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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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 α-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. [ABSTRACT FROM AUTHOR]

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, nonprofessional 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-c 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 a-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular a-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized. [ABSTRACT FROM AUTHOR]

Amyloid formation is a nucleation-dependent process that is accelerated dramatically in vivo and in vitro upon addition of appropriate fibril seeds. A potent species barrier can be effective in this reaction if donor and recipient come from different biological species. This species barrier is thought to reflect differences in the amino acid sequence between seed and target polypeptide. Here we present an in vitro mutagenic cross-seeding analysis of Alzheimer's Aβ(1-40) peptide in which we mapped out the effect of systematically varied amino acid replacements on the propensity of seed-dependent amyloid fibril formation. We find that the susceptibility of different peptides toward cross-seeding relates to the intrinsic aggregation propensity of the respective polypeptide chain and, therefore, to properties such as β-sheet propensity and hydrophobicity. These data imply that the seed-dependent formation of amyloid-like fibrils is affected by the intrinsic properties of the polypeptide chain in a manner that is similar to what has been described previously for aggregation reactions in general. Hence, the nucleus acts in this case as a catalyst that promotes the fibrillation of different polypeptide chains according to their intrinsic structural predilection. [ABSTRACT FROM AUTHOR]

Caveolae are specialised vesicular microdomains of the plasma membrane. Using freeze-fracture immunogold labelling and stereoscopic imaging, the distribution of labelled caveolin 1 in caveolae of 3T3-L1 mouse fibroblast cells was shown. Immunogold-labelled caveolin structures surrounded the basolateral region of deeply invaginated caveolae like a belt whereas in the apical region distal to the plasma membrane, the caveolin labelling was nearly absent. Shallow caveolar membranes showed a dispersed caveolin labelling. After membrane cholesterol reduction by methyl-ß-cyclodextrin treatment, a dynamic re-distribution of labelled caveolin 1 and a flattening of caveolar structures was found. The highly curved caveolar membrane got totally flat, and the initial belt-like caveolin labelling disintegrated to a ring-like structure and later to a dispersed order. Intramembrane particle-free domains were still observable after cholesterol depletion and caveolin re-distribution. These results indicate that cholesterol interacting with caveolin structures at the basolateral part of caveolae is necessary for the maintenance of the deeply invaginated caveolar membranes. [ABSTRACT FROM AUTHOR]