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U Seydel

Publications and source records attributed to U Seydel.

At least 91 records · Page 5Linked to original sources
PubMed1992

Polymyxin B induces transient permeability fluctuations in asymmetric planar lipopolysaccharide/phospholipid bilayers.

The interaction of the polycationic decapeptide polymyxin B with asymmetric planar bilayers from lipopolysaccharide and phospholipid monolayers, which resemble the lipid matrix of the outer membrane of Gram-negative bacteria, was investigated. The addition of polymyxin B in micromolar amounts to the lipopolysaccharide side of the asymmetric bilayers resulted, under voltage-clamp conditions, in a fast macroscopic increase of their ionic conductance, whereas the polymyxin B nonapeptide induced no significant conductance changes. The polymyxin B induced macroscopic conductance exhibited large fluctuations and was strongly dependent on the amplitude and polarity of the transmembrane potential. The temporal pattern and amplitudes of the fluctuations were characterized by power spectra of the membrane currents and their variances, respectively. In the initial phase following peptide addition, the conductance changes appeared to be channellike discrete fluctuations. The lifetimes of the fluctuations were exponentially distributed, and the mean lifetimes were strongly voltage-dependent, ranging from approximately 30 ms at +80 mV (positive at the side opposite to peptide addition) to less than 5 ms at reverse polarity. The conductance amplitudes of the single fluctuations exhibited a broad distribution with a mean of 2 nS. A comparison of the features of the macroscopic conductance and of the discrete fluctuations showed that the former can basically be understood as a superposition of a large number of the latter. From the amplitudes of the fluctuations, the diameter of the polymyxin-induced lesions was estimated to about 3 nm. The experimental findings can be understood by assuming a detergent-like action of polymyxin B.

Cardiolipins↗
PubMed1991

Chemical structure of glycosphingolipids isolated from Sphingomonas paucimobilis.

Two novel glycosphingolipids were isolated from Sphingomonas paucimobilis and their structures were completely elucidated. The glycosyl portion of the glycosphingolipid consists of an alpha-D-Manp-[1----2)-alpha-D-Galp-(1----6)-alpha-D-GlcpN-(1 ----4)-alpha-D- GlcpA-R tetrasaccharide. The hydrophobic residue R was found to be heterogeneous with respect to the dihydrosphingosine residue. Erythro-1,3-dihydroxy-2-amino-octadecane and erythro-1,3-dihydroxy-2-amino-cis-13,14-methyleneoctadecane were identified in comparable amounts. Both dihydrosphingosine derivatives were quantitatively substituted by an (S)-2-hydroxymyristic acid in amide linkage.

Glycosphingolipids↗
PubMed1991

A comment on the preparation of liposomes from and on the beta in equilibrium alpha acyl chain melting behaviour of rough mutant lipopolysaccharide.

We would like to comment on the investigations of Vaara, M., Plachy, W.Z. and Nikaido, H. (Vaara, M. et al. (1990) Biochim. Biophys. Acta 1024, 152-158) on the partitioning of hydrophobic probes in lipopolysaccharide bilayers. These authors reported that they did not succeed in preparing closed vesicles (liposomes) from rough mutant lipopolysaccharide. We describe the conditions under which lipopolysaccharide liposomes are formed most readily. We, furthermore, summarize data which strongly support the existence of thermotropic phase transitions of lipopolysaccharides (with transition temperatures lying in the range of 30-36 degrees C) contradictory to Vaara et al. who argue that such transitions are artefacts. Exemplary measurements of the beta in equilibrium alpha acyl chain melting for lipopolysaccharide from Escherichia coli deep rough mutant (strain F515) as compared to synthetic and natural phospholipids are presented using fluorescence spectroscopy, Fourier-transform infrared spectroscopy and differential scanning calorimetry. These results unequivocally prove the necessity to perform experiments at 37 degrees C for a determination of the outer membrane permeability under physiological conditions.

Hot Temperature↗
PubMed1991

Chemical structure of the carbohydrate backbone of Vibrio parahaemolyticus serotype 012 lipopolysaccharide.

The chemical structure of the saccharide portion of Vibrio parahaemolyticus serotype 012 lipopolysaccharide was studied. Using chemical degradation and modification, as well as methylation analysis in combination with GLC-MS, laser-desorption mass spectrometry and 1H-NMR and 13C-NMR spectroscopy, the carbohydrate backbone of the lipopolysaccharide was characterized as a branched decasaccharide with the following structure: (formula; see text) In the native lipopolysaccharide two additional phosphate groups are present and 3-deoxy-D-threo-hexulosonic acid and D-galacturonic acid are bound via acid-labile linkages.

Carbohydrate Conformation↗
PubMed1991

Structural analysis of the lipid A component of Campylobacter jejuni CCUG 10936 (serotype O:2) lipopolysaccharide. Description of a lipid A containing a hybrid backbone of 2-amino-2-deoxy-D-glucose and 2,3-diamino-2,3-dideoxy-D-glucose.

The chemical structure of Campylobacter jejuni CCUG 10936 lipid A was elucidated. The hydrophilic backbone of the lipid A was shown to consist of three (1----6)-linked bisphosphorylated hexosamine disaccharides. Neglecting the phosphorylation pattern, a D-glucosamine (2-amino-2-deoxy-D-glucose) disaccharide [beta-D-glucosaminyl-(1----6)-D-glucosamine], a hybrid disaccharide of 2,3-diamino-2,3-dideoxy-D-glucose and D-glucosamine [2,3-diamino-2,3-dideoxy-beta-D-glucopyranosyl-(1----6)-D-glucosamine], and a 2,3-diamino-2,3-dideoxy-D-glucose disaccharide were present in a molar ratio of 1:6:1.2. Although the backbones are bisphosphorylated, heterogeneity exists in the substitution of the polar head groups. Phosphorylethanolamine is alpha-glycosidically bound to the reducing sugar residue of the backbone, though C-1 is also non-stoichiometrically substituted by diphosphorylethanolamine. Position 4' of the non-reducing sugar residue carries an ester-bound phosphate group or is non-stoichiometrically substituted by diphosphorylethanolamine. By methylation analysis it was shown that position 6' is the attachment site for the polysaccharide moiety in lipopolysaccharide. These backbone species carry up to six molecules of ester- and amide-bound fatty acids. Four molecules of (R)-3-hydroxytetradecanoic acid are linked directly to the lipid A backbone (at positions 2, 3, 2', and 3'). Laser desorption mass spectrometry showed that both (R)-3-hydroxytetradecanoic acids linked to the non-reducing sugar unit carry, at their 3-hydroxyl group, either two molecules of hexadecanoic acid or one molecule of tetradecanoic and one of hexadecanoic acid. It also suggested that the (R)-3-(tetradecanoyloxy)-tetradecanoic acid was attached at position 2', whereas (R)-3-(hexadecanoyloxy)-tetradecanoic acid was attached at position 3', or at positions 2' and 3'. Therefore, the occurrence of three backbone disaccharides differing in amino sugar composition and presence of a hybrid disaccharide differentiate the lipid A of this C. jejuni strain from enterobacterial and other lipids A described previously.

Campylobacter jejuni↗
PubMed1991

A 28,000-Da GDP/GTP-binding protein specific to the nuclear envelope.

Using a photoaffinity labeling approach to characterize GDP/GTP-binding proteins of the nuclear envelope, we found that several polypeptides of isolated rat liver nuclear envelopes were photolabeled with [alpha-32P]GDP. One of these labeled proteins, which had an apparent molecular mass of 28 kDa (p28), was highly enriched in nuclear envelopes relative to unfractionated nuclei and was not detectable in fractions of mitochondria, cytosol, and endoplasmic reticulum membranes by two-dimensional gel electrophoresis. Thus p28 is specific to the nuclear envelope. Chemical extractions showed that p28 was tightly associated with a subfraction of the nuclear envelope enriched in nuclear pore complexes and lamina. Solubilization of p28 required buffers containing 1% octylglucoside and at least 300 mM KCl. Photolabeling of p28 was specific for GTP and GDP, since no other guanine or adenine nucleotide tested was able to compete efficiently with [alpha-32P]GDP for photolabeling. Added magnesium or manganese cations were not required for photolabeling of p28, and labeling was substantially inhibited with 10 mM MgCl2, 1 mM MnCl2, and KCl concentrations greater than 150 mM. Sucrose density centrifugation showed that detergent-solubilized p28 had a S value of 2.5. The properties of p28 described in this study suggest that it may have a role in functions of the pore complex or lamina.

Affinity Labels↗
PubMed1991

Bacterial endotoxin: molecular relationships between structure and activity.

The significance of endotoxins in bacterial infection and their role as bacterial surface antigens (O antigens) have stimulated investigations into their chemical nature and the mechanisms of their biologic action during the last few decades. This article summarizes some of the recent results and emphasizes structure-activity relationships.

Animals↗
PubMed1991

Intrabacterial sodium-to-potassium ratios and ATP contents of Mycobacterium leprae from ofloxacin-treated patients.

In a clinical trial including 17 multibacillary leprosy patients the in vivo effectiveness of ofloxacin on Mycobacterium leprae was tested via mass spectrometric determination of intrabacterial ratios of the concentrations of the sodium and potassium ions of individual organisms and of the ATP content per 10(6) bacteria isolated from skin biopsies. After 3 months of treatment, the in vivo drug effect could be determined with at least one of the two methods in 14 cases. Both methods revealed that in two cases the bacteria definitely did not respond to a 3-month ofloxacin monotherapy (200 mg twice daily). In three further cases a nonresponse of the M. leprae organisms was suspected from the mass spectrometric measurements. In the responder cases, the M. leprae were severely impaired. From the intrabacterial cation ratios the percentage of viable organisms averaged over all untreated biopsies was determined to be 58% and the percentage-killing during the first 3 months of treatment was 72%.

Adenosine Triphosphate↗
PubMed1991

Determination of in vivo and in vitro drug effects of mycobacteria from the mass spectrometric analysis of single organisms.

Laser microprobe mass analysis of single bacterial organisms allows the determination of their intrabacterial ratio of sodium-to-potassium ions and the registration of fragment ions originating from their organic bacterial cell matrices as mass fingerprint spectra. It has been established previously that the intrabacterial cation ratio provides information on the physiological state of an individual bacterial cell. In the present experiments it is also shown, with different cultivable mycobacterial species and strains (drug sensitive and resistant) exposed to various drugs, that data derived from the evaluation of the mass fingerprint spectra reflect changes in the degree of impairment. The analysis of Mycobacterium leprae derived from a limited number of skin biopsies of lepromatous/borderline lepromatous leprosy patients under World Health Organization-recommended multiple-drug therapy (WHO/MDT) showed impairment of the organisms with both of the methods of measurement in proportion to the duration of treatment except in one case. In one M. leprae population from a patient who had been treated for 19 months, the fingerprint evaluation gave the first evidence for an insufficient response to treatment. This was further confirmed by the unusual frequency distribution of the Na+,K+ ratios which revealed the existence of two subpopulations, one impaired and one unimpaired.

Anti-Bacterial Agents↗
PubMed1990

Investigation into the fluidity of lipopolysaccharide and free lipid A membrane systems by Fourier-transform infrared spectroscopy and differential scanning calorimetry.

The phase behaviour, particularly the fluidity within each phase state and the transitions between them, of lipopolysaccharides and of their lipid moiety, free lipid A, of various species of Gram-negative bacteria, especially of Salmonella minnesota and Escherichia coli, has been investigated by applying mainly Fourier-transform infrared spectroscopy and differential scanning calorimetry. For enterobacterial strains, the transition temperatures of the gel----liquid crystalline (beta----alpha) phase transition of the hydrocarbon chains in dependence on the length of the sugar moiety are highest for free lipids A (around 45 degrees C) and lowest for deep rough mutant lipopolysaccharides (around 30 degrees C). Evaluating certain infrared active vibration bands of the hydrocarbon moiety, mainly the symmetric stretching vibration of the methylene groups around 2850 cm-1, it was found that, in the gel state, the acyl chains of lipopolysaccharides and free lipid A have a higher fluidity as compared with saturated and the same fluidity as compared with unsaturated phospholipids. This 'partial fluidization' of lipopolysaccharide below the transition temperature correlates with its reduced enthalpy change at that temperature compared to phospholipids with the same chain length. The fluidity depends strongly on ambient conditions, i.e. on the Mg2+ and H+ content: higher Mg2+ concentrations and low pH values make the acyl chains of free lipid A and lipopolysaccharide preparations significantly more rigid and also partially increase the transition temperature. The influence of Mg2+ is highest for free lipid A and decreases with increasing length of the sugar side chain within the lipopolysaccharide molecules, whereas the effect of a low pH is similar for all preparations. At basic pH, a fluidization of the lipopolysaccharide and lipid A acyl chains and a decrease in transition temperature take place. Free lipid A and all investigated rough mutant lipopolysaccharides exhibit an extremely strong lyotropic behaviour in the beta----alpha melting enthalpy but not in the value of the transition temperature. The phase transition is distinctly expressed only at water concentrations higher than 50-60%. A further increase of the water content still leads to an increase in the phase-transition enthalpy, particularly for lipopolysaccharides with a more complete sugar moiety. The fluidity of the hydrocarbon chains is shown to be an important parameter with respect to the expression of biological activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗
PubMed1990

Pore formation by complement in the outer membrane of gram-negative bacteria studied with asymmetric planar lipopolysaccharide/phospholipid bilayers.

The interaction of complement with an asymmetric planar lipopolysaccharide/phospholipid bilayer system as a model for the lipid matrix of the outer membrane of Gram-negative bacteria has been studied. The addition of whole human serum to the aqueous solution at the lipopolysaccharide side of the asymmetric membrane resulted in a rapid increase of the bilayer conductance in discrete steps, indicating the formation of transmembrane pores, which were not observed in the case of pure phospholipid membranes. The amplitudes of the discrete conductance steps varied over a range of more than one order of magnitude. The mean single step conductance was (0.39 +/- 0.24) nS for a subphase containing (in mM): 100 KCl, 5 MgCl2 and 5 HEPES buffer. The steps were grouped into bursts of typically 9 +/- 3 events per burst and the conductance change within one burst was (8.25 +/- 4.00) nS. The pore-forming activity of serum at the asymmetric membrane system was independent of the presence of specific antibodies against the lipopolysaccharide but was dependent on calcium ions. Furthermore, the pore-forming activity required complement component C9. A model for the mode of pore formation by complement is proposed: The complement pore is generated in discrete steps by insertion of C9 monomers into the membrane and their irreversible aggregation to water-filled channels with a diameter of approximately 7 nm assuming a circular geometry.

Bacterial Outer Membrane Proteins↗
PubMed1990

Comparative investigations of regional lymph nodes and pseudocapsules after implantation of joint endoprostheses.

Morphological alterations of pseudocapsules and regional lymph nodes were studied by light and electron microscopy and by Laser Microprobe Mass Analysis (LAMMA). The tissue specimens originated from 32 autopsies of patients with hip joint endoprostheses (time in situ: 3 weeks - 15 years, average: 6 years) and two cases with knee joint endoprostheses. Characteristic changes of the lymph nodes as well as of the pseudocapsules consisted in an infiltration by monocytic histiocytes with various intracytoplasmatic wear particles. The foreign material consisted mainly of the components of bone cement: polymethylmethacrylate (PMMA) and zirconium oxide, to a lesser degree of polyethylene from the articulating surfaces. In two cases with special types of prostheses ceramic or metallic wear particles could be detected too. Most of the wear particles were found in the ipsilateral parailiac lymph nodes and in the paraaortic lymph nodes bilaterally. In the cases with mostly stable prostheses small amounts of wear particles were found in the lymph nodes as soon as 1.5 years after insertion and their number increased in all groups of lymph nodes after longer duration of the implant. The phagocytosing histiocytes showed degenerative changes. At present it is not clear, if the cell damage is caused by the amount of phagocytosed wear particles alone or if specific toxic effects of certain substances are of importance.

Granulation Tissue↗
PubMed1990

[Morphological demonstration of function-dependent cellular calcium redistributions in secretory cells by x-ray microanalysis, LAMMA and fluorescence cytometry].

Cytosolic calcium and its parathyroid hormone induced increase was evaluated in rat osteosarcoma cells by quin 2 fluorometry. Electron microscopic calcium detection in depot organelles (i.e. the recently defined calciosomes) is improved by a new precipitation method with hydroxylamine naphthoic acid and fluoride combined with X-ray microanalysis. As shown in submandibulary gland and pancreatic B cells, a new combination of GBHA staining with laser microprobe mass analysis (LAMMA) for the first time enables direct kinetical studies of calcium at the microscopical level using stable calcium isotopes (i.e. Ca44) as tracers.

Animals↗
PubMed1989

Supramolecular structure of lipopolysaccharide and free lipid A under physiological conditions as determined by synchrotron small-angle X-ray diffraction.

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria, may assume various three-dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small-angle X-ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia-coli-type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (greater than 90%) at different temperatures (20, 37, and 55 degrees C) and at different lipid/divalent cation molar ratios (20:1 to 1:1). We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20 degrees C, i.e. below the beta----alpha acyl-chain-melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55 degrees C), 4.60 nm for Re lipopolysaccharide (at 37 degrees C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37 degrees C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20 degrees C. At higher concentrations of divalent cations (e.g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1: square root of 2, 1: square root of 3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration.

Escherichia coli↗
PubMed1989

Structural characterization of the lipid A component of Bacteroides fragilis strain NCTC 9343 lipopolysaccharide.

The chemical structure of Bacteroides fragilis NCTC 9343 lipid A was characterized by using conventional chemical procedures, methylation analysis, and laser desorption mass spectrometry. It was found that B. fragilis lipid A consists of a beta-D-glucosaminyl-(1-6)-D-glucosaminyl-1-O-phosphate backbone whose hydroxyl groups in positions 4, 4' and 6' are free, the latter serving as the attachment site for the polysaccharide component in lipopolysaccharide. This backbone molecule carries up to of five molecules of ester- and amide-bound long chain non-hydroxylated and (R)-3-hydroxy fatty acids. With regard to the distribution on the fatty acids on the lipid A backbone, a considerable heterogeneity was revealed by laser desorption mass spectrometry. Despite this heterogeneity, a major species of B. fragilis lipid A could be defined in which the hydroxyl group at position 3' of the distal GlcN carries (R)-3-hydroxyhexadecanoic acid and the hydroxyl group at position 3 of the reducing GlcN is acylated by (R)-3-hydroxypentadecanoic acid. The amino group of the distal GlcN residue carries (R)-3-(13-methyltetradecanoyloxy)-15-methylhexadecanoic acid and that of the reducing GlcN group (R)-3-hydroxyhexadecanoic acid. The absence of ester-bound phosphate and ester-linked 3-acyloxyacyl groups, the presence of not more than five acyl residues and the predominance of fatty acids possessing 15-17 carbon atoms are unique features of B. fragilis lipid A which differentiate it from enterobacterial and other lipids A and which are likely to be related to its low endotoxic activity.

Acylation↗
PubMed1989

Structural analysis of the nontoxic lipid A of Rhodobacter capsulatus 37b4.

Lipid A from Rhodobacter capsulatus 37b4 consists of a D-glucosaminyl-(beta 1-6)-D-glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C-1 of the reducing glucosamine and phosphorylethanolamine at C-4' of the nonreducing glucosamine. 1,4'-Bisphosphorylated lipid A, lacking the polar head groups, was also encountered and contributed to the observed microheterogeneity in the phosphate substitution. The amino functions of both glucosamines are substituted almost entirely by the rare 3-oxotetradecanoic acid, which is a characteristic constituent of lipid A in the genus Rhodobacter. 3-Hydroxydecanoic acid is ester-bound at C-3 and C-3' of the glucosamine disaccharide and the one at the nonreducing glucosamine (C-3') is partially substituted by dodecenoic acid to form an ester-bound diester. In free lipid A, hydroxy groups at C-4 and C-6' of the glucosamine disaccharide are unsubstituted. C-6' being the putative attachment point of the lipopolysaccharide core. The nontoxic Rhodobacter capsulatus lipid A shows extensive serological cross-reaction with the toxic Salmonella lipid A. Structural similarities in the hydrophilic part of both types of lipid A, dissimilarities in the hydrophobic part and their impacts on serologic properties are discussed.

Binding Sites↗
PubMed1989

Structural and physicochemical requirements of endotoxins for the activation of arachidonic acid metabolism in mouse peritoneal macrophages in vitro.

Lipopolysaccharides of different wild-type and mutant gram-negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep-rough mutants of Salmonella minnesota and Escherichia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC4) and prostaglandin E2 (PGE2). Lipopolysaccharides of wild-type strains (S. abortus equi, S. friedenau) only induced PGE2 and not LTC4 formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC4 and PGE2 production. Deacylated Re-mutant lipopolysaccharide preparations were inactive. However, co-incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC4 and PGE2, similar to those obtained with Re-mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin-low-responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re-mutant lipopolysaccharide and that polymyxin B prevented the Re-lipopolysaccharide-induced LTC4 and PGE2 release. Physical measurements showed that the phase-transition temperatures of both free lipid A and S-form lipopolysaccharide were above 37 degrees C while those of R-mutant lipopolysaccharides were significantly lower (30-35 degrees C). Thus, with the materials investigated, an inverse relationship between the phase-transition temperature and the capacity to elicit LTC4 production was revealed.

Animals↗