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![Amide I vibrational band in the wavenumber range 1700 to 1600 cm -1 for 0.1 mM Hb (top) and in the presence of LPS Re, [LPS]:[Hb]= 10:1 molar (bottom). The band position is typical for the occurrence of an -helical structure.](profile/Malte-Hammer/publication/6537195/figure/fig6/AS:394635257106445@1471099785913/Fig-8-Amide-I-vibrational-band-in-the-wavenumber-range-1700-to-1600-cm-1-for-01-mM.png)
Amide I vibrational band in the wavenumber range 1700 to 1600 cm -1 for 0.1 mM Hb (top) and in the presence of LPS Re, [LPS]:[Hb]= 10:1 molar (bottom). The band position is typical for the occurrence of an -helical structure.
Source publication
Bacterial endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of the outer membrane in gram-negative bacteria. During severe infections, bacteria may reach the blood circuit of humans, and endotoxins may be released from the bacteria due to cell division or cell death. In particular enterobacterial forms of LPS represent...
Context in source publication
Context 1
... wavenumber range of the amide I-band is presented in Fig. 8 for pure Hb (top) and in the presence of LPS ( [LPS]: [Hb] 10:1 molar, bottom). There is a strong domi- nance of the band at 1653 cmP -1 P corresponding to a nearly completely -helical structure, in accordance with crystallo- graphic data (Protein Data Bank, structure 1A06), and there is no change in the presence of LPS. This means ...
Citations
... Other groups have established that free adult hemoglobin can enhance the production of tumor necrosis factor-α (TNFα) induced by LPS in human mononuclear cells [5], and increase the rate of mortality by free LPS in rats [6,7]. Previous studies have led to the hypothesis that this may in part reflect the effect of hemoglobin on the molecular conformation of LPS/ MPLA [8]. As we have noted in an earlier publication, in mice, unlike in man, there is no clear-cut fetal hemoglobin chain corresponding to human Hgbγ, whose expression curtails rapidly following embryonic life, although a (Hgbε) chain is expressed transiently in utero [9]. ...
Injections of a crude fetal sheep liver extract (FSLE) containing fetal hemoglobin, MPLA, and glutathione (GSSH) reversed cytokine changes in aged mice. To investigate the role of fetal hemoglobin we derived mice with homzygous deletions for either of the two major βchains, HgbβmaKO or HgbβmiKO. Hgbβmi is the most prominent fetal Hgbβ chain, with Hgbβma more prominent in adult mice. Mice lacking another fetal Hgb chain, HgbεKO, died in utero. CHO cells transfected with cloned Hgb chains were used to produce proteins for preparation of rabbit heteroantibodes. Splenocytes from HgbβmaKO mice stimulated in vitro with Conconavalin A showed a higher IL-2:IL-4 ratio than cells from HgbβmiKO mice. Following immunization in vivo with ovalbumin in alum, HgbβmaKO mice produced less IgE than HgbβmiKO mice, suggesting that in the absence of HgbβmiKO mice had a predeliction to heightened allergic-type responses. Using CHO cells transfected with cloned Hgb chains, we found that only the fetal Hgb chain, Hgbε, was secreted at high levels. Secretion of Hgbβma or Hgbβmi chains was seen only after genetic mutation to introduce the two N-linked glycosylation sites present in Hgbε, but absent in the Hgbβ chains. We speculated that a previously unanticipated biological function of a naturally secreted fetal Hgb chain may be partly responsible for the effects reported following injection of animals with fetal, not adult, Hgb. Mice receiving injections of rabbit anti-Hgbε but not either anti-Hgbβma or anti-Hgbβmi from day 14 gestation also showed a bias towards the higher IL-2:IL-4 ratios seen in HgbβmiKO mice.
... 16,17,19,21 This enhancement of the biological response was associated with the conversion of LPS aggregates into a rather nonlamellar form, especially into cubic structures. 22,23,24 Furthermore, Hb has been assessed as an important subject of clinical investigations in the resuscitation, cancer therapy, sickle cell anaemia, coronary balloon, angioplasty, stroke and myocardial infraction, and has an adjunctive therapeutic treatment. 18,19 However, using cell-free Hb involves the risk of possible contamination with other blood components or even LPS, representing a barrier for the successful use of Hb as an adjuvant. ...
... 11,39,40 The interaction between Hb and LPS enhances the biological response, which was found to result from a conversion of LPS aggregates into a more pronounced nonlamellar, cubic aggregate structure and to a disaggregation. 22,24 Furthermore, the diverging phase transition behaviour of bioactivity increasing and bioactivity decreasing compounds has also been reported for the biological weak or inactive pentaacyl lipid A and LPS preparations. Hb, for which a decrease in mobility of the hydrocarbon chains of LPS was also observed, stands in contrast to the system LPS and polymyxin B, which decreases the bioactivity and leads to a fluidisation at 37 C. 41 These findings are in direct accordance to the data presented here concerning the influence on the TNF-a production in MNCs by the two different peptides Hbg-35 and Pep19-2.5 ( Figure 1). ...
Endotoxins (LPS) are highly potent immune stimulatory molecules and are mainly known for triggering Gram-negative sepsis. However, besides their toxic effects, this stimulatory function may be advantageous, for example when used as an adjuvant during vaccination. Thus, there is always a narrow range between the useful wake-up of the immune system and its overwhelming reaction, which can lead to diseases like sepsis. This raises the question of which conformational properties are responsible for making the LPS aggregates more or less potent. As described previously, the size, type and form of LPS aggregates play a major role in their immune stimulatory activity. In this study we investigate the role of these parameters. On the one hand, we use a peptide (Pep19-2.5; Aspidasept) that causes a change of the LPS aggregate structure into a less toxic state; on the other hand, we use a potent immune stimulating peptide (Hbγ-35), leading to higher toxicity. We have found opposing effects on LPS aggregate conformations allowing a better understanding of the processes of immune stimulation.
... LPS can oxidize Hb and convert metHb, which facilitates iron release and generation of free radicles, a condition known to contribute to Hb related antibacterial activity. Recently it has also been shown that Hb can synergize with LPS and LTA during macrophage activation and enhance innate immune responses by inducing expression of cytokines and chemokines [42,43]. We hypothesize that the expression of Hb in these cells increases due to inflammation and might be required to neutralize the cytotoxic effects of H 2 O 2 and LPS. ...
Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). Red blood cells (RBCs) contain maximum amount of Hb and because of their unique structure and plasticity they transport O2 to various tissues of the body at an optimal concentration. Recently, it has been reported that, apart from RBCs, Hb is also expressed by nonerythroid cells such as epithelial cells of different origin. The cells expressing Hb are from the tissues where maintenance of O2 homeostasis is of paramount importance. Hb expression has been observed in the epithelial cells from human tissues including lungs, neurons, retina, and endometrium. Our group has recently demonstrated that Hb is expressed by the cervicovaginal epithelial cells. We further showed that, apart from maintaining O2 homeostasis, Hb and the peptides derived from it play an indispensable role in the protection of vaginal epithelium by exhibiting antimicrobial activity. In this review, we discuss the significance of Hb expression in vaginal epithelial cells and its role in the recognition of pathogens thereby reducing the risk and/or severity of inflammation and/or infections and the possible mechanism by which Hb exhibits antimicrobial and antioxidative functions.
... It is clear that free Hb and a growing number of bacterial molecules can synergize during macrophage activation to result in enhanced innate immune responses [52][53][54][55][56][57]. This observation is biologically important because even though the body has highly efficient systems for scavenging Hb and heme under physiological conditions, there are a myriad of pathological states in which these systems can be overwhelmed, exposing the tissues to free Hb [58][59][60]. ...
Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-β mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.
Structure of the Bacterial Cell Phylum Tenericutes, Order Mycoplasmatales Order Entomoplasmatales Order Incertae Sedis Phylum Proteobacteriae, Order Rickettsiales Order Rhizobiales Order Burkholderiales Order Neisseriales Order Campylobacterales Order Aeromonadales Order Enterobacteriales Order Legionellales Order Pasteurellales Order Pseudomonadales Order Thiotricales Order Vibrionales Phylum Chlamydiae, Order Chlamydiales Order Actinomycetales Phylum Firmicutes, Order Bacillales Order Lactobacillales Order Clostridiales Order Spriochaetales Order Bacteroidales Order Fusobacteriales Other Bacteria
Selected recent applications of synchrotron radiation in the study of non-crystalline matter are reviewed. During the period under consideration, 2006–2007, new instrumentation developments made studies with microbeams, ultrafast measurements and imaging applications possible. These provided many of the highlights, but the bulk of the scientific and technical results on specific materials were obtained using well-established techniques. Although significant progress has also been made in the development of data analysis and modelling software, this remains an area where further advances are still very much needed, especially for time-resolved measurements.
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.
Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and β subunits possess high affinity LPS-binding sites, with K(D) in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59-95 of Hbβ attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.
Endotoxins as amphiphilic components of the outer layer of the outer membrane of Gram-negative bacteria exert their immunostimulatory activity after release from bacterial cells. Thus, the characterization of the physicochemical properties of this glycolipid in physiological fluids is of utmost importance for an understanding of cell activation processes. Here, the essential physicochemical parameters describing endotoxins such as critical micellar concentration, acyl chain fluidity, intramolecular conformation, supramolecular structures, and size as well as morphology of the aggregates are discussed and assessed with respect to their importance for an understanding of the interaction mechanisms with immunorelevant cells. The reviewed data clearly indicate that knowledge of these parameters is essential for understanding the bioactivity of not only endotoxins, but also endotoxin-like amphiphiles.
