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Biomedical subjects

A Herrmann

Publications and source records attributed to A Herrmann.

At least 91 records · Page 5Linked to original sources
PubMed1997

Interactions of the nicotinic acetylcholine receptor transmembrane segments with the lipid bilayer in native receptor-rich membranes.

Proper ion channel function of the nicotinic acetylcholine receptor (nAChR) requires the interaction of the protein with distinct lipid species present in the receptor's membrane microenvironment. Two classes of lipid binding sites present at the protein-membrane interface have been postulated: annular binding sites primarily occupied by phospholipids and non-annular binding sites mainly occupied by cholesterol [Jones & McNamee (1988) Biochemistry 27, 2364-2374]. We investigated the binding of these lipids to the nAChR and potential dynamics of these interactions during events associated with signal transduction by electron spin resonance spectroscopy (ESR) using spin-labeled analogues of phospholipids, androstane, and stearic acid. Protein-lipid interactions were characterized in receptor-rich membranes prepared from Torpedo californica electric tissue preserving the native lipid environment of the nAChR. We found a strong preference of the receptor for the phosphatidylserine (PS) analogue as compared to the other probes. Up to 57% of PS were perturbed by the membrane protein, while the fraction of motionally restricted lipid for the other analogues was on the order of 30%. After removal of the extramembrane portions of the membrane-bound receptor, we observed a loss of binding sites for the spin-labeled analogue of androstane and for stearic acid, but not for phospholipids and sphingomyelin analogues. Our results demonstrate the existence of topologically distinct lipid binding sites for different lipid species. In the case of cholesterol, extramembrane portions of the receptor are involved, whereas the transmembrane segments meet the requirements for the binding of phospholipids. Tyrosine phosphorylation of the nAChR did not affect protein-lipid interactions in samples of intact nAChR. Similarly, no significant changes were observed in the presence of carbamoylcholine at concentrations that caused rapid and quantitative desensitization of the nAChR.

Animals↗
PubMed1997

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach.

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement. By means of stopped-flow electron paramagnetic resonance (EPR) spectroscopy, we have studied the kinetics of incorporation of the spin-labeled phosphatidylcholine (PC) analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) and of its accessibility to chemical reduction and to back-exchange at room temperature. Incorporation of SL-PC into the outer leaflet of egg phosphatidylcholine (EPC) and red cell ghost membranes was essentially completed within 5 s. Ninety percent of the SL-PC molecules located in the outer membrane leaflet of those membranes were extracted by BSA within 15 s. All exterior-facing SL-PC molecules were reduced by ascorbate in a pseudo-first-order reaction within 60 s in EPC membranes and within 90 s in red cell ghost membranes. The rate of the reduction process could be enhanced by approximately 30-fold when 6-O-phenyl-ascorbic acid was used instead of ascorbate as the reducing agent. The results are discussed in light of assaying rapid transbilayer movement of spin-labeled analogs in biological membranes.

Animals↗
PubMed1997

Meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin.

Fusion between influenza virus and target membranes is mediated by the viral glycoprotein hemagglutinin (HA). Replacement of the transmembrane domain of HA with a glycosylphosphatidylinositol (GPI) membrane anchor allows lipid mixing but not the establishment of cytoplasmic continuity. This observation led to the proposal that the fusion mechanism passes through an intermediate stage corresponding to hemifusion between outer monolayers. We have used confocal fluorescence microscopy to study the movement of probes for specific bilayer leaflets of erythrocytes fusing with HA-expressing cells. N-Rh-PE and NBD-PC were used for specific labeling of the outer and inner membrane leaflet, respectively. In the case of GPI-HA-induced fusion, different behaviors of lipid transfer were observed, which include 1) exclusive movement of N-Rh-PE (hemifusion), 2) preferential movement of N-Rh-PE relative to NBD-PC, and 3) equal movement of both lipid analogs. The relative population of these intermediate states was dependent on the time after application of a low pH trigger for fusion. At early time points, hemifusion was more common and full redistribution of both bilayers was rare, whereas later full redistribution of both probes was frequently observed. In contrast to wild-type HA, the latter was not accompanied by mixing of the cytoplasmic marker Lucifer Yellow. We conclude that 1) the GPI-HA-mediated hemifusion intermediate is meta-stable and 2) expansion of an aqueous fusion pore requires the transmembrane and/or cytoplasmic domain of HA.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Transbilayer motion of spin-labelled phospholipids in the plasma membrane of epididymal and ejaculated ram spermatozoa.

The transbilayer movement and distribution of spin-labelled phospholipid analogues were studied in the plasma membrane of ram sperm cells isolated from functionally different regions of the epididymis (caput, cauda) and from the ejaculate. As already shown for ejaculated cells, (i) a rapid movement of aminophospholipid analogues phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet, and (ii) a slow transbilayer movement of phosphatidylcholine were observed in the plasma membrane of maturating ram sperm cells. This suggests an asymmetric steady state transbilayer distribution of phospholipids, with a preferential enrichment of aminophospholipids on the cytoplasmic leaflet of those cells. The fast inward redistribution of the aminophospholipid analogues is consistent with the presence of an aminophospholipid translocase activity in all three sperm cell preparations. The translocase activity was enhanced slightly during the epididymal transit of spermatozoa. However, compared with epididymal sperm cells, a marked increase in the aminophospholipid translocase activity and a more pronounced phospholipid asymmetry for phosphatidylethanolamine was established for ejaculated spermatozoa. The physiological relevance of the rapid removal of aminophospholipids from the exoplasmic leaflet of the plasma membrane of ram sperm cells is discussed. The quality of sperm cell fractions was characterized by the cell morphology, membrane integrity and the cellular ATP concentration.

Adenosine Triphosphate↗
PubMed1997

Photopenia in chronic vertebral osteomyelitis with technetium-99m-antigranulocyte antibody (BW 250/183).

UNLABELLED: Photon-deficient areas in 99mTc/111 in white blood cell (WBC) images for diagnosing vertebral osteomyelitis have been published often. This study retrospectively evaluated whether the use of 99mTc-labeled monoclonal antigranulocyte antibodies (BW 250/183) is superior to WBC and whether it offers higher specificity. METHODS: The study included 81 patients (46 men, 35 women; mean age 55 +/- 2 yr; from 1989 to 1995) with clinically suspected vertebral osteomyelitis who underwent scintigraphic imaging after intravenous injection of 555 MBq 99mTc-labeled monoclonal antigranulocyte antibodies. Forty patients suffered from osteomyelitis (20 men, 20 women; mean age 56 +/- 6 yr), 6 patients had metastases, 28 patients had spondylosis and disk herniation and 5 patients vertebral compression fractures. Diagnosis was not histologically verified in 2 patients. Planar imaging was performed at 4 and 24 hr postinjection. Histology of osteomyelitis was available in 30 patients, clinical follow-up in 10 patients. Visual uptake scores and quantitative uptake scores of the suspected areas were calculated. The results were compared to a semiquantitative histological score (high, medium, low grade) as well as to the scintigraphic scores. RESULTS: Scintigraphy showed photopenia in all patients with histologically proven vertebral osteomyelitis, independent of the grade of infection. A quantitative evaluation of 4 and 24 hr postinjection demonstrated a 58% increase of the uptake score in cases of histologically proven high-grade infections. This increase was seen predominantly in the thoracic spine but not in lumbar spine. All nonosseous paravertebral abscesses (n = 2) showed positive images and an increasing uptake over 24 hr. CONCLUSION: Paravertebral soft tissue infections can be differentiated excellently, whereas vertebral osteomyelitis, vertebral tumors or fractures can be localized, but no differentiation is possible.

Aged↗
PubMed1997

Altered bone metabolism in inflammatory bowel disease.

UNLABELLED: A reduced bone mineral density has been reported in inflammatory bowel disease (IBD). OBJECTIVE: To assess the mechanisms of bone disease in IBD. METHODS: We studied in 90 patients (61 with Crohn's disease, 22 with ulcerative colitis, 7 with indeterminate colitis) biochemical markers of bone metabolism in serum and bone mineral density by peripheral quantitative computed tomography at the forearm. RESULTS: Forty-five percent of the patients had a reduced bone density (Z score < -1). Serum calcium was normal in most patients, vitamin D deficiency was documented in 17%. Osteocalcin, a serum marker of bone formation, was decreased in 26% (1.2 +/- 0.1 ng/ml), whereas the carboxyterminal cross-linked telopeptide of type I collagen (ICTP), a recently described serum parameter of bone breakdown, was stimulated in 38% (10.4 +/- 2.3 microg/L). Of 33 patients with increased ICTP levels, 19 showed a decreased bone density (Z score < -1), and 2 of them never received steroids. An active status of the underlying disease in most patients with increased ICTP levels suggests a direct effect of the underlying IBD. In the whole series of patients with a history of active disease (n = 34), 47% had signs of an increased bone degradation (ICTP > 5 microg/L; mean, 12.9 +/- 4.7 microg/L). Data derived from a retrospective survey of 245 patients with IBD suggest that the prevalence of bone fractures in IBD is unexpectedly high, particularly in patients with a long duration of disease, frequent active phases, and high cumulative doses of corticosteroid intake. CONCLUSIONS: Several mechanisms may be involved in IBD-associated bone disease: (1) a high inflammatory activity directly induces bone degradation via yet unknown pathways, (2) treatment with corticosteroids may exert catabolic effects on the bone, or (3) malabsorption and vitamin D deficiency may activate bone turnover.

Adolescent↗
PubMed1996

Interactions of the dye Evans Blue and GYKI 52466, a 2,3-benzodiazepine, with (S)- alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured rat cortical neurons: electrophysiological evidence for at least two different binding sites for non-competitive antagonists.

The effects of the dye Evans Blue and GYKI 52466, a 2,3-benzodiazepine, on (S)- alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors in primary cultured rat cortical neurons were investigated using the patch-clamp technique. Evans Blue and GYKI 52466 reduced the currents induced by the application of 100 microM kainate with IC50 values of 10.6 +/- 1.4 microM and 12.1 +/- 0.4 microM, respectively. In contrast to the similar potencies of the two compounds, their kinetics of block were quite different with those of Evans Blue being more complex. The on-, as well as the off-reaction of the block by GYKI 52466 could be described by single exponential functions, whereas two different time-constants for binding and one time-constant for the unbinding of Evans Blue were found. The block of AMPA receptors by Evans Blue was not completely reversible under the experimental conditions applied in this study. GYKI 52466 was not able to augment the recovery after inhibiting AMPA receptors with Evans Blue. Moreover, preapplication of a high concentration of GYKI 52466 did not prevent the inhibition of AMPA receptors by Evans Blue. We therefore conclude that GYKI 52466 and Evans Blue bind to two different sites at AMPA receptors in primary cultured cortical neurons.

Animals↗
PubMed1996

MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions.

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrifugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.

Bronchitis↗
PubMed1996

ATP-dependent redistribution of phosphatidylethanolamine in the plasma membrane of an epithelial and a hepatocytic cell line.

The redistribution of spin-labeled phospholipid analogues of sphingomyelin (SM) and phosphatidylethanolamine (PE) from the outer to the inner leaflet of the plasma membrane of an epithelial (CaCo-2) and a hepatocytic cell line (HepG2) was investigated. The amount of analogues in the outer leaflet was determined by their back-exchange to bovine serum albumin (BSA). For both cell lines a fast ATP-dependent inward movement of spinlabeled PE (SL-PE) was found while SL-SM redistributed only slowly by a passive mechanism. After depletion of intracellular ATP transverse diffusion of SL-PE was similar to that of SL-SM. The data are compatible with the presence of an aminophospholipid translocase in both cell lines.

Adenosine Triphosphate↗
PubMed1996

Transverse mobility of aminophospholipids in the plasma membrane of ram sperm cells.

The transbilayer movement of aminophospholipids in the plasma membrane of ram sperm cells was investigated using spin-labeled lipid analogues. After incorporation, spinlabeled phosphatidylserine (SL-PS) and phosphatidyl-ethanolamine (SL-PE) rapidly disappeared from the exoplasmic monolayer. Even at lower temperatures (10 degrees C) the inward movement of SL-PE is fast. The initial velocities of the internalization of SL-PS and SL-PE were compared with those of ram and human erythrocytes.

Acrosome↗
PubMed1996

Deacylation of influenza virus hemagglutinin does not affect the kinetics of low pH induced membrane fusion.

The relevance of palmitoylation of cysteine residues of influenza virus hemagglutinin (HA) for the HA-mediated membrane fusion triggered at low pH is investigated. Either wild-type HA (subtype H7) or mutant HA devoid of fatty acids were expressed in insect cells. The kinetics as well as the extent of fusion of HA-expressing cells with human erythrocyte ghosts were measured by a membrane mixing assay. Fusion was measured continuously at different pH by fluorescence dequenching of the lipid-like fluorophore R18 initially incorporated into the erythrocyte membrane. No significant difference between fusion of wild-type and mutant HA expressing cells with ghosts could be detected showing that deacylation does affect neither the extent nor the kinetics of fusion.

Animals↗
PubMed1996

Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease.

Previous studies on the frequency of intestinal mast cells and eosinophils in patients with inflammatory bowel disease yielded conflicting results. In the present morphometric study, we quantified mast cells and eosinophils in the lamina propria by histological and immunohistochemical methods in 64 patients suffering from Crohn's disease (33 cases) or ulcerative colitis (31 cases), and in 29 controls. Histological data from 206 biopsies were related to the presence of mucosal inflammation and clinical parameters. The number of eosinophils was increased in patients with inflammatory bowel conditions (mean +/- SE: 331 +/- 44/mm2) as compared to controls (258 +/- 27/mm2), and was dependent on disease activity and drug treatment. Mean mast cell numbers did not differ between patients and controls. However, a reduced mast cell number was found in toluidine blue-stained sections of actively inflamed tissue areas (143 +/- 16/mm2, versus 206 +/- 18/mm2 in non-inflamed tissue). Immunohistochemical studies using antibodies against the granule proteins tryptase and chymase suggest that this decrease in mast cell numbers is due to mast cell degranulation. The present data show that the number of intestinal mast cells and eosinophils is altered in patients with inflammatory bowel diseases, suggesting that both cell types are involved in the pathogenesis of chronic intestinal inflammation.

Adult↗
PubMed1996

Transverse movement of spin-labeled phospholipids in the plasma membrane of a hepatocytic cell line (HepG2): implications for biliary lipid secretion.

The redistribution of spin-labeled phospholipid analogs across the plasma membrane of HepG2 cells, either in suspension or grown as monolayers, was investigated. After incorporation into the outer membrane leaflet spin-labeled aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) moved rapidly to the inner monolayer, whereas the analog of phosphatidylcholine (PC) disappeared more slowly from the outer leaflet. The fast, inward movement of the aminophospholipids was abolished after adenosine triphosphate (ATP)-depletion of cells, suggesting the presence of an aminophospholipid translocase in the plasma membrane of these cells. Compared with human red blood cells, the activity of the aminophospholipid translocase is two orders of magnitude higher in HepG2 cells. From these data, a transverse phospholipid asymmetry can be inferred with the aminophospholipids mainly concentrated on the inner monolayer and the choline-containing phospholipids on the outer leaflet. The relevance of the enrichment of PC in the outer membrane leaflet for the formation and composition of the bile is discussed.

Adenosine Triphosphate↗
PubMed1996

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach.

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

Adenosine Triphosphate↗