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Biomedical subjects

U Seydel

Publications and source records attributed to U Seydel.

At least 73 records · Page 4Linked to original sources
PubMed1996

Lipopolysaccharide-binding protein mediates CD14-independent intercalation of lipopolysaccharide into phospholipid membranes.

Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CD14-dependent whereas at high LPS concentrations it is CD14-independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CD14-independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide-binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14-independent cell activation at high endotoxin concentrations.

Acute-Phase Proteins↗
PubMed1996

Zinc enhances lipopolysaccharide-induced monokine secretion by alteration of fluidity state of lipopolysaccharide.

Elevated zinc serum concentrations have been shown to restore impaired immune response. Therefore, pharmacologic zinc supplementation has been used to improve immune function, particularly in intensive care patients. In these patients, Gramnegative sepsis, the symptoms of which are predominantly caused by LPS-induced release of monokines, represents a serious problem. We have recently shown that zinc enhances induction of TNF-alpha and IL-1 beta in cultures of PBMC by LPS. By fluorescence polarization and infrared spectroscopic measurements we found that zinc addition leads to decreased fluidity of the hydrocarbon chains of LPS. Experiments at different temperatures showed that the less fluid gel (beta) phase of LPS is more effective in cytokine induction than the more fluid liquid-crystalline (alpha) phase. Our studies suggest that the synergistic effect of zinc on monokine induction by LPS is caused by direct interaction of zinc with LPS altering the fluidity of the hydrocarbon chains. Although this effect is zinc specific, other divalent ions, like cobalt and nickel, with a complex structure and size comparable to those of zinc also enhance LPS-induced monokine secretion but to a much lesser extent. Our data indicate that the zinc level represents a relevant clinical parameter in the treatment of Gram-negative infection. This reveals potential risks in the therapeutic application of zinc.

Adjuvants, Immunologic↗
PubMed1996

Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport.

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import.

Animals↗
PubMed1996

Planar asymmetric lipid bilayers of glycosphingolipid or lipopolysaccharide on one side and phospholipids on the other: membrane potential, porin function, and complement activation.

We have determined some physicochemical properties of the monosaccharide-type fraction (GSL-1) of glycosphingolipids, the major glycolipid components of the outer leaflet of the Gram-negative species Sphingomonas paucimobilis. These properties included the state of order of the hydrocarbon moiety, the effective molecular area, surface charge density, and intrinsic transmembrane potential profile of reconstituted planar asymmetric GSL-1/phospholipid bilayer membranes. We have, furthermore, investigated the insertion into and the function of porin channels in the reconstituted bilayers and the complement-activating capability of GSL-1 surfaces. All results were compared with respective data for deep rough mutant lipopolysaccharide of Salmonella minnesota R595. We found a remarkable agreement in most functional properties of the two glycolipids.

Biophysical Phenomena↗
PubMed1996

Targeted delivery of an antisense oligonucleotide in the retina: uptake, distribution, stability, and effect.

In this article, we describe the preliminary results of the development of an animal model that will enable us to study the effect of photoreceptor-derived debris accumulation on the normal function of the retina in vivo. An antisense oligonucleotide (Cat 5), saline, and two control oligonucleotides were injected into the vitreous of 7-week-old RCS-rdy+ rats. The uptake, distribution, and persistence of the antisense oligonucleotide in the retina was demonstrated by fluorescent confocal microscopy, and the stability of the oligonucleotide was shown by GeneScan analysis using a fluorescein-labeled derivative of Cat 5 (Cat 5F). The accumulation of photoreceptor-derived debris was monitored by the number of undigested phagosomes in the RPE layer by light microscopy. Following intravitreal injection of Cat 5F, penetration of the oligonucleotide was observed in the ganglion cell layer in 2 hours and in the photoreceptor and pigment epithelial layers 3 days later. However, at 7, 28, and 56 days postinjection, only the RPE layer had significant amounts of Cat 5F present. Using GeneScan analysis, it was demonstrated that the fluorescein-labeled oligonucleotide present in the RPE layer was not degraded and it retained its original 19-mer length. There was no statistically significant difference in the number of phagosomes found in the RPE layer of control uninjected, saline-injected, and two sense and two antisense oligonucleotides-injected animals at 7 and 28 days postinjection. In contrast, the number of phagosomes was significantly higher (p < 0.001) in the RPE layer of Cat 5 antisense oligonucleotide-injected animals at 7 and 28 days postinjection. This difference, however, disappeared by 56 days postinjection. The inner nuclear layers of the retina of control and experimental animals were not affected by the injections.

Adolescent↗
PubMed1996

Drug effects on intracellular mycobacteria determined by mass spectrometric analysis of the Na(+)-to-K+ ratios of individual bacterial organisms.

The successful establishment of a drug screening system for intracellular cultivable and noncultivable mycobacteria based on the mass spectrometric determination of bacterial viability is described. To compare drug efficacies on intra- and extracellular mycobacteria, the mycobacteria were subjected to drug treatment either after phagocytosis by the mouse macrophage cell line RAW 264.7 or in cell-free medium. After reisolation, their viability was monitored by analyzing the intrabacterial sodium-to-potassium ratios for a limited number of individual organisms. This approach offers a reliable and quick tool for monitoring the influence of intracellular growth and of additional permeation barriers on intracellular drug efficacy and will thus provide useful information for the rational development and testing of optimized antimycobacterial drugs. In particular, the methodology is applicable to the noncultivable species Mycobacterium leprae, because the mass spectrometric analysis of the intrabacterial sodium-to-potassium ratio allows the determination of bacterial viability independent from their ability to multiply in vitro. Because of the improved metabolic activity of intracellularly growing M. leprae compared with that of extracellularly growing M. leprae, the spectrum of antileprosy drugs that can be tested in vitro could even be extended to those interfering with DNA replication and cell division.

Animals↗
PubMed1995

Lipopolysaccharide of Rhodospirillum salinarum 40: structural studies on the core and lipid A region.

The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) of Rhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4'bisphosphorylated beta (1 --> 6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlcN --> GlcN, 2,3-diamino-2,3-dideoxy-D-Glc-(DAG --> DAG, and DAG --> GlcN. Lipid A of R. salinarum contained preferentially 3-OH-18:0 and 3-OH-14:0 as amide-linked and cis delta 11-18:1 and c19:0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cis delta 11-18:1)-18:0 and 3-O-(c19:0)-14:0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine, phosphorylethanolamine, nor by 4-amino-4-deoxy-L-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1 --> 4)HexA(1 --> 5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS of R. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10(-1)-10(-2) of that reported for Salmonella abortus equi LPS, and it was also capable of inducing TNF alpha and IL6 in macrophages of C57BL/10ScSN mice.

Animals↗
PubMed1994

Chemical structure of the lipid A component of lipopolysaccharides of the genus Pectinatus.

The chemical structure of the lipid A components of smooth-type lipopolysaccharides isolated from the type strains of strictly anaerobic beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were analyzed. The hydrophilic backbone of lipid A was shown, by controlled degradation of lipopolysaccharide combined with chemical assays and 31P-NMR spectroscopy, to consist of the common beta 1-6-linked disaccharide of pyranosidic 2-deoxy-glucosamine (GlcN), phosphorylated at the glycosidic position and at position 4'. In de-O-acylated lipopolysaccharide, the latter phosphate was shown to be quantitatively substituted with 4-amino-4-deoxyarabinose, whereas the glycosidically linked phosphate was present as a monoester. Laser-desorption mass spectrometry of free dephosphorylated lipid A revealed that the distal (non-reducing) GlcN was substituted at positions 2' and 3' with (R)-3-(undecanoyloxy)tridecanoic acid, whereas the reducing GlcN carried two unsubstituted (R)-3-hydroxytetradecanoic acids at positions 2 and 3. The lipid A of both Pectinatus species were thus of the asymmetric hexaacyl type. The linkage of lipid A to polysaccharide in the lipopolysaccharide was relatively resistant to acid-catalyzed hydrolysis, enabling the preparation of a dephosphorylated and deacylated saccharide backbone. Methylation analysis of the backbone revealed that position 6' of the distal GlcN of lipid A was the attachment site of the polysaccharide. Despite the quantitative substitution of the lipid A 4'-phosphate by 4-amino-4-deoxyarabinose, which theoretically should render the bacteria resistant to polymyxin, P. cerevisiiphilus was shown to be susceptible to this antibiotic. P. cerevisiiphilus was, however, also susceptibile to vancomycin and bacitracin, indicating that the outer membrane of this bacterium does not act as an effective permeability barrier.

Amino Sugars↗
PubMed1994

Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans.

We have studied the role of lipopolysaccharide (LPS) for the insertion of LPS-free porin from Paracoccus denitrificans into planar lipid bilayers and its function therein. For this, we reconstituted the porin into different asymmetric planar lipid bilayers with or without LPS and into symmetric phospholipid bilayers. LPS-free porin added to the various bilayer systems was found to induce a step-wise increase in membrane conductance with different incorporation rates, depending on the presence of LPS in the bilayer leaflet opposite to porin addition. The incorporation rate into asymmetric LPS/phospholipid membranes from the phospholipid side was more than 10-fold higher than that observed for pure phospholipid membranes. The porin formed general diffusion pores without any salt specificity. The mean single-channel conductance did not depend on the presence of LPS and was about 4.2 nS for a subphase containing 1 M KCl in all systems tested. At certain applied transmembrane voltages, which depended on membrane composition and were approximately greater than 100 mV for the LPS/phospholipid system, single-channel closing in three steps was observed. Differences in the voltage dependence of porin-channel closing could be correlated with the surface charge of the bilayer. From the voltage-dependent gating behaviour proof for an oriented incorporation of the porin molecules, depending on the side of porin addition, and evidence for their orientation could be derived. Measurements at temperatures above and below the beta<==>alpha phase transition temperature of LPS gave evidence for the influence of membrane rigidity on the gating behaviour.

Carbohydrate Sequence↗
PubMed1994

Bacterial endotoxin: molecular relationships of structure to activity and function.

Endotoxins of Gram-negative microbes fulfill as components of the outer membrane a vital function for bacterial viability and, if set free, induce in mammalians potent pathophysiological effects. Chemically, they are lipopolysaccharides (LPS) consisting of an O-specific chain, a core oligosaccharide, and a lipid component, termed lipid A. The latter determines the endotoxic activities and, together with the core constituent Kdo, essential functions for bacteria. The primary structure of lipid A of various bacterial origin has been elucidated and lipid A of Escherichia coli has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique primary structure and a peculiar endotoxic conformation. The biological lipid A effects are mediated by macrophage-derived bioactive peptides such as tumor necrosis factor alpha (TNF). Macrophages possess LPS receptors, and the lipid A regions involved in specific binding and cell activation have been characterized. Synthetic lipid A partial structures compete the specific binding of LPS or lipid A and antagonistically inhibit the production of LPS-induced TNF. LPS toxicity, in general, and the ability of LPS to induce TNF are also suppressed by a recently developed monoclonal antibody (IgG2a), which is directed against an epitope located in the core oligosaccharide. At present we determine molecular and submolecular details of the specificity of the interaction of lipid A with responsive host cells with the ultimate aim to provide pharmacological or immunological therapeutics that reduce or abolish the fatal inflammatory consequences of endotoxicosis.

Animals↗
PubMed1994

A case for an endotoxic conformation.

Gram-negative bacteria express on their surface endotoxins which are essential for bacterial growth and survival. If released from the bacterial cell, endotoxins induce, in higher organisms, a variety of pathophysiological effects known as manifestations of bacterial sepsis. Chemically, endotoxins are lipopolysaccharides (LPS) composed of a polysaccharide and a lipid component, termed lipid A, which is responsible for the induction of endotoxin effects. Lipid A of all endotoxically active LPS have a similar architecture. The structural and conformational parameters endowing lipid A with its potent bioactivity have been well characterized. The toxic effects of endotoxins or their lipid A component, respectively, are initiated by the specific interaction of lipid A with macrophages/monocytes resulting in the production of peptide or lipid mediators like tumor necrosis factor alpha (TNF), the leukotrienes, and oxygen radicals. This interaction requires a unique "endotoxic conformation" of lipid A on the one hand, and specific cellular receptors on the other. The interaction and subsequent mediator production can be specifically and antagonistically inhibited by lipid A partial structures.

Animals↗
PubMed1993

Influence of the supramolecular structure of free lipid A on its biological activity.

The three-dimensional supramolecular structures and the states of order of the acyl chains of lipid A from different Gram-negative species were investigated at 40 degrees C, high water content (80-90%), and different [lipid A]/[Mg2+] molar ratios using synchrotron radiation X-ray diffraction and Fourier-transform infrared spectroscopy. Measurements were made on free lipid A from Salmonella minnesota R595, mono- and bi-phosphoryl, as well as those from the non-enterobacterial strains Rhodobacter capsulatus 37b4, Rhodopseudomonas viridis F, and Rhodocyclus gelatinosus 29/1. Parallel to differences in their chemical primary structure, the structural polymorphisms and states of order at 37 degrees C of the non-enterobacterial lipid A were found to be different from those of enterobacterial lipid A. A clear correlation between the supramolecular structure and previously determined biological activities was found. Lipid A with a strong preference for lamellar structures (Rb. capsulatus and Rp. viridis) are endotoxically inactive and lack cytokine-inducing capacity; the compounds assuming a mixed lamellar/nonlamellar structure (monophosphoryl lipid A from S. minnesota) are of lower toxicity in vivo, but may induce cytokines in vitro; those lipid A with a strong tendency to form non-lamellar inverted structures (lipid A from S. minnesota and Rc. gelatinosus) exhibit full endotoxicity in vitro and in vivo. In contrast, anti-complementary activity is most pronounced for compounds with lamellar and least expressed for those with inverted structures. The states of order at 37 degrees C vary non-systematically, exhibiting the highest values for lipid A of S. minnesota and the lowest for that of Rc. gelatinosus. We propose to extend the term 'endotoxic conformation', which is used to describe the conformation of a single lipid A molecule required for optimal triggering of biological effects, to 'endotoxic supramolecular conformation' which denotes the particular organization of lipid A aggregates in physiological fluids causing biological activity.

Carbohydrate Sequence↗
PubMed1993

Defective biosynthesis of the lipid A component of temperature-sensitive firA (omsA) mutant of Escherichia coli.

The biosynthesis of lipid A component was shown to be defective in a temperature-sensitive firA mutant of Escherichia coli. Cells were biosynthetically labelled with [14C]acetate and incorporation of radioactivity into the glycerophospholipid compared to lipid A fractions was measured. The lipid A/glycerophospholipid biosynthesis ratio of the firA mutant at 37 degrees C was approximately 50%, and at the nonpermissive temperature of 42 degrees C was less than 20% of that observed in the corresponding wild-type strain. Analysis of radiolabelled lipid A 4'-monophosphate derivatives and glycerophospholipids by thin-layer chromatography revealed that the firA mutant at 42 degrees C elaborated an altered lipid A, and its phosphatidylglycerol content was low. The chemical composition of the extracted lipopolysaccharides differed significantly between the firA and the wild-type strain only in the proportion of hexadecanoic acid, which was minimal in the wild type grown at 37 degrees C and 42 degrees C and in firA lipopolysaccharide grown at 37 degrees C. In the firA mutant lipopolysaccharide produced at 42 degrees C, hexadecanoic acid was present in approximately every third molecule, attached to the hydroxyl group of the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing glucosamine of lipid A. Inspection of dephosphorylated free lipid A preparations by laser-desorption mass spectrometry confirmed that significant amounts of heptaacyl lipid A was elaborated by the firA strain grown at 42 degrees C.

Acyltransferases↗
PubMed1993

Dialysis-associated encephalopathy: light and electron microscopic morphology and topography with evidence of aluminum by laser microprobe mass analysis.

Recently we described silver-staining variants for the demonstration of beta/A4 amyloid and neurofibrillary tangles in senile dementia of Alzheimer type (SDAT). The same methods allowed, for the first time, the visualization of characteristic patterns and distinct morphological changes in human dialysis-associated encephalopathy. Light and electron microscopy demonstrated typical silver-stained inclusions in the cytoplasm of choroidal epithelium, glia and neurons. Performing laser microprobe mass analysis on en-bloc silver-stained semithin sections, evidence for significant amounts of aluminum was obtained within the lesions. Prominent aluminum-signals were obtained additionally in adjacent structures and nuclei of sections which were stained with toluidine-blue exclusively. Silver-stained paraffin sections of ten patients with a history of long-term hemodialysis were evaluated. The choroidal epithelium--obviously the most sensitive structure--showed black inclusions ranging from a few dots to a complete black staining of cells. Glial cells presented massive silver-stained deposits, which were restricted to the gray matter. Finally, neurons revealed numerous fine-granular black inclusions, scattered throughout the cytoplasm. Brain stem nuclei were primarily affected, but neurons within cortex, subcortical gray matter and spinal cord were also involved to various degrees; inclusions were not evident in the nucleus dentatus and the oliva inferior. Vessel-related deposits were found frequently. By electron microscopy the cytoplasm of neurons was filled either with large amounts of small electron-dense granules, or with lipofuscin granules, containing numerous irregular, non-membrane-bound inclusions. Massive electron-dense depositions were seen in the cytoplasm of choroidal epithelia and in proximity to nuclei of cortical astro- and oligodendroglia. The described neuronal changes and, in particular, alterations of choroidal epithelium and glia are completely different from characteristic plaques and tangles in SDAT.

Adult↗
PubMed1993

[Bacterial endotoxins: relationship between chemical structure and biological effect].

Gram-negative bacteria carry on their surface endotoxins, which are essential for bacterial growth and survival. If released from the bacterial cell, endotoxins induce in higher organisms a great variety of pathophysiological effects. Chemically, endotoxins constitute lipopolysaccharides (LPS), the lipid component (termed lipid A) of which is responsible for the induction of endotoxin effects. The structural and conformational parameters, endowing lipid A with its potent bioactivity, have been well characterized. The toxic effects of endotoxins are initiated by the specific interaction of lipid A with macrophages/monocytes resulting in the production of peptide or lipid mediators. This interaction is governed by a unique (toxic) conformation of lipid A on the one hand, and by specific cellular receptors on the other. The interaction and subsequent mediator production can be specifically and antagonistically inhibited by lipid A partial structures. A recently developed monoclonal anti-LPS-antibody cross-reacts with endotoxins of various bacterial origin, and it cross-protects against harmful endotoxin effects such as pyrogenicity and lethality.

Animals↗