Biomedical subjects

K Brandenburg

Publications and source records attributed to K Brandenburg.

At least 19 recordsLinked to original sources

PubMed2006

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

Aging↗

PubMed2004

Structural polymorphism and endotoxic activity of synthetic phospholipid-like amphiphiles.

The physicochemical characteristics and in vitro biological activity of various synthetic hexaacyl phospholipid dimers were compared with the respective behavior of bacterial endotoxins (lipopolysaccharide, LPS). The structural variations of the synthetic amphiphiles include different stereochemical (R,S) configurations about their ester- and amide-linkages for the acyl chains and differences in the length of the serine backbone spacer. The temperature of the gel to liquid crystalline phase transition of the acyl chains (T(c)) lies between 10 and 15 degrees C for the compounds with the shortest backbone and decreases rapidly for the compounds with longer backbones. The phase transition enthalpies (8-16 kJ x mol(-1)) are considerably lower than those of lipid A from hexaacyl endotoxins (28-35 kJ x mol(-1)). In contrast, the dependence of T(c) on Mg(2+) and water content shows a behavior typical for endotoxins: a significant increase with increasing Mg(2+) and decreasing water concentrations. The aggregate structure is sensitively dependent not only on the length of the backbone spacer but also on the different stereochemical variations. It can be directly correlated with the biological activity of the compounds. Thus, as with natural lipid A, the capacity to induce cytokine production in mononuclear cells is directly related to the affinity to form nonlamellar cubic or inverted hexagonal H(II) aggregate structures. Together with the data on the transport and intercalation of the dimers into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP), our conformational concept of endotoxicity and cell activation can be applied to these non-LPS structures: endotoxically active compounds incorporate into membranes of immune cells and cause conformational changes at the site of signaling proteins such as Toll-like receptors or K(+)-channels due to their conical molecular shape.

Animals↗

PubMed2003

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure.

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.

Acylation↗

PubMed2002

A study on the C-terminal membrane anchoring of Escherichia coli penicillin-binding protein 5.

Escherichia coli penicillin-binding protein 5 (PBP5) anchors to the inner membrane in a pH-dependent manner via a C-terminal amphiphilic alpha-helix. Low pH was found to enhance both levels of PBP5 membrane anchoring and levels of alpha-helicity in an aqueous PBP5 C-terminal homologue, which led to the suggestion that levels of PBP5 membrane anchoring are related to levels of PBP5 C-terminal alpha-helicity. Here we have used Fourier-transformed infrared spectroscopy (FTIR) and a peptide homologue of the PBP5 C-terminal sequence to investigate the effect of pH on the conformational behavior of this sequence at a lipid interface and on its ability to interact with lipid. Our results suggest that the membrane-anchoring mechanism of PBP5 is unlikely to involve conformational change in the protein's C-terminal region and may therefore involve conformational changes in the protein's ectomembranous domain.

Bacterial Proteins↗

PubMed2002

Lack of interaction of fluoroquinolones with lipopolysaccharides.

Fluoroquinolones are known to chelate with di- and trivalent cations, and it has accordingly been claimed that they perturb the integrity of the outer membrane (OM) of gram-negative bacteria. So far, chelation has not been assessed in biologically relevant test systems. Therefore, we investigated the interaction of ciprofloxacin and moxifloxacin in the absence and presence of Mg2+ with whole bacteria and isolated lipopolysaccharide (LPS) from various rough mutant strains of Salmonella enterica chemotypes by applying different biophysical techniques. We found that the fluoroquinolones did not disturb the integrity of the OM and neither were incorporated into LPS monolayers nor displaced Ca2+ from LPS monolayers, suggesting that chelation of fluoroquinolones with divalent cations does not contribute to the antibacterial effect of fluoroquinolones.

Anti-Infective Agents↗

PubMed2001

An investigation into the membrane-interactive potential of the Escherichia coli KpsE C-terminus.

Membrane binding via C-terminal amphiphilic alpha-helical structure is a novel anchoring mechanism, which has been characterised in a number of prokaryotic carboxypeptidases. Here, we have used graphical and DWIH analyses to ascertain if a similar anchoring mechanism may be utilised by the Escherichia coli KpsE protein in its binding to the periplasmic face of the inner membrane. The results of these analyses have been compared to those obtained for similar analyses of the C-terminal sequences of E. coli penicillin-binding proteins (PBPs) PBP5 and PBP6 which, are known to function as amphiphilic alpha-helical membrane anchors, and of melittin, a known membrane-interactive toxin. We have also used FTIR spectroscopy and lipid phase transition temperature analysis to investigate the interaction of a peptide homologue of KpsE C-terminal region with membrane lipid. Our results suggest that the KpsE C-terminal sequence has the potential to form an amphiphilic alpha-helix and that this alpha-helix could feature in the membrane binding of the protein.

Amino Acid Sequence↗

PubMed2001

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

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

Air↗

PubMed2001

Differential environment alters ontogeny of dopamine innervation of the orbital prefrontal cortex in gerbils.

In the present study, the influence of postnatal environmental conditions on the structural ontogeny of the orbital prefrontal cortex of adult gerbils (Meriones unguiculatus) was examined. The animals were bred and reared either isolated in standard laboratory cages or grouped in an object-filled environment. At the age of postnatal day 90, dopamine fibers were stained immunocytochemically and innervation density was determined in the orbital prefrontal cortex. By comparison, restricted rearing produced a restraint of the subsequent maturation of orbital prefrontal dopamine innervation, leading to adult fiber densities that were approximately 38% below those in seminaturally reared gerbils. Results are discussed in terms of activity-dependent postnatal maturation of the cortex and adaptive neuroplasticity with regard to previously published data concerning diminished dopamine innervation in the medial prefrontal cortex (Winterfeld et al. [1998]

Animals↗

PubMed2001

New insights into endotoxin-induced activation of macrophages: involvement of a K+ channel in transmembrane signaling.

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 Ca(2+)- 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.

Antibodies, Blocking↗

PubMed2001

Laparoscopic management of 641 adnexal tumors in Kiel, Germany.

STUDY OBJECTIVE: To evaluate the effectiveness and safety of laparoscopic and laparotomic management of ovarian tumors. DESIGN: Retrospective analysis (Canadian Task Force classification II-2). SETTING: University-affiliated hospital. PATIENTS: Six hundred forty-one women with benign and malignant ovarian tumors. INTERVENTIONS: Laparoscopy and laparotomy. MEASUREMENTS AND MAIN RESULTS: Between January 1997 and December 1998, 493 (76.9%) ovarian tumors were treated laparoscopically and 138 (21.5%) by laparotomy. Criteria for laparotomy were high suspicion of malignancy and tumors larger than 10 cm that were technically too difficult for the laparoscopic approach. The mean size of tumors treated laparoscopically was 4.5 cm (range 1.1-11 cm) and by laparotomy 8.2 cm (range 3-20 cm). Mean operating times were 75.7 minutes (range 30-200 min) and 126 minutes (range 30-235 min), respectively, and mean blood loss was 193 ml (range 50-1200 ml) and 431 ml (range 50-2500 ml), respectively. Twelve laparoscopies were converted to laparotomy, six because of technical reasons such as severe adhesions, bleeding, or tumor size, and six for intraoperative suspicion of malignancy. Of the latter, four (66.7%) turned out to be ovarian carcinoma and two (33.3%) borderline tumors. Histologic evaluation clearly revealed predominance of functional ovarian cysts, endometriomas, and dermoid cysts in the group treated by laparoscopy, whereas ovarian carcinomas, large endometriomas, and serous cysts prevailed in the laparotomy group. CONCLUSION: With careful preoperative screening, the rate of laparoscopies for treatment of benign ovarian cysts can be increased. (J Am Assoc Gynecol Laparosc 8(1):74-82, 2001)

Adolescent↗

PubMed2001

Interaction of hemoglobin with enterobacterial lipopolysaccharide and lipid A. Physicochemical characterization and biological activity.

The interaction of hemoglobin (Hb) with endotoxins [i.e. with enterobacterial deep rough mutant lipopolysaccharide (LPS) Re and the "endotoxic principle" of LPS, lipid A] was investigated using a variety of physical techniques and with two biological assays, tumor necrosis factor (TNF)-alpha induction in human mononuclear cells and the Limulus amebocyte lysate (LAL) assay. Fourier-transform IR-spectroscopic experiments indicate nonelectrostatic binding to the hydrophobic moiety with a slight rigidification of the lipid A acyl chains, and an increase in the inclination of the lipid A backbone with respect to the membrane surface from 35 degrees to more than 40 degrees due to Hb binding, but no change of the predominantly alpha-helical secondary structures of Hb due to LPS binding. From isothermal titration calorimetry, the molar [Hb] : [endotoxin] binding ratio lies between 1 : 3 and 1 : 5 molar. Synchrotron radiation X-ray diffraction measurements indicate a reorientation of the lipid A aggregates from one cubic structure to another, the final structure belonging to space group Q224. The LPS-induced TNF-alpha production of mononuclear cells is enhanced by Hb, whereas in the LAL assay an LPS concentration-dependent increase or decrease was observed. Although a detailed mechanism of action cannot be given, the enhancement of LPS bioactivity can be understood in the light of the previously presented conformational concept; Hb induces an increase in the conical shape of the lipid A moiety of LPS, higher cross-section of the hydrophobic than the hydrophilic part, and of the inclination angle of the diglucosamine backbone with respect to the direction of the acyl chains.

Animals↗

PubMed2001

Lyotropic phase transitions in phospholipids as evidenced by small-angle synchrotron X-ray scattering.

Hydration is an important factor in regulating the phase behaviour of lipids and besides affects their interactions with other compounds relevant for biological membranes. We present a reliable and fast method to detect and characterise hydration-induced phase transitions in phospholipids by means of small-angle synchrotron X-ray scattering. Films consisting of aggregations of representatives of the two important lipid classes lecithins (DPPC a, POPC and OPPC,a for abbreviations, see below) and cephalins (DPPE and DOPE) were investigated at room temperature in dependence on relative humidity. Qualitative changes in the sets of the diffraction patterns obtained in dynamic hydration/dehydration scans were taken as markers indicating the existence of lyotropic phase transitions. The efficiency of this methodology is demonstrated by illustrating the course of hydration-driven phase transitions between lamellar as well as nonlamellar phases. In detail, this was realised for chain melting in the mixed-chain lipids, POPC and OPPC, and for a novel nonlamellar-phase transition for DOPE between a disordered inverted ribbon phase designated as Palpha and the canonical H(II), phase, respectively.

Macromolecular Substances↗

PubMed2001

Analysis of motor vehicle ejection victims admitted to a level I trauma center.

OBJECTIVES: The objective of this study was to compare the injuries and outcomes of ejected victims who reached a Level I trauma center with nonejected MVC occupants. METHODS: Data from 6,909 MVC victims admitted to a Level I trauma center, over a 91/2-year period, were retrospectively reviewed. Three mutually exclusive groups were studied: ejected, nonejected nonrestrained, and nonejected restrained. RESULTS: The patient distribution was as follows: ejected 6.4% (n = 443), nonrestrained 50.1% (n = 3,461), and restrained 43.5% (n = 3,005). Ejected patients were younger, required ICU care more frequently, and a higher percentage were males compared with nonrestrained or restrained patients. Injury Severity Score (ISS) and length of stay (LOS) were significantly higher in ejected patients. Ejected patients suffered more injuries per anatomic region, and had a higher number of severe injuries in the head and neck region. The overall in-hospital mortality was 3.9% (272/6,909), and 10.8% (48/443) for the ejected group. The incidence of restrained patients increased during the study period but was not associated with a change in the incidence of ejected patients. CONCLUSION: Patients who were ejected after motor vehicle collisions were more severely injured and had a worse outcome than those not ejected. Efforts should be concentrated on enforcement and enactment of better seat belt laws, as well as the development of new strategies that will prevent ejection regardless of occupant behavior.

Accidents, Traffic↗

PubMed2001

Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin.

The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as monitored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. X-ray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharide-binding protein (LBP)-induced intercalation of LPS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.

Animals↗

PubMed2001

A K+ channel is involved in LPS signaling.

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

Cells, Cultured↗

PubMed2000

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides. Part II. Disaccharide headgroups.

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.

Carbohydrate Conformation↗

PubMed2000

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

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

Calorimetry, Differential Scanning↗

PubMed2000

Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity.

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 degrees ) 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 (< 25 degrees ) and a cylindrical shape and are endotoxically inactive, but may be antagonistic.

Carbohydrate Conformation↗