Supramolecular assembly of micellar aggregates is the basis of low endotoxin recovery (LER) in a drug formulation that can be resolved by a whole blood assay.

Lipopolysaccharide (LPS, endotoxin) is ubiquitous and represents a harmful contaminant of pharmaceutical compounds, recombinant biologicals and drug products. The pyrogen can induce severe immune responses and pathology in vitro and in vivo. Health authorities require strict control of endotoxin in parenteral drugs. However, for research and pre-clinical compound analysis, endotoxin testing is not a required quality control, which may cause potential drawbacks in the translational pipeline. Endotoxin testing is usually performed by the Limulus amebocyte lysate (LAL) assay, which is hampered by the so-called low endotoxin recovery (LER) effect when certain drug formulations are tested. A comprehensive study including structural, biophysical, and biological analyses was conducted to identify LER root cause for phosphate- and polysorbate-containing parenteral drug products. LPS in water showed extended ribbon-like aggregate structures. In placebo (formulation buffer without drug) and in drug product (drug in formulation buffer), a reaggregation of LPS into a network of interlinked micelles with hidden head group charges, and a strong reduction of the negative surface potential was observed. The non-accessibility of the LPS backbone has a direct impact leading (i) to a loss of activation of the LAL-cascade, (ii) reduced activation of the TLR4/MD-2 receptor system, and (iii) increased survival in a mouse model of endotoxemia. These data provide a structure-based explanation of the LER-underlying mechanisms. A human whole blood assay is shown to resolve LER and detect the pyrogenic activity of endotoxin with high sensitivity. This may open new test options to improve quality control in drug development and drug safety.

Enhancing actions of peptides derived from the γ-chain of fetal human hemoglobin on the immunostimulant activities of monophosphoryl lipid A.

Hemoglobin and its structures have been described since the 1990s to enhance a variety of biological activities of endotoxins (LPS) in a dose-dependent manner. To investigate the interaction processes in more detail, the system was extended by studying the interactions of newly designed peptides from the γ-chain of human hemoglobin with the adjuvant monophosphoryl lipid A (MPLA), a partial structure of lipid A lacking its 1-phosphate. It was found that some selected Hbg peptides, in particular two synthetic substructures designated Hbg32 and Hbg35, considerably increased the bioactivity of MPLA, which alone was only a weak activator of immune cells. These findings hold true for human mononuclar cells, monocytes and T lymphocytes. To understand the mechanisms of action in more detail, biophysical techniques were applied. These showed a peptide-induced change of the MPLA aggregate structure from multilamellar into a non-lamellar, probably inverted, cubic structure. Concomitantly, the peptides incorporated into the tightly packed MPLA aggregates into smaller units down to monomers. The fragmentation of the aggregates was an endothermic process, differing from a complex formation but rather typical for a catalytic reaction.

Cellular distribution of lipid A and LPS R595 after in vitro application to isolated human monocytes by freeze-fracture replica immunogold-labelling.

We have performed freeze-fracture replica immunogold labelling of endotoxin preparations (lipid A and deep rough mutant LPS Re from Salmonella enterica sv. Minnesota), i.e. adding the endotoxins to human monocytes, labelling with monoclonal Abs recognizing either lipid A or LPS Re (A6 and A20 respectively), and fixing with immunogold secondary Ab. We have found that the endotoxins intercalated into the cell membranes with subsequent internalization by the cells. Surprisingly, membrane uptake took place only in the inner, plasmic leaflet of the plasma membrane, but there was no uptake of the outer leaflet for both compounds. Remarkable labelling could be also found for the two membranes of the nuclear envelope-in the case of lipid A only at the plasmic leaflet, but in the case of LPS Re on both leaflets. Isothermal calorimetric titration of the AB A20 with LPS and phospholipids showed that the Ab may bind not only to LPS but also to negatively charged phosphatidylserine. These results are discussed in the frame of the published concepts of cell activation induced by the endotoxins, i.e. how they are able to cause a conformational change of signalling proteins, such as the TLR4/MD2 complex.

Thermoresponsive hydrogel of diblock methylcellulose: formation of ribbonlike supramolecular nanostructures by self-assembly.

This article provides detailed insight into the thermoresponsive gelation mechanism of industrially produced methylcellulose (MC), highlighting the importance of diblock structure with a hydrophobic sequence of 2,3,6-tri-O-methyl-glucopyranosyl units for this physicochemical property. We show herein, for the first time, that well-defined diblock MC self-assembles thermoresponsively into ribbonlike nanostructures in water. A cryogenic transmission electron microscopy (cryo-TEM) technique was used to detect the ribbonlike nanostructures formed by the diblock copolymers consisting of hydrophilic glucosyl or cellobiosyl and hydrophobic 2,3,6-tri-O-methyl-cellulosyl blocks, methyl ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-celluloside 1 (G-236MC, DP(n) = 10.7, DS = 2.65), and methyl ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-celluloside 2 (GG-236MC, DP(n) = 28.2, DS = 2.75). Rheological measurements revealed that the gel strength of a dispersion of GG-236MC (2, 2.0 wt %) in water at 70 °C was 3.0 times stronger than that of commercial MC SM-8000, although the molecular weight of GG-236MC (2) having M(w) = 8 × 10(3) g/mol was 50 times smaller than that of SM-8000 having M(w) = 4 × 10(5) g/mol. Cryo-TEM observation suggested that the hydrogel formation of the diblock copolymers could be attributed to the entanglement of ribbonlike nanostructures self-assembled by the diblock copolymers in water. The cryo-TEM micrograph of GG-236MC (2) at 5 °C showed rectangularly shaped nanostructures having a thickness from 11 to 24 nm, although G-236MC (1) at 20 °C showed no distinct self-assembled nanostructures. The ribbonlike nanostructures of GG-236MC (2) having a length ranging from 91 to 864 nm and a thickness from 8.5 to 27.1 nm were detected above 20 °C. Small-angle X-ray scattering measurements suggested that the ribbonlike nanostructures of GG-236MC (2) consisted of a bilayer structure with a width of ca. 40 nm. It was likely that GG-236MC (2) molecules were oriented perpendicularly to the long axis of the ribbonlike nanostructure. In addition, wide-angle X-ray scattering measurements revealed that GG-236MC (2) in its hydrogel formed the same crystalline regions as 2,3,6-tri-O-methylcellulose. The influence of the DP of diblock MC with a DS of around 2.7 on the gelation behavior will be discussed.

Biophysical investigations into the interactions of endotoxins with bile acids.

The interaction of selected endotoxin preparations (lipid A from Erwinia carotovora and LPS Re and Ra from Salmonella enterica sv. Minnesota strains R595 and R60, respectively) with selected bile acids was investigated biophysically. Endotoxin aggregates were analyzed for their gel-to-liquid crystalline phase behavior, the type of their aggregates, the conformation of particular functional groups, and their Zeta potential in the absence and presence of the bile acids by applying Fourier-transform infrared spectroscopy, differential scanning calorimetry, measurements of the electrophoretic mobility, and synchrotron radiation X-ray scattering. In addition, the ability of the endotoxins to induce cytokines in human mononuclear cells was tested in the absence and presence of varying concentrations of bile acids. The data show that the endotoxin:bile acid interaction is not governed by Coulomb forces, rather a hydrophobic interaction takes place. This leads to an enhanced formation of the inherent cubic aggregate structures of the endotoxins, concomitant with a slight disaggregation, as evidenced by freeze-fracture electron microscopy. Parallel to this, the addition of bile acids increased the bioactivity of lipid A and, to a lower degree, also that of the tested rough mutant LPS at lower concentrations of the endotoxin preparation, a finding similar as reported for the interaction of other agents such as hemoglobin. These data imply that there are general mechanisms that govern the expression of biological activities of endotoxins.

Dynamic properties of microemulsions in the single-phase channels.

We have studied the dynamic and rheological properties in the single-phase channels of a microemulsion system with a mixed anionic/nonionic surfactant system and decane from the aqueous to the oil phase. One isotropic channel, called the "upper" channel, begins at the L(3) phase (sponge-like phase) of the binary surfactant mixture on the water side and passes with a shallow minimum for the surfactant composition to the oil side. The other "lower" single-phase channel begins at the micellar L(1) phase and ends in the middle of the phase diagram. Both isotropic channels are separated by a huge anisotropic single phase L(α) channel that reaches from the water side to 90% of oil in the solvent mixture. The structural relaxation time of the viscous fluids could be measured with electric birefringence (EB) measurements, where a signal is caused by the deformation of the internal nanostructure of the fluids by an electric field. For the L(3) phase, the EB signal can be fitted with a single time constant. With increasing oil in the upper channel, the main structural relaxation time passes over a maximum and correlates with the viscosity. Obviously, this time constant controls the viscosity of the fluid (η(o) = G'·τ). It is remarkable that the longest structural relaxation time increases three decades, and the viscosity increases two decades when 10% of oil is solubilized into the L(3) phase. Conductivity data imply that the fluid in the upper channel has a bicontinuous structure from the L(3) phase to the microemulsion with only 10% oil. In this oil range, the conductivity decreases three decades, and the electric birefringence signals are complicated because of a superposition of up to three processes. For higher oil ratios, the structure obviously changes to a HIPE (high internal phase emulsion) structure with water droplets in the oil matrix.

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.

Morphology, size distribution, and aggregate structure of lipopolysaccharide and lipid A dispersions from enterobacterial origin.

Lipopolysaccharides (LPSs) from Gram-negative bacteria are strong elicitors of the human immune systems. There is strong evidence that aggregates and not monomers of LPS play a decisive role at least in the initial stages of cell activation of immune cells such as mononuclear cells. In previous reports, it was shown that the biologically most active part of enterobacterial LPS, hexa-acyl bisphosphorylated lipid A, adopts a particular supramolecular conformation, a cubic aggregate structure. However, little is known about the size and morphology of these aggregates, regarding the fact that LPS may have strong variations in the length of the saccharide chains (various rough mutant and smooth-form LPS). Thus, in the present paper, several techniques for the determination of details of the aggregate morphology such as freeze-fracture and cryo-electron microscopy, analytical ultracentrifugation, laser backscattering analysis, and small-angle X-ray scattering were applied for various endotoxin (lipid A and different LPS) preparations. The data show a variety of different morphologies not only for different endotoxins but also when comparing different applied techniques. The data are interpreted with respect to the suitability of the single techniques, in particular on the basis of available literature data.

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation.

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted "neck" region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.

Comparison of Alzheimer Abeta(1-40) and Abeta(1-42) amyloid fibrils reveals similar protofilament structures.

We performed mass-per-length (MPL) measurements and electron cryomicroscopy (cryo-EM) with 3D reconstruction on an Abeta(1-42) amyloid fibril morphology formed under physiological pH conditions. The data show that the examined Abeta(1-42) fibril morphology has only one protofilament, although two protofilaments were observed with a previously studied Abeta(1-40) fibril. The latter fibril was resolved at 8 A resolution showing pairs of beta-sheets at the cores of the two protofilaments making up a fibril. Detailed comparison of the Abeta(1-42) and Abeta(1-40) fibril structures reveals that they share an axial twofold symmetry and a similar protofilament structure. Furthermore, the MPL data indicate that the protofilaments of the examined Abeta(1-40) and Abeta(1-42) fibrils have the same number of Abeta molecules per cross-beta repeat. Based on this data and the previously studied Abeta(1-40) fibril structure, we describe a model for the arrangement of peptides within the Abeta(1-42) fibril.

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation.

Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.

Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins.

An understanding of details of the interaction mechanisms of bacterial endotoxins (lipopolysaccharide, LPS) with the oxygen transport protein hemoglobin is still lacking, despite its high biological relevance. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is presented which comprises the use of various rough mutant LPS as well as free lipid A; in addition to the complete hemoglobin molecule from fetal sheep extract, also the partial structure alpha-chain and the heme-free sample are studied. The investigations comprise the determination of the gel-to-liquid crystalline phase behaviour of the acyl chains of LPS, the ultrastructure (type of aggregate structure and morphology) of the endotoxins, and the incorporation of the hemoglobins into artificial immune cell membranes and into LPS. Our data suggest a model for the interaction between Hb and LPS in which hemoglobins do not react strongly with the hydrophilic or with the hydrophobic moiety of LPS, but with the complete endotoxin aggregate. Hb is able to incorporate into LPS with the longitudinal direction parallel to the lipid A double-layer. Although this does not lead to a strong disturbance of the LPS acyl chain packing, the change of the curvature leads to a slightly conical molecular shape with a change of the three-dimensional arrangement from unilamellar into cubic LPS aggregates. Our previous results show that cubic LPS structures exhibit strong endotoxic activity. The property of Hb on the physical state of LPS described here may explain the observation of an increase in LPS-mediating endotoxicity due to the action of Hb.

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages.

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

Burkholderia rhizoxinica sp. nov. and Burkholderia endofungorum sp. nov., bacterial endosymbionts of the plant-pathogenic fungus Rhizopus microsporus.

Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic cyclopeptide rhizonin. R. microsporus and isolated endobacteria were selected for freeze-fracture electron microscopy, which allowed visualization of bacterial cells within the fungal cytosol by their two parallel-running envelope membranes and by the fine structure of the lipopolysaccharide layer of the outer membrane. Two representatives of bacterial endosymbionts were chosen for phylogenetic analyses on the basis of full 16S rRNA gene sequences, which revealed that the novel fungal endosymbionts formed a monophyletic group within the genus Burkholderia. Inter-sequence similarities ranged from 98.94 to 100%, and sequence similarities to members of the Burkholderia pseudomallei group, the closest neighbours, were 96.74-97.38%. In addition, the bacterial strains were distinguished from their phylogenetic neighbours by their fatty acid profiles and other biochemical characteristics. The phylogenetic studies based on 16S rRNA gene sequence data, together with conclusive DNA-DNA reassociation experiments, strongly support the proposal that these strains represent two novel species within the genus Burkholderia, for which the names Burkholderia rhizoxinica sp. nov. (type strain, HKI 454T=DSM 19002T=CIP 109453T) and Burkholderia endofungorum sp. nov. (type strain, HKI 456T=DSM 19003T=CIP 109454T) are proposed.

Effect of different salt ions on the propensity of aggregation and on the structure of Alzheimer's abeta(1-40) amyloid fibrils.

The formation of amyloid fibrils and other polypeptide aggregates depends strongly on the physico-chemical environment. One such factor affecting aggregation is the presence and concentration of salt ions. We have examined the effects of salt ions on the aggregation propensity of Alzheimer's Abeta(1-40) peptide and on the structure of the dissolved and of the fibrillar peptide. All salts examined promote aggregation strongly. The most pronounced effect is seen within the cationic series, i.e. for MgCl2. Evaluation of different possible explanations suggests that Abeta(1-40) aggregation depends on direct interaction between ions and Abeta(1-40) peptide, and correlates with ion-induced changes of the surface tension. Salts have profound effects on the fibril structure. In the presence of salts, fibrils are associated with smaller diameters, narrower crossover distances and lower amide I maxima. Since Abeta(1-40) aggregation responds to salts in a manner unlike that for other polypeptides, such as glucagon, beta2-microglobulin or alpha-synuclein; these data argue that there is no fully uniform way in which salts affect aggregation of different polypeptide chains. These observations are important for understanding and predicting aggregation on the basis of simple physico-chemical properties.

Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins.

To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.

Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamic, biophysical and microbiological analysis.

On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from LALF [Limulus anti-LPS (lipopolysaccharide) factor], a protein from Limulus polyphemus, we have designed and synthesized peptides of different lengths with the aim of obtaining potential therapeutic agents against septic shock syndrome. For an understanding of the mechanisms of action, we performed a detailed physicochemical and biophysical analysis of the interaction of rough mutant LPS with these peptides by applying FTIR (Fourier-transform infrared) spectroscopy, SAXS (small-angle X-ray scattering), calorimetric techniques [DSC (differential scanning calorimetry) and ITC (isothermal titration calorimetry)] and FFTEM (freeze-fracture transmission electron microscopy). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our results indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease in the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which becomes saturated at a 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. The results obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumour necrosis factor alpha in human mononuclear cells, but not with the action of the peptides on bacteria.

Swelling of Lalpha-phases by matching the refractive index of the water-glycerol mixed solvent and that of the bilayers in the block copolymer system of (EO)15-(PDMS)15-(EO)15.

The swelling of Lalpha-phases from the block copolymer polyethylenoxide-b-polydimethylsiloxane-polyethylenoxide (EO)15-(PDMS)15-(EO)15 in water/glycerol mixtures is reported. At low and medium polymer concentrations (<60%), the block copolymer forms a turbid vesicular dispersion in water. With time, the small unilamellar vesicles (SUV) and the large multilamellar vesicles (MLV) separate into a two phase L1/Lalpha-system. The turbid dispersions of the Lalpha-phase below 60% of the compound become more and more transparent with increasing glycerol and at 60% of glycerol become completely clear. Replacement of water by the solvent glycerol thus lowers the turbidity of the dispersion and swells the interlamellar distance between the bilayers. A 20% aqueous L1/Lalpha-dispersion can thus be transformed into a single birefringent transparent Lalpha-phase. The swelling of the Lalpha-phase in water and the decrease of the turbidity of the dispersion by the addition of glycerol is explained by the matching of the refractive index of the solvent to the refractive index of the bilayers of the block copolymer. The matching of a refractive index lowers the Hamaker constant in the DLVO theory between the bilayers and therefore decreases the attraction between the bilayers what allows them to swell to a larger separation. The microstructures in the phases were determined by cryo- and FFR-TEM. The interlamellar distance between the bilayers was determined by SAXS measurements. The viscous properties of the Lalpha-phases were determined by oscillatory rheological measurements. In comparison to other Lalpha-phases from normal surfactants, the Lalpha-phases from the block copolymer (EO)15-(PDMS)15-(EO)15 have low shear moduli. This is probably due to the high flexibility of the poly dimethylsiloxane block in the bilayers what can be recognized on the non-spherical shapes of the SUV's.

Improved antigen retrieval in freeze-fracture cytochemistry by evaporation of carbon as first replication layer.

The recently developed freeze-fracture replica immunolabeling technique uses sodium dodecyl sulfate to clean replicas obtained from chemically unfixed, rapidly frozen cells by evaporation of platinum as first and carbon as second replication layer. The detergent dissolves remains of cellular material with the exception of components which are in direct contact to the replica film. Membrane lipids and membrane protein complexes of the protoplasmic and the exoplasmic membrane halves remain attached to the replica film and are accessible for cytochemical localization. We immunolabeled the membrane proteins caveolin-1 and connexin 43 in mouse cell lines as well as the membrane attached protein tetrachloroethene reductive dehalogenase (PceA) in bacterial cells at freeze-fracture replicas generated by different evaporation parameters. The labeling experiments for caveolin-1 and the PceA showed that freeze-fracture replication of cellular membranes accomplished with thin platinum layers as well as replication with carbon as first evaporation layer lead in these cases to an improved antigen retrieval, whereas the labeling efficiency of connexin 43 was not affected by different evaporation conditions.