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Biomedical subjects

A Herrmann

Publications and source records attributed to A Herrmann.

At least 37 records · Page 2Linked to original sources
PubMed2001

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.

Animals↗
PubMed2001

Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential.

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130 has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.

Animals↗
PubMed2001

Stochastic simulation of hemagglutinin-mediated fusion pore formation.

Studies on fusion between cell pairs have provided evidence that opening and subsequent dilation of a fusion pore are stochastic events. Therefore, adequate modeling of fusion pore formation requires a stochastic approach. Here we present stochastic simulations of hemagglutinin (HA)-mediated fusion pore formation between HA-expressing cells and erythrocytes based on numerical solutions of a master equation. The following elementary processes are taken into account: 1) lateral diffusion of HA-trimers and receptors, 2) aggregation of HA-trimers to immobilized clusters, 3) reversible formation of HA-receptor contacts, and 4) irreversible conversion of HA-receptor contacts into stable links between HA and the target membrane. The contact sites between fusing cells are modeled as superimposed square lattices. The model simulates well the statistical distribution of time delays measured for the various intermediates of fusion pore formation between cell-cell fusion complexes. In particular, these are the formation of small ion-permissive and subsequent lipid-permissive fusion pores detected experimentally (R. Blumenthal, D. P. Sarkar, S. Durell, D. E. Howard, and S. J., J. Cell Biol. 135:63-71). Moreover, by averaging the simulated individual stochastic time courses across a larger population of cell-cell-complexes the model also provides a reasonable description of kinetic measurements on lipid mixing in cell suspensions (T. Danieli, S. L. Pelletier, Y.I. Henis, and J. M. White, 1996, J. Cell Biol. 133:559-569).

Cell Fusion↗
PubMed2001

Dynamics of membrane penetration of the fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group attached to an acyl chain of phosphatidylcholine.

Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long- and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells.

We have investigated the transport and canalicular enrichment of fluorescent phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and beta-BODIPY-PC. Fluorescent PC was efficiently transported to the biliary canaliculus (BC) and became enriched on the lumenal side of the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recycling compartment (ARC) in polarized HepG2 cells as shown by colocalization with fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respectively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of PC analogs from the canalicular membrane were found. Evidence for nonvesicular transport included enrichment of the PC-analog beta-BODIPY-PC in the BC (t1/2 = 3.54 min) prior to its accumulation in the SAC/ARC (t1/2 = 18.5 min) at 37 degrees C. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton and at 2 degrees C. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in hepatocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Lipid composition of virosomes modulates their fusion efficiency with cryopreserved bull sperm cells.

Virosomes derived from different fusogenic enveloped viruses have been generated for potential application in gene targeting to sperm cells. Comparative characterization of reconstitution products revealed that virosomes derived from influenza viruses are superior to those generated from Sendai viruses, with respect to the fusion rates with cryopreserved bull sperm cells and to sperm cell vitality after fusion. Modulation of the lipid composition during virosome reconstitution affects fusion sites on target sperms and allows optimization of the fusion rate and sperm cell vitality. A fluorescence-based microscopic fusion assay combined with a vital cell stain revealed that about 90% of sperm cells fused with influenza virosomes containing exogenous cholesterol, sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. About 85% of the fused sperm cells remained vital. Such optimized influenza-derived virosomes provide the basis for ongoing experiments, which aim at eventually generating biologically active transgenic sperms.

Animals↗
PubMed2000

Zinc plays a key role in human and bacterial GTP cyclohydrolase I.

The crystal structure of recombinant human GTP cyclohydrolase I was solved by Patterson search methods by using the coordinates of the Escherichia coli enzyme as a model. The human as well as bacterial enzyme were shown to contain an essential zinc ion coordinated to a His side chain and two thiol groups in each active site of the homodecameric enzymes that had escaped detection during earlier studies of the E. coli enzyme. The zinc ion is proposed to generate a hydroxyl nucleophile for attack of imidazole ring carbon atom eight of the substrate, GTP. It may also be involved in the hydrolytic release of formate from the intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, and in the consecutive Amadori rearrangement of the ribosyl moiety.

Amino Acid Sequence↗
PubMed2000

Light-induced reorganization of phospholipids in rod disc membranes.

The transbilayer redistribution of spin-labeled phospholipid analogues (SL-PL) with choline, serine, and ethanolamine head groups (PC, PS, and PE, respectively) was studied on intact disc vesicles of bovine rod outer segment membranes in the dark and after illumination. Redistribution was measured by the extraction of spin-labeled lipid analogues from the outer leaflet of membrane using the bovine serum albumin back-exchange assay. In the dark, PS was distributed asymmetrically, favoring the outer leaflet, whereas PC and PE showed small if any asymmetry. Green illumination for 1 min caused lipid head group-specific reorganization of SL-PL. Extraction of SL-PS by bovine serum albumin showed a fast transient (<10 min) enhancement, which was further augmented by a peptide stabilizing the active metarhodopsin II conformation. The data suggest a direct release of 1 molecule of bound PS per rhodopsin into the outer leaflet and subsequent redistribution between the two leaflets. SL-PE and SL-PC showed more complex kinetics, in both cases consistent with a prolonged period of reduced extraction (2 phospholipids per rhodopsin in each case). The different phases of SL-PL reorganization after illumination may be related to the formation and decay of the active rhodopsin species and to their subsequent regeneration process.

Animals↗
PubMed2000

Membrane transport: deciphering fusion.

Membrane fusion events that occur in yeast have been reconstituted with a minimal set of SNARE protein components. This system has been exploited to establish the syntax underlying specificity of intracellular fusion events from yeast to mammals.

Animals↗
PubMed2000

Binding properties of agonists and antagonists to distinct allosteric states of the nicotinic acetylcholine receptor are incompatible with a concerted model.

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.

Acetylcholine↗
PubMed2000

Transmembrane movement of diether phospholipids in human erythrocytes and human fibroblasts.

We have synthesized spin-labeled (SL) and fluorescently labeled diacyl, 1-alkyl-2-acyl-, and di-alkyl glycerophospholipids. The sn-2 chain was a short chain with either a nitroxide group or a 7-nitro-2, 1,3-benzoxadiazol-4-yl (NBD). After incorporation in the exoplasmic leaflet of human erythrocytes, we found that SL-phosphatidylcholine (PC) redistributed very slowly across the plasma membrane, less than 20% reaching the cytoplasmic leaflet in 3 h at 37 degrees C. In contrast, SL-phosphatidylserine (PS) accumulated on the cytoplasmic leaflet with the same plateau corresponding to 90% of the probes inside. The characteristic times for inward redistribution were different for the three PS analogues: at 37 degrees C, the t(1/2) for the diacyl, alkyl-acyl, and dialkyl compounds were 2.3, 3.5, and 41 min, respectively. ATP depletion or incubation with N-ethylmaleimide inhibited the rapid translocation of the PS derivatives. The diether PS bearing an NBD group translocated very slowly in human erythrocytes and no acceleration by ATP could be measured. On the other hand, in human fibroblasts, the diether NBD-PS and SL-PS were both transported from the exoplasmic to the cytoplasmic monolayer of the plasma membrane as it is the case for the transport of the respective diester PS analogues. These results prove that the ether bonds do not prevent completely PS binding and translocation by the aminophospholipid translocase despite a probable hindrance due to the ether linkage on the sn-2 chain. Because of the high stability of the ether linkage, SL and NBD diether analogues should be useful to investigate lipid traffic in cultured cells.

Biological Transport↗
PubMed2000

Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes.

A gene encoding a cyclomaltodextrinase (neopullulanase) was cloned from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 and its nucleotide sequence was determined. The encoded CdaA protein lacked an N-terminal signal sequence and aligned well with a family of bacterial proteins described as maltogenic alpha-amylases, neopullulanases or cyclomaltodextrinases. Escherichia coli cells harboring the cloned cdaA gene produced a 66-kDa protein that degraded pullulan in a sodium dodecyl sulfate-polyacrylamide gel. A. acidocaldarius cells grown on maltose, soluble starch or pullulan synthesized the same protein. Neopullulanase activity of the protein was cytoplasmic and its pH optimum of 5.5 was close to the pH value of the cytoplasm. CdaA degraded cyclomaltodextrins rapidly and pullulan (to panose) more slowly. It is proposed that CdaA functions as a cytoplasmic cyclomaltodextrinase (EC 3.2.1.54).

Amino Acid Sequence↗
PubMed2000

[Acne inversa (hidradenitis suppurativa): early deetection and curative surgery].

Acne inversa (synonyms are hidradenitis suppurativa, pyodermia fistulans sinifica) is a chronic inflammatory disorder of the terminal hair follicles of the intertrigines. Abscesses and fistules develop mostly in both axillae, in the anogenital region, and under the breasts. The disease is common, but the right diagnosis is often missed. Local incision gives only a short relief of pain. The therapy of choice is wide local excision. We report on 12 patients with acne inversa who were surgically treated from 1991-1999 in our department. Surgical results and patient satisfaction were assessed on an average of 4.2 years. In all, 20 excisions in the axillae and 5 excisions anogenital were done. Seven patients were treated preoperatively with isotretinoin over at least 2 months. All patients could move their limbs without trouble. The cosmetic result was good in nine patients and three expressed dissatisfaction. Nine patients did not develop new lesions in the treated area. Three patients had new inflammations in both treated and previously uninvolved and therefore not resected regions. Wide local excision gives very good functional and cosmetic results. It usually heate long-lasting disease for nearly disabled patients. Isotretinoin reduces the disease activity and allows radical surgical treatment.

Adolescent↗
PubMed2000

Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach.

The transbilayer movement of short-chain spin-labeled and fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) phospholipid analogs in rat liver microsomes is measured by stopped-flow mixing of labeled microsomes with bovine serum albumin (BSA) solution. Extraction of analogs from the outer leaflet of microsomes to BSA can be directly monitored in conjunction with electron paramagnetic resonance or fluorescence spectroscopy by taking advantage of the fact that the signal of spin-labeled or fluorescent analogs bound to BSA is different from that of the analogs inserted into membranes. From the signal kinetics, the transbilayer movement and the distribution of analogs in microsomal membranes can be derived provided the extraction of analogs by BSA is much faster in comparison to the transbilayer movement of analogs. Half-times of the back-exchange for spin-labeled and fluorescent analogs were <3.5 and <9.5 s, respectively. The unprecedented time resolution of the assay revealed that the transbilayer movement of spin-labeled analogs is much faster than previously reported. The half-time of the movement was about 16 s or even less at room temperature. Transmembrane movement of NBD-labeled analogs was six- to eightfold slower than that of spin-labeled analogs.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

Continuous measurement of rapid transbilayer movement of a pyrene-labeled phospholipid analogue.

The excimer forming capacity of the fluorescent moiety pyrene is employed to measure continuously the transbilayer (re)distribution of a pyrene-labeled phosphatidylcholine analogue (pyPC) in liposomal membranes. pyPC with a lauroyl residue (sn-1 position) and a short (butyroyl) fatty acid chain (sn-2 position) bearing the pyrene moiety incorporates rapidly into the outer leaflet of liposomes. The fluorescence intensities of excimers (I(E)) and of monomers (I(M)) of pyPC depend on the concentration of the analogue in a membrane leaflet. Therefore, the redistribution of pyPC from the outer to the inner leaflet can be followed by changes of the ratio I(E)/I(M). The transbilayer movement of pyPC in pure phospholipid vesicles is very slow indicated by a constant I(E)/I(M). However, addition of membrane active peptides (melittin, magainin 2 amide or a mutant of magainin 2 amide) induced a rapid translocation of pyPC from the outer to the inner leaflet. An approach is presented which allows estimating the transbilayer distribution of pyPC from the measured ratio I(E)/I(M).

Antimicrobial Cationic Peptides↗
PubMed2000

The role of the transmembrane and of the intraviral domain of glycoproteins in membrane fusion of enveloped viruses.

Fusion of enveloped viruses with their target membrane is mediated by viral integral glycoproteins. A conformational change of their ectodomain triggers membrane fusion. Several studies suggest that an extended, triple-stranded rod-shaped alpha-helical coiled coil resembles a common structural and functional motif of the ectodomain of fusion proteins. From that, it is believed that essential features of the fusion process are conserved among the various enveloped viruses. However, this has not been established so far for the highly conserved transmembrane and intraviral sequences of fusion proteins. The article will focus on the role of both sequences in the fusion process. Recent studies from various enveloped viruses strongly imply that a transmembrane domain with a minimum length is required for later steps of membrane fusion, i.e., the formation and enlargement of the aqueous fusion pore. Although no specific sequence of the TM is necessary for pore formation, distinct properties and motifs of the domain may be obligatory to ascertain full fusion activity. However, with some exceptions, the intraviral domain seems to be not required for fusion activity of viral fusion proteins.

Amino Acid Sequence↗