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K Brandenburg

Publications and source records attributed to K Brandenburg.

67 records · Page 4Linked to original sources
PubMed1989

Reconstitution of the lipid matrix of the outer membrane of gram-negative bacteria as asymmetric planar bilayer.

This paper is a report on the reconstitution of the lipid matrix of the outer membrane of Gram-negative bacteria as an asymmetric planar bilayer. This is the first time that a planar membrane is described, which consists on one side of a phospholipid (PL) mixture and on the other side of lipopolysaccharide (LPS). Therefore, strong emphasis is placed on a physical characterization of this membrane via its electrical properties. The membranes were prepared from spread monolayers or from vesicle-derived monolayers. Contrary to observations for symmetric phospholipid membranes, specific capacitances of (0.67 +/- 0.02) mu F.cm-2, breakdown voltages between 200 and 400 mV and specific conductances between 10(-8) and 2 x 10(-7) S.cm-2 were obtained independent of the preparation method. The LPS-containing membranes were stable up to 3 hr if they were formed and kept at temperatures above the hydrocarbon chain melting temperature of the LPS. For the specific capacitance, a dependence on the aperture radius was observed. This is explained by assuming a toroidal transition zone at the rim of the aperture. First results on the action of the pore-forming alpha-toxin from Staphylococcus aureus on bilayers of different composition demonstrate particular characteristics of this asymmetric bilayer system. The pore-formation rate is highest in symmetric phospholipid bilayers, considerably lower in asymmetric PL/LPS systems and fully inhibited in LPS/LPS systems.

Bacterial Toxins↗
PubMed1988

Orientation measurements on membrane systems made from lipopolysaccharides and free lipid A by FT-IR spectroscopy.

Here we report on investigations into the orientational behaviour of hydrated membrane systems made from lipopolysaccharide and its lipid component, free lipid A, using Fourier-transform infrared spectroscopy and applying attenuated total reflectance. For the investigated lipopolysaccharides--extracted from mutants of Salmonella minnesota and Escherichia coli, and differing in the length of the polysaccharide moiety--the dependence of dichroic ratios on temperature for several vibrations of the hydrophilic and hydrophobic portions was measured, from which the order parameter for the lipid assembly can be calculated. In the lower temperature range (less than 40 degrees C) for all lipopolysaccharide preparations the evaluation of the dichroic ratios clearly shows the existence of a highly ordered phase, i.e. the gel state of the hydrocarbon chains within lamellar structures. For this phase an order parameter S = 0.70 +/- 0.05 could be calculated which is lower than that of typical phospholipids in the same phase state (S = 0.80 +/- 0.05 for e.g. phosphatidylethanolamines). For deep rough mutant lipopolysaccharides at higher temperatures (greater than 40 degrees C) a transition into a disordered, isotropic phase can usually be observed for which an order parameter S = 0.25 +/- 0.05 could be approximated. The other rough mutant lipopolysaccharides at higher temperatures predominantly form lamellar structures. Only in special cases, under the influence of divalent cations like Mg2+, could isotropic phases also be observed. Free lipid A preparations over the whole temperature range exhibited no unequivocal orientational behaviour. However, the existence of a pure L beta-phase even at lower temperatures may be excluded for these compounds. The observed structural preferences might be of great importance with respect to the expression of biological activities of lipopolysaccharide and free lipid A systems in vivo and in vitro.

Escherichia coli↗
PubMed1987

Investigations into the polymorphism of lipid A from lipopolysaccharides of Escherichia coli and Salmonella minnesota by Fourier-transform infrared spectroscopy.

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5 degrees and 58 degrees C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41 degrees, 46 degrees, 44 degrees and 47 degrees C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4'-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52 degrees C for E. coli lipid A). The analysis of greater than CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22 degrees, 30 degrees, 27 degrees and 25.5 degrees C (first transition) and at about 34 degrees, 42 degrees, 38.5 degrees and 40.5 degrees C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: Up to about 30 degrees C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of greater than CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry↗
PubMed1987

The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment.

We investigated the immunogenicity and antigenicity of synthetic lipid A and partial structures thereof. Included in the study were compounds which varied in the position of phosphate (1-mono-, 4'-mono-, and 1,4'-bisphosphates) and in the acylation (type, number, and distribution of fatty acids) and, in the case of monosaccharide compounds, the nature of the backbone sugar (D-glucosamine, D-glucose, 3-amino-3-deoxy-D-glucose, and 2,3-diamino-2,3-dideoxy-D-glucose). With the aid of the passive-hemolysis and passive-hemolysis-inhibition assays and by absorption experiments, five distinct antibody specificities were detected in polyclonal rabbit antisera raised against sheep erythrocyte-coated lipid A and lipid A incorporated into the membrane of liposomes (liposome-incorporated immunogens). Three antibody specificities reacted with disaccharide antigens specific for a 1-mono-, 4'-mono-, and 1,4'-bisphosphorylated beta-1,6-linked D-glucosamine disaccharide. Two antibodies reacted with either 1- or 4-phosphates of acylated D-gluco-configured monosaccharides and exhibited no cross-reaction with each other. However, they cross-reacted with disaccharide antigens with phosphate groups in the appropriate positions. We found that the physicochemical state and the environment of lipid A modulated its immunoreactivity. The immunogenicity was best expressed by erythrocyte-coated and liposome-incorporated immunogens. The antigenicity of lipid A was also greatly influenced by its physical surroundings. The reaction pattern of the above antibodies was highly specific in the hemolysis assay and in absorption experiments (the antibody reacted with antigen embedded in a cell membrane), whereas some cross-reactivities were observed in inhibition studies (the antibody reacts with antigen in aqueous solution). By using liposome-incorporated antigens as inhibitors, nonspecific reactions were avoided and specific ones were enhanced. Thus the antibodies described above against lipid A recognize epitopes in the hydrophilic backbone, the exposure of which depends on the intrinsic physicochemical properties of lipid A on the one hand and the physical environment on the other.

Animals↗
PubMed1986

1% permethrin cream rinse vs 1% lindane shampoo in treating pediculosis capitis.

The therapeutic efficacy and tolerance of a single application of 1% permethrin cream rinse, applied for ten minutes, and a single application of 1% lindane shampoo applied, as recommended by the manufacturer, for four minutes, against the head louse Pediculus humanus var capitis were compared in a single-blinded, randomized, controlled clinical trial. Of 573 patients enrolled at eight centers, 559 were assessable for tolerance and 508 for efficacy. Of the 257 patients treated with 1% permethrin cream rinse, 99% were lice free at 14 days; of the 251 patients treated with 1% lindane shampoo, 85% were lice free at 14 days. The difference is statistically significant. For both treatments, adverse experiences were infrequent, mild, and usually difficult to distinguish from the symptoms of head lice infestation. A single ten-minute application of 1% permethrin cream rinse was well tolerated, highly effective, and therapeutically superior to a single four-minute application of 1% lindane shampoo.

Clinical Trials as Topic↗
PubMed1986

Orientation measurements on ordered multibilayers of phospholipids and sphingolipids from synthetic and natural origin by ATR Fourier transform infrared spectroscopy.

Fourier-transform infrared spectroscopy was applied to natural and synthetic planar membrane multibilayer systems made from phospholipids and sphingolipids to perform orientational measurements utilizing the attenuated total reflectance technique. Applying polarized infrared light, the dichroic ratios and the positions of the peak maxima of various infrared-active bands in dependence on temperature and degree of hydration as well as the phase behaviour were studied. Under certain assumptions - which are discussed - made for the order parameter S, the orientation of various functional groups with respect to the molecular axis or to the membrane normal can be determined. For the gel phase values for S between 0.80 +/- 0.05 for the complete molecule and 0.40 +/- 0.05 for the end methyl group and for the liquid crystalline phase a value of 0.50 +/- 0.05 were approximated. Applying these data, a relatively precise determination of the angles of the oscillating dipole moments of various functional groups could be performed, which - for some vibrations - deviate considerably from those angles assumed earlier. Regarding the phase behaviour, it was found that not only the degree of hydration but the amount of bulk water of the sample has a strong influence on the phase transition temperature Tc (lyotrophism), but only a weak influence on the orientational data.

Fourier Analysis↗
PubMedDate not supplied

Structural polymorphisms of rough mutant lipopolysaccharides Rd to Ra from Salmonella minnesota.

The structural polymorphisms of rough mutant lipopolysaccharides (LPS) Rd, Rc, Rb, and Ra from Salmonella minnesota (strains R4, R7, Rz, R5, R345, and R60, respectively) were investigated as a function of temperature, water content, and Mg2+ concentration. The gel to liquid crystalline (beta<==>alpha) phase transition temperature (Tc) and the state of order within each phase were measured by Fourier transform infrared spectroscopy. The amount of bound water was determined by differential scanning calorimetry and the three-dimensional structures in each phase state were characterized by synchrotron radiation X-ray diffraction. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The beta<==>alpha acyl chain melting temperatures at high water contents (95-97%), Tc = 31 to 32 degrees C for LPS Rd, 33 to 35 degrees C for LPS Rc to Rb, and 36 degrees C for LPS Ra, increase with decreasing water content and in the presence of Mg2+ cations with a concomitant broadening of the transition range. Below 30 to 50% water content, no distinct phase transitions can be observed. These effects are most pronounced for LPS with the shortest sugar chains. Below 50% water content, only lamellar structures can be observed in the temperature range 5 to 80 degrees C, independent of the Mg2+ concentration. Above 50% water concentration, for large [LPS]:[Mg2+] molar ratios the predominant structure above Tc is nonlamellar; for smaller [LPS]:[Mg2+] molar ratios a superposition of lamellar and nonlamellar structures is found. For all LPS Rd to Rb at low Mg2+ concentrations, the phase transition is connected with a change in the three-dimensional structure from lamellar or mixed lamellar/nonlamellar to a pure nonlamellar, probably cubic structure. The tendency to form non-lamellar structures decreases with the length of the core oligosaccharide. At an equimolar ratio of [LPS] and [Mg2+] a multibilayered organization is observed. Some of the nonlamellar structures are cubic phases with periodicities between 12 and 16 nm. The molecular dimensions of the single endotoxin molecules in the absence and the presence of external water are estimated from the different lamellar periodicities, including those of free lipid A and deep rough mutant LPS Re. These observations are discussed with respect to the biological importance of LPS as a potent inducer of biological effects in mammals.

Bacterial Toxins↗
PubMedDate not supplied

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide.

We have reported here on the structural polymorphism of lipid A, the "endotoxic principle" of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg2+] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70 degrees C. These data were correlated with measurements of the beta----alpha phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges--in the absence of Mg2(+)-from 45 degrees C at high to 56 degrees C at low water content, and-at an equimolar content of Mg2(+)-from 52 degrees C at high to 59 degrees C at low water concentrations. In the gel phase-in which the lipid A acyl chains are more disordered than those from saturated phospholipids-cubic phases are adopted at high water content (greater than 60%) and at high [lipid A]:[Mg2+] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal HII state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg2+ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.

Escherichia coli↗
PubMedDate not supplied

Phase diagram of deep rough mutant lipopolysaccharide from Salmonella minnesota R595.

The structural polymorphism of deep rough mutant lipopolysaccharide--in many biological systems the most active endotoxin--from Salmonella minnesota strain R595 was investigated as function of temperature, water content, and Mg2+ concentration. Differential scanning calorimetry was used to determine the amount of bound water and the enthalpy change at the beta<==>alpha gel to liquid crystalline acyl chain melting. The onset, midtemperature Tc, and completion of the beta<==>alpha phase transition were studied with Fourier-transform infrared spectroscopy. Synchrotron radiation X-ray diffraction was used to characterize the supramolecular three-dimensional structures in each phase state. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The beta<==>alpha acyl chain melting temperature Tc lying at 30 degrees C at high water content (95%) increases with decreasing water content reaching a value of 50 degrees C at 30% water content. Concomitantly, a broadening of the transition range takes place. At still lower water content, no distinct phase transition can be observed. This behavior is even more clearly expressed in the presence of Mg2+. In the lower water concentration range (< 50%) at temperatures below 70 degrees C, only lamellar structures can be observed independent of the Mg2+ concentration. This correlates with the absence of free water. Above 50% water concentration, the supramolecular structure below 70 degrees C strongly depends on the [LPS]:[Mg2+] ratio. For large [LPS]:[Mg2+] ratios, the predominant structure is nonlamellar, for smaller [LPS]:[Mg2+] ratios there is a superposition of lamellar and nonlamellar structures. At an equimolar ratio of LPS and Mg2+ a multibilayered organization is observed. The nonlamellar structures can be assigned in various cases to structures with cubic symmetry with periodicities between 12 and 16 nm. Under all investigated conditions, a transition into the hexagonal II structure takes place between 70 and 80 degrees C. These observations are discussed in relation to the biological importance of LPS as constituent of the outer membrane of gram-negative bacteria and as potent inducer of biological effects in mammals.

Carbohydrate Conformation↗
PubMedDate not supplied

Chemical structure and biologic activity of bacterial and synthetic lipid A.

The chemical structure of the lipid A component of enterobacterial lipopolysaccharide (LPS) is now known in some detail. For example, lipid A of Escherichia coli consists of a beta(1----6)-linked D-glucosamine disaccharide that carries four (R)-3-hydroxytetradecanoyl groups in positions 2, 3, 2', and 3' and two phosphoryl residues in positions 1 and 4'. The hydroxy fatty acids at positions 2' and 3' are acylated at their 3-hydroxyl groups by dodecanoic acid and tetradecanoic acid, respectively. The hydroxyl groups in positions 4 and 6' are free, the latter serving as the attachment site for the polysaccharide component in intact LPS. On the basis of this structure, E. coli-type lipid A and partial structures thereof have been chemically synthesized (group of T. Shiba, Osaka University, Osaka, Japan) and analyzed for endotoxic activity. In all in vivo and in vitro test systems employed (including lethal toxicity, pyrogenicity, local Shwartzman reactivity, B lymphocyte mitogenicity, macrophage activation, and serologic cross-reactivity with lipid A antiserum), synthetic lipid A has activity identical to that of E. coli lipid A. These findings support the structural proposal for lipid A and prove the previous hypothesis that the endotoxic principle is embedded in lipid A.

Animals↗
PubMedDate not supplied

The dual role of lipopolysaccharide as effector and target molecule.

Lipopolysaccharides (LPS) are major integral components of the outer membrane of Gram-negative bacteria being exclusively located in its outer leaflet facing the bacterial environment. Chemically they consist in different bacterial strains of a highly variable O-specific chain, a less variable core oligosaccharide, and a lipid component, termed lipid A, with low structural variability. LPS participate in the physiological membrane functions and are, therefore, essential for bacterial growth and viability. They contribute to the low membrane permeability and increase the resistance towards hydrophobic agents. They are also the primary target for the attack of antibacterial drugs and proteins such as components of the host's immune response. When set free LPS elicit, in higher organisms, a broad spectrum of biological activities. They play an important role in the manifestation of Gram-negative infection and are therefore termed endotoxins. Physico-chemical parameters such as the molecular conformation and the charges of the lipid A portion, which is responsible for endotoxin-typical biological activities and is therefore termed the 'endotoxic principle' of LPS, are correlated with the biological activity of chemically different LPS.

Anti-Bacterial Agents↗
PubMedDate not supplied

Electrostatic potential barrier in asymmetric planar lipopolysaccharide/phospholipid bilayers probed with the valinomycin-K+ complex.

Using the carrier-ion complex valinomycin-K+, current/voltage (I/U) characteristics were registered for planar asymmetric lipid bilayers composed on one side of a phospholipid mixture and on the other side of rough mutant lipopolysaccharide. This system resembles the lipid matrix of the outer membrane of Gram-negative bacteria. The evaluation of the current/voltage curves yielded a highly asymmetric electrical potential barrier. The total potential difference between the phospholipid and the lipopolysaccharide was -85 mV, a result which cannot be explained by contributions of Gouy-Chapman potentials alone. The possible contribution of dipole potentials and influences of headgroup effects are discussed. It is shown that the asymmetry of the I/U-characteristic results from the differences of the surface charge densities of the two monolayers but not from those of the states of order of their hydrocarbon chains.

Carbohydrate Sequence↗