of LPS and is also responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS. By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A. Bisphosphoryl-hexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (> 45 °) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active. Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape. This correlates with decreasing endotoxic activity. Penta- and tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (

Lipopolysaccharide (LPS) represents a major virulence factor of Gram-negative bacteria ('endotoxin') that can cause septic shock in mammals including man. The lipid anchor of LPS to the outer membrane, lipid A, has a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against LPS, a property that can be utilized in antisepsis treatment. We show here that these different biological activities are directly correlated with the molecular shape of lipid A. Only (hexaacyl) lipid A with a conical/concave shape, the cross-section of the hydrophobic region being larger than that of the hydrophilic region, exhibited strong interleukin-6 (IL-6)-inducing capacity. Most strikingly, a correlation between a cylindrical molecular shape of lipid A and antagonistic activity was established: IL-6 induction by enterobacterial LPS was inhibited by cylindrically shaped lipid A except for compounds with reduced headgroup charge. The antagonistic action is interpreted by assuming that lipid A molecules intercalate into the cytoplasmic membrane of mononuclear cells, and subsequently blocking of the putative signaling protein by the lipid A with cylindrical shape.

Two glycosphingolipids, GSL-1 and GSL-3, were isolated from Sphingomonas capsulata and studied by methylation analysis, laser desorption mass spectrometry, and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY and heteronuclear 13C,1H COSY experiments. GSL-1 and GSL-3 differ in their carbohydrate part, their structures being alpha-D-GlcpA-(1-->1)-Cer and alpha-D-Galp-(1-->6)-alpha-D-GlcpN-(1-->4)-alpha-D-GlcpA(1-- >1)Cer, respectively. Variations occur in the ceramide of GSL-1 and GSL-3, both having the same long-chain bases, erythro-2-amino-1, 3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1, 3-diol and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol, in the ratios 2.6 : 1 : 3.5 in GSL-1 and 1 : 1.2 : 1.5 in GSL-3. All bases are quantitatively substituted by amide-linked (S)-2-hydroxymyristic acid.

Investigations are reported into the interaction of lipid A, the ‘endotoxic principle’ of bacterial lipopolysaccharide (LPS), with phospholipid membranes in the absence and presence of an acute-phase lipid transport protein, lipopolysaccharide-binding protein (LBP) applying Fourier-transform infrared (FTIR) and fluorescence resonance energy transfer (FRET) spectroscopy. In the absence of LBP, intermixing of phospholipids with lipid A takes place on the time-scale of hours, while in the presence of LBP this process takes place in the order of minutes. A comparison of chemically different lipid A shows that a prerequisite for the intercalation of lipid A into the phospholipid membrane is a sufficiently high negative charge density of lipid A. Variations in the lipid A acyl chain fluidity may modulate the intercalation, whereas the type of lipid A aggregate structure has no influence on the intercalation.The intercalation is a necessary, but not sufficient prerequisite for cell activation. Only lipid A with a conical molecular shape and a tilt angle of more than 40° of the backbone with respect to the direction of the acyl chains induces cytokine induction in human mononuclear cells, while lipid A with a cylindrical shape and a small tilt angle does not exhibit this biological activity but may act antagonistically. This antagonistic effect may be explained by blocking of the binding-sites of the putative signal-transducing protein, possibly an ion channel, by the antagonist.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.

The conventional view of the effects of 1,25- (OH)2D3on osteoclastogenesis and bone resorption are thought to be mediated by stromal cells or osteoblastic cell. Giant cell tumor of bone (GCT) is characterized by multinuclear giant cells (osteoclast-like cells) distributed among a mass of mononuclear cells. Because it is very difficult to obtain normal human osteoclasts, many investigators, including ourselves, have used GCT as a source of human osteoclasts to enhance the understanding of normal skeletal remodeling and especially the hormone regulation of osteoclast functions. In this report, expression of human vitamin D receptor (VDR) mRNA was examined in the various components of GCT using the fluorescence in situ hybridization (FISH) technique. The results showed that VDR gene transcripts were expressed in both multinuclear cells (osteoclast-like cells) and mononuclear macrophage-like cells but not in stromal-like tumor cells in two cases of GCT examined. Measurement of VDR gene transcripts in situ by confocal microscopy showed that the level of VDR mRNA in osteoclast-like giant cells is greater than that in macrophage-like cells (p < 0.05). These findings suggest that 1,25- (OH)2D3mediated osteoclastic bone resorption or osteoclastogenesis could at least, in part, occur by direct action on osteoclasts/their precursor cells.

The purpose of our studies is to define the physicochemical parameters involved in the activation of host cells by endotoxin and to characterize the processes operative during endotoxin/membrane interaction with the aim of understanding transmembrane signal transduction mechanisms. To this end, we determined the molecular conformation of the lipid A component of various endotoxins (endotoxic conformation) using X-ray small angle diffraction, their intercalation into reconstituted macrophage membranes with fluorescence resonance energy transfer spectroscopy, and their IL-6 inducing capacity in whole blood. We also investigated their influence on ion channels as a possible primary event in cell activation applying patch-clamp techniques to macrophages. We found a strong influence of the molecular charge on the molecular conformation, and we could show that the presence of charged groups and a cone- or wedge-like molecular conformation of lipid A are prerequisites for the expression of bioactivity. We also obtained strong evidence supporting the idea that the interaction of endotoxin with ion channels is one of the very early events in the interaction with the cell and, most likely, in signal transduction.

Planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the outer membrane of Gram-negative bacteria to study complement (C) activation by various bacterial surface glycolipids with the aim of defining the C activation pathway. As glycolipids the lipopolysaccharides of Salmonella enterica serovar Minnesota R mutant strains R595 (Re LPS) and R4 (Rd2 LPS), pentaacyl lipid A from the LPS of the Escherichia coli Re mutant F515, and glycosphingolipid GSL-1 of Sphingomonas paucimobilis IAM 12576 were used. Methylester and carboxyl-reduced derivatives of GSL-1 were used to elucidate the role of the carboxyl group as common functional group of LPS and GSL-1 for C activation. The formation of lytic pores was monitored via the measurement of changes in membrane current. For all glycolipids we observed a considerable increase in membrane current soon after addition of whole human serum due to the formation of lytic pores in the membranes. Pore formation was dependent on the presence of C9, indicating that the observed current changes were due to C activation. We found that in our reconstitution system of the outer membrane lipid A, Re LPS, and Rd2 LPS activated the classical pathway, the activation being independent of specific anti-LPS antibodies. In contrast, GSL-1 and the methylester derivative of GSL-1 activated the alternative pathway even at the low serum concentrations used in this study (about 0.2% v/v). Interestingly, the carboxyl reduced GSL-1 activated the classical pathway.

Osteoclasts are multinuclear bone-resorbing cells. Little is known about regulation of gene expression in these cells and how it changes during the aging of an osteoclast, which is indicated by an increasing number of nuclei. Silver staining of nucleolar organizer regions, so-called Ag-NORs, allows us to assess the activity of rRNA expression. Using this technique, we show that individual nuclei of osteoclasts can be stained specifically and that the majority of individual nuclei contained Ag-NORs. The number and size of Ag-NORs varied, indicative of difference in the progression of the cell cycle through the G1 phase. The relative activity of Ag-NORs was not synchronized on the level of individual nuclei, regardless of the number of nuclei per osteoclast. Surprisingly, the total activity of Ag-NOR appeared to be correlated to the total nuclear area of an osteoclast. We conclude that regulation of gene expression in multinuclear osteoclasts most likely occurs on the level of individual nuclei.

The activation of cells by endotoxin (LPS) is one of the early host responses to infections with Gram-negative bacteria. The lipid A part of LPS molecules is known to represent the endotoxic principle; however, the specific requirements for the expression of biologic activity are still not fully understood. We previously found that a specific molecular conformation (endotoxic conformation) is a prerequisite for lipid A to be biologically active. In this study, we have investigated the interdependence of molecular charge and conformation of natural and chemically modified LPS and lipid A and its transport and intercalation into phospholipid membranes mediated by human LPS-binding protein, as well as IL-6 production after stimulation of whole blood or PBMCs. We found that the number, nature, and location of negative charges strongly modulate the molecular conformation of endotoxin. In addition, the LPS-binding protein-mediated transport of LPS into phospholipid membranes depends on the presence of net negative charge, yet charge is only a necessary, but not a sufficient, prerequisite for transport and intercalation. The biologic activity is determined mainly by the molecular conformation: only conical molecules are highly biologically active, whereas cylindrical ones are largely inactive. We could demonstrate that the net negative charge of the lipid A component and its distribution within the hydrophilic headgroup strongly influence the molecular conformation and, therefore, also the biologic activity.

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.

Lipopolysaccharides (LPS, endotoxin) of gram-negative bacteria are among the main causes of sepsis and septic shock. In the present study, the influence of temperature on the biological activity of LPS was investigated. Lowering the temperature from 37 degrees C to 34.5 degrees C or to 30 degrees C significantly enhances in vitro tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-6 release induced by different LPS chemotypes and heat-inactivated Escherichia coli. This cytokine-increasing effect of lowering the temperature is highly mediated by serum proteins, particularly by LPS-binding protein (LBP) and low-density lipoproteins (LDL). In contrast, cytokine production induced by the superantigen toxic shock syndrome toxin-1 (TSST-1) from Gram-positive Staphyloccoccus aureus decreases by around 70% at 30 degrees C as compared with 37 degrees C, corresponding to the expected effect of change in temperature and regardless of the presence of serum proteins. In order to explain the unexpected biological hypothermia effect with regard to LPS, the fluidity state of the lipid A portion of LPS as one important physico-chemical property possibly involved was investigated. The fluidity, determined by fluorescence polarization measurements, was found to decrease with decreasing temperature. These data suggest that a low fluid LPS chemotype is biologically more active than a more fluid one (and vice versa). Statistical analysis of the results shows a strong correlation between cytokine secretion and fluidity state of a given LPS chemotype (0.71r0.89, all P0.01). As a clinical consequence, these data may be one possible explanation for the higher mortality rate of hypothermic Gram-negative sepsis.

The action of bactericidal polycationic peptides was compared in Yersinia spp. by testing peptide binding to live cells and changes in outer membrane (OM) morphology and permeability. Moreover, polycation interaction with LPS was studied by measuring the dependence of dansylcadaverine displacement and zeta potential on polycation concentration. When grown at 37 °C, Yersinia pestis and Yersinia pseudotuberculosis bound less polymyxin B (PMB) than pathogenic or non-pathogenic Yersinia enterocolitica, regardless of virulence plasmid expression. Y. pseudotuberculosis OMs were unharmed by PMB concentrations causing extensive OM blebbing in Y. enterocolitica. The permeability to Iysozyme caused by PMB was greater in Y. enterocolitica than in Y. pseudotuberculosis or Y. pestis and differences increased at 37 °C. Similar observations were made with other polycations using a polymyxin/novobiocin permeability assay. With LPS of cells grown at 26 °C, polycation binding was highest for Y. pseudotuberculosis and lowest for Y. pestis, with Y. enterocolitica yielding intermediate results which were lower for pathogenic than for non-pathogenic strains. With LPS of cells grown at 37 °C, polycation binding remained unchanged for Y. pastis and pathogenic Y. enterocolitica, increased for non-pathogenic Y. enterocolitica and decreased for Y. pseudotuberculosis to Y. pestis levels. Polycation binding related in part to differences in charge density (zeta potential) of LPS aggregates, suggesting similar effects at bacterial surfaces. It is suggested that species and temperature differences in polycation resistance relate to infection route, invasiveness and intracellular multiplication of Yersinia spp.

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.

Chylomicron remnants transport cholesterol from the intestine, and are removed from the circulation principally by the liver. While hepatic receptors, including the low density lipoprotein (LDL) receptor account for endocytosis, heparan sulfate proteoglycans (HSPG) participate in the initial binding of remnants to liver cells. To explore the interactions between HSPG and endocytosis of remnants, in the present study the expression of HSPG was inhibited in HepG2 cells transfected by a synthetic antisense oligodeoxynucleotide SYN5. Immunofluorescent staining by a monoclonal anti-syndecan antibody showed significant reduction in the expression of syndecan in SYN5-treated cells compared with control cells. Remnant binding decreased by about 50–70% in SYN5-transfected cells. Monoclonal antibodies to either heparan sulphate or the LDL receptor decreased binding by about 60–65%. The glycosylation inhibitor β-nitrophenyl-xylopyranoside inhibited remnant uptake by 25%, whereas 4-nitrophenyl-β-d-galactopyranoside had no effect on remnant binding. Heparinase completely abolished binding at appropriate concentrations. Heparitinase was less effective than heparinase in inhibiting remnant binding. Suramin completely abolished the remnant binding. Poly-arginine, poly-lysine, and protamine all reduced remnant uptake by the cells, as did polybrene, a synthetic polycation, suggesting a role of cation–anion interactions in remnant binding. Brefeldin A, colchicine, and monensin caused the fluorescence associated with remnants to persist within the cells, confirming that blockers of tubulovesicular processes and Golgi function inhibit the intracellular transport and degradation of the remnants. Our results show that remnant binding to liver cells depends on the LDL receptor, on the expression of HSPG core proteins, and on the functionality of heparan sulfate in HSPG.—Zeng, B-J., B-C. Mortimer, I. J. Martins, U. Seydel, and T. G. Redgrave. Chylomicron remnant uptake is regulated by the expression and function of heparan sulfate proteoglycan in hepatocytes. J. Lipid Res. 1998. 39: 845–860.

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.

Osteoclasts resorb bone by a complex dynamic process that initially involves attachment, polarization and enzyme secretion, followed by their detachment and migration to new sites. In this study, we postulated that mineralized and osteoid bone matrix signal osteoclasts differently, resulting in the resorption of mineralized bone matrix only. We, therefore, compared the cytoplasmic distribution of cytoskeletal proteins F-actin and vinculin using confocal laser-scanning microscopy in osteoclasts cultured on mineralized and demineralized bone slices and correlated the observations with their functional activity. Our results have demonstrated significant differences in F-actin and vinculin staining patterns between osteoclasts cultured on mineralized bone matrix and those on demineralized bone matrix. In addition, the structural variations were accompanied by significant differences in bone resorbing activity between osteoclasts grown on mineralized bone matrix and those on demineralized bone matrix after 24 h of culture --resorption only occurring in mineralized bone but not in demineralized bone. These results indicated that failure of osteoid bone resorption is caused by perturbation of osteoclast polarization.

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 β ⇔ α 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.

The pivotal role of the endothelial cell in the regulation of vascular tone has been well demonstrated in many vascular beds, including the retina. However, in the retina, little is known about how the structural elements of the endothelial cells are arranged along the arborisation pathway from artery to vein, the nature of which has been linked to functional heterogeneity in other vascular beds. The relative vulnerability of the retina to vascular based diseases, and the heavy reliance on local regulation of the retinal vasculature makes an improved understanding of such local regulatory mechanisms of significant clinical importance. The present study focuses on identifying differences in endothelial cells along the arborisation pathway in the porcine retinal vasculature. Enucleated pig eyes were arterially cannulated and perfused with fixative followed by double staining for F-actin microfilaments (rhodamine phalloidin) and nucleic acid (YO-PRO-1). The intact retina was then viewed by confocal microscopy. The distribution of F-actin, vessel diameter, endothelial cell size and shape, nucleus size and shape, and position within the cell were determined as a function of location along the vascular tree. The main retinal arterioles (A1) contained full length F-actin internal stress fibers which lay parallel to the long axis of the endothelial cell. Subsequent branches from the A1 arteriole (A2 and A3) showed fewer, shorter fibers, with none visible in the A4 and A5 branches, the capillaries, or in the venous side of the vasculature. All endothelial cells showed peripheral border staining of F-actin microfilaments which allowed the shape of the cell to be determined. All endothelial cells were elongated with the long axis parallel to the vessel, but the mean aspect ratio decreased from 10.9+/-0.5, s.e.m. in the A1 arterioles to 3.2+/-0.2 in the major veins (V1). The position of the endothelial cell nucleus relative to the cell was eccentric in the downstream direction in the A2-A5 arterioles, whilst centrally placed in the A1 arterioles and veins. The structural heterogeneity of endothelial cells along the pig retinal circulation suggests that functional heterogeneity of the endothelium may be involved in regulation of retinal blood flow.