Biomedical subjects

Andreas Herrmann

Publications and source records attributed to Andreas Herrmann.

At least 55 records · Page 3Linked to original sources

PubMed2003

Early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: stability and energetics of the hemagglutinin.

A conformational change of the homotrimeric glycoprotein hemagglutinin (HA) of influenza virus mediates fusion between the viral envelope and the endosome membrane. The conformational change of the HA ectodomain is triggered by the acidic pH of the endosome lumen. An essential step of the conformational change is the formation of an extended coiled-coil motif exposing the hydrophobic fusion peptide toward the target membrane. The structures of the neutral-pH, non-fusion active conformation of the HA ectodomain and of a fragment of the ectodomain containing the coiled-coil motif are known. However, it is not known by which mechanism protonation triggers the conformational change of the stable neutral-pH conformation of the ectodomain. Here, recent studies on the stability of the HA ectodomain at neutral pH, the energetics of the conformational change toward the fusion-active state and of the unfolding of the HA ectodomain are summarised. A model for the early steps of the conformational change of the HA ectodomain is presented. The model implicates that protonation leads to a partial dissociation of the distal domains of the HA monomers that is driven by electrostatic repulsion. The opening of the ectodomain enables water to enter the ectodomain. The interaction of water with respective sequences originally shielded from contact with water drives the formation of the coiled-coil structure.

Endosomes↗

PubMed2003

Rapid transport of phospholipids across the plasma membrane of Leishmania infantum.

The internalization of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) in Leishmania infantum promastigotes was studied. We observed a rapid inward redistribution of NBD-PC, -PE, and -PS across the plasma membrane at 28 and 4 degrees C. This internalization was shown to be independent of the endocytic activity of parasites. Rapid inward movement was coupled to an energy-dependent transporter because it was almost inhibited by depletion of cellular ATP and was blocked after pretreatment with N-ethylmaleimide (NEM). In contrast, NBD-SM traverses the plasma membrane by passive flip-flop. By comparing this pattern of phospholipid transbilayer movement with those known from other eukaryotic cells, candidate lipid transporters are discussed.

4-Chloro-7-nitrobenzofurazan↗

PubMed2003

Signal transduction in the visual cascade involves specific lipid-protein interactions.

In retinal rod photoreceptor cells, transducin (Gt) and cyclic GMP phosphodiesterase (PDE) are peripherally anchored to the cytoplasmic surface of the disk saccules. We have examined the role of specific phospholipids in the interaction of these proteins with native osmotically intact disk vesicles, employing spin-labeled phospholipid analogues (2% of total phospholipids) and bovine serum albumin back-exchange assay. Inactive GDP-bound transducin exclusively reduced the extraction of negatively charged phosphatidylserine. The effect disappeared upon activation of the G-protein with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). PDE affected the extraction of the zwitterionic phosphatidylcholine and, to a smaller extent, of phosphatidylethanolamine. When active GtGTPgammaS interacted with the PDE to form the active effector, the interaction with phosphatidylcholine was specifically enhanced. Each copy of the G-protein bound 3 +/- 1 molecules of phosphatidylserine, whereas the PDE bound a much larger amount (70 +/- 10) of a mixture of phosphatidylcholine and ethanolamine. The results are interpreted as a head group-specific and state-dependent interaction of the signaling proteins with the phospholipids of the photoreceptor membrane.

Animals↗

PubMed2003

A fusion protein of the gp130 and interleukin-6Ralpha ligand-binding domains acts as a potent interleukin-6 inhibitor.

Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an alpha-subunit (IL-6Ralpha) that binds IL-6 with low affinity and a beta-subunit (gp130) that binds the IL-6.IL-6Ralpha complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6.soluble IL-6Ralpha complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Ralpha (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies.

Animals↗

PubMed2003

Preclinical evaluation of inducible nitric oxide synthase lipoplex gene therapy for inhibition of stent-induced vascular neointimal lesion formation.

Several reports have established the concept of nitric oxide synthase (NOS) gene transfer for inhibiting smooth muscle cell (SMC) proliferation after vascular injury. To minimize potential risks associated with viral gene transfer, we developed a liposome-based gene transfer approach employing inducible NOS (iNOS) overexpression for inhibition of stent-induced neointimal lesion formation. Therapeutic lipoplexes were transferred to femoral or coronary arteries of Goettingen minipigs, using the Infiltrator local drug delivery device. Efficiency of local iNOS lipoplex transfer was analyzed by iNOS-specific immunohistochemistry. NO-mediated inhibition of stent-induced neointimal lesion formation was analyzed by intravascular ultrasound (IVUS) and computerized morphometry. Gene transfer efficiency increased dose dependently to a maximum of 44.3 +/- 4.2% iNOS-positive vessel area (dose, 2 microg of iNOS lipoplex). Proliferating cell nuclear antigen (PCNA) expression of medial SMCs (immunohistochemistry) was inhibited significantly by transfer of 2 microg of iNOS lipoplexes (111 +/- 27 cells [iNOS] versus 481 +/- 67 cells [control; PCNA-positive medial cells]). IVUS analysis demonstrated that local transfer of iNOS lipoplexes resulted in a significant reduction of femoral in-stent plaque area (control, 40.85 +/- 6.37 mm(2); iNOS, 24.69 +/- 1.8 mm(2); p = 0.03). Coronary in-stent lesion formation was reduced by about 45% as determined by histologic morphometry (control, 4.0 +/- 0.29; iNOS, 2.2 +/- 0.30; p < 0.01). In conclusion, this study demonstrates that local intramural delivery of iNOS lipoplexes can exert therapeutic effects in inhibiting stent-induced neointimal lesion formation. Together with the nonviral character of this gene therapy approach, these findings may have important impact on the transition of NOS-based gene therapy to clinical practice.

Animals↗

PubMed2003

Analysis of the lipid composition of bull spermatozoa by MALDI-TOF mass spectrometry--a cautionary note.

In this study we demonstrated the combination of MALDI-TOF MS and TLC as a fast and powerful tool to investigate the phospholipid (PL) composition of organic extracts of bull spermatozoa. Since phosphatidylcholine (PC) is the dominant PL species, an adequate resolution of MALDI-TOF spectra for sphingomyelin (SM) or phosphatidylethanolamine (PE) was achieved only after previous PL separation by TLC. We found a poor diversity especially for PE and PC, mainly containing ether-linked fatty acids which were 1-palmityl-2-docosahexaenoyl-PL and the corresponding alkenyl-acyl compound (plasmalogen) 1-palmitenyl-2-docosahexaenoyl-PL. For PC, both lipids were quantified after phospholipase A2 digestion to represent 44.2 and 37.2%, respectively, of the total PC. In contrast, the diacyl-PC content of bull spermatozoa was comparatively low (18.6% of total PC). In the presence of trifluoroacetic acid (TFA), which is routinely added to the MALDI-TOF matrix to improve the signal to noise ratio, a high lysophospholipid (LPL) content was detected in the PL extracts of bull spermatozoa, whereas TLC did not reveal significant amounts of LPL. The TFA mediated hydrolysis of the acid-labile alkenyl-acyl PL to the corresponding LPL was shown to cause this discrepancy. This assumption was verified by analysing the PL composition by MALDI-TOF MS before and after (i) digestion of sperm cell lipids with phospholipase A2 and (ii) exposition of spermatozoa to HCl fumes. We conclude that the analysis of samples containing alkenyl-acyl-PL by MALDI-TOF has to be performed with great caution.

Animals↗

PubMed2003

Protein-mediated transbilayer movement of lipids in eukaryotes and prokaryotes: the relevance of ABC transporters.

Lipid distribution across cellular membranes is regulated by specific membrane proteins controlling transbilayer movement of lipids. Flippases facilitate flip-flop of lipids and allow them to equilibrate between the two membrane leaflets independent of ATP. Distinct P-Type-ATPases transport specific lipids unidirectionally across the membrane at the expense of ATP. A group of ATP-dependent lipid transporters, the ATP-binding cassette (ABC) transporter family, was identified in studies originally related to multidrug resistance (MDR) in cancer cells. Meanwhile, lipid transport activity has been shown for full and half size ABC proteins in eukaryotic and prokaryotic cells. This activity may not only modify the organisation of lipids in membranes, but could also be of significant consequence for cell homeostasis. The various types of lipid movement mediating proteins and their cellular localisation in eukaryotes and prokaryotes are reviewed.

ATP-Binding Cassette Transporters↗

PubMed2003

Generation dependent singlet-singlet annihilation within multichromophoric dendrimers studied by polychromatic transient absorption.

Intramolecular kinetic processes in a series of shape-persistent meta- and para-substituted polyphenylene dendrimers bearing different peryleneimide chromophores at the rim have been investigated using time-resolved polychromatic transient absorption measurements. The influence of the generation number and different substitution patterns upon these processes was revealed by comparing different compounds. In particular, in multichromophoric systems a singlet-singlet annihilation process was detected. The corresponding time constant was dependent on the generation number.

Journal Article↗

PubMed2003

The synaptophysin/synaptobrevin interaction critically depends on the cholesterol content.

Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol-binding protein. Depleting the membranous cholesterol content by filipin or beta-methylcyclodextrin (beta-MCD) decreased the solubility of synaptophysin in Triton X-100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon beta-MCD treatment as revealed by chemical cross-linking. Mice with a genetic mutation in the Niemann-Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo- or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down-regulated after depleting the endogenous cholesterol content by the HMG-CoA-reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up-regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.

Animals↗

PubMed2003

Antibody-mediated targeting of an adenovirus vector modified to contain a synthetic immunoglobulin g-binding domain in the capsid.

Adenovirus vectors have been targeted to different cell types by genetic modification of the capsid or by using recombinant or chemically engineered adaptor molecules. However, both genetic capsid modifications and bridging adaptors have to be specifically tailored for each particular targeting situation. Here, we present an efficient and versatile strategy allowing the direct use of monoclonal antibodies against cell surface antigens for targeting of adenovirus vectors. A synthetic 33-amino-acid immunoglobulin G (IgG)-binding domain (Z33) derived from staphylococcal protein A was inserted into the adenovirus fiber protein. The fiber retained the ability to assemble into trimers, bound IgG with high affinity (Kd = 2.4 nM), and was incorporated into vector particles. The transduction efficiency of the Z33-modified adenovirus vector in epidermal growth factor receptor (EGFR)-expressing cells was strongly and dose-dependently enhanced by combination with an EGFR-specific monoclonal antibody. The antibody-mediated increase in cellular transduction was abolished in the presence of competing protein A. In targeting experiments with differentiated primary human muscle cells, up to a 77-fold increase in reporter gene transfer was achieved by preincubation of the vector with monoclonal antibodies directed against neuronal cell adhesion molecule or integrin alpha(7), respectively. The IgG-binding adenovirus vector holds promise for directed gene transfer to a wide variety of cell types by simply changing the target-specific antibody.

Adenoviridae↗

PubMed2002

Dynamics of lipid chain attached fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) in negatively charged membranes determined by NMR spectroscopy.

We have determined the average location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) attached to a C12 sn-2 chain of a phosphatidylserine (PS) analogue (C12-NBD-PS) in zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylserine (PS) host membranes. (1)H magic angle spinning nuclear Overhauser enhancement spectroscopy indicates a highly dynamic reorientation of the aromatic molecule in the membrane. The average location of NBD is characterized by a broad distribution function along the membrane director with a maximum indicating the location of the probe in the lipid/water interface of the lipid membrane. This behavior can be explained by a backfolding of the sn-2 chain towards the aqueous phase. Small differences in the distribution profiles of the NBD group along the membrane normal between PC and PS host membranes were found: in a PC host membrane, the NBD distribution has its maximum in the glycerol region; in a PS host membrane, NBD resides mostly in the upper chain region. These differences may be accounted for by packing differences in the PC versus PS host membranes. As seen by (2)H NMR order parameters, PS bilayers show a much higher packing density compared to PC membranes. Consequently, backfolding of the sn-2 chain with the NBD group attached causes a larger decrease of molecular order of the sn-1 chain in PS than in PC membranes. The broad distributions obtained for lipid chain attached NBD molecules reflect the motional freedom and molecular disorder in the liquid-crystalline lipid membrane.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Transbilayer movement of monohexosylsphingolipids in endoplasmic reticulum and Golgi membranes.

The transbilayer movement of glycosphingolipids has been characterized in Golgi, ER, plasma, and model membranes using spin-labeled and fluorescent analogues of the monohexosylsphingolipids glucosylceramide and galactosylceramide and of the dihexosylsphingolipid lactosylceramide. In large unilamellar lipid vesicles, monohexosylsphingolipids underwent a slow transbilayer diffusion (half-time between 2 and 5 h at 20 degrees C). Similarly, the inward redistribution of these sphingolipids in the plasma membrane of the hepatocyte-like cell line HepG2 and of erythrocytes was slow. However, in rat liver ER and Golgi membranes, we found a rapid transbilayer movement of spin-labeled monohexosylsphingolipids (half-time of approximately 3 min at 20 degrees C), which suggests the existence of a monohexosylsphingolipid flippase. The transbilayer movement of glucosylceramide in the Golgi and the ER displayed a saturable behavior, was inhibited by proteolysis, did not require Mg-ATP, and occurs in both directions. Treatment with DIDS inhibited the flip-flop of glucosylceramide but not that of phosphatidylcholine. These data suggest that the transbilayer movement of monoglucosylceramide in the ER and in the Golgi involves a protein that could be distinct from that previously evidenced for glycerophospholipids in the ER. In vivo, transbilayer diffusion should promote a symmetric distribution of monohexosylsphingolipids which are synthesized in the cytosolic leaflet. This should allow glucosylceramide rapid access to the lumenal leaflet where it is converted to lactosylceramide. No significant transbilayer movement of lactosylceramide occurred in both artificial and natural membranes over 1 h. Thus, lactosylceramide, in turn, is unable to diffuse to the cytosolic leaflet and remains at the lumenal leaflet where it undergoes the subsequent glycosylations.

Animals↗

PubMed2002

[Severe electrolyte imbalance and edema in therapy with rosiglitazone].

CASE REPORT: A case of a 49-year-old male with preexisting liver damage is reported. The patient was admitted to hospital with severe electrolyte disorder and face edema after therapy first with 4 mg for 2 months and later for 5 months with 8 mg rosiglitazone. The initial electrolyte values were: sodium 110 mmol/l, potassium 3.3 mmol/l, calcium 2.0 mmol/l, chloride 81 mmol/l. An already known hypercholesterolemia worsened substantially to values up to 28.5 mmol/l. Under substitution therapy with sodium chloride infusion and potassium, the electrolyte level normalized rapidly. The hypercholesterolemia improved over several weeks after stopping the drug, and the general condition of the patient improved clearly. CONCLUSION: Rosiglitazone has been certified in Germany since July 2000. Although a liver toxicity with rosiglitazone has been denied, the administration of this drug in patients with liver damage is contraindicated. Especially when prescribing new drugs one has to pay special attention to contraindications and comedication since often not all therapeutic mechanisms and side effects are fully known/understood. Interaction between different drugs and their influences on existing diseases are only noticed after a widespread application of the drug.

Chemical and Drug Induced Liver Injury↗

PubMed2002

Transport of phosphatidylserine via MDR1 (multidrug resistance 1)P-glycoprotein in a human gastric carcinoma cell line.

The ATP-binding cassette transporter multidrug resistance 1 P-glycoprotein (MDR1 Pgp) has been implicated with the transport of lipids from the inner to the outer leaflet of the plasma membrane. While this has been unambigously shown for the fluorescent lipid analogues [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (C6-NBD)-phosphatidylcholine, -phosphatidylethanolamine, -sphingomyelin and -glucosylceramide, by using a novel approach we have now found significantly increased outward transport also for C6-NBD-phosphatidylserine (C6-NBD-PS) in EPG85-257 human gastric carcinoma cells overexpressing MDR1 (coding for MDR1 Pgp). The increased transport of C6-NBD-PS is mediated by MDR1 Pgp, shown by transport reduction nearly to the level of controls in the presence of MDR1 Pgp inhibitors [PSC 833, cyclosporin A and dexniguldipine hydrochloride (Dex)]. Addition of MK 571, a specific inhibitor of the MDR protein MRP1, does not decrease transport in either of the two cell lines. The plasma-membrane association of FITC-annexin V, a fluorescent protein conjugate binding PS, is significantly increased in MDR1-overexpressing cells as compared with controls, and can be reduced by an MDR1 Pgp inhibitor. This suggests that MDR1 Pgp transports endogenous PS, the lipid exhibiting the most pronounced transverse asymmetry in the plasma membrane.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Influence of the bovine seminal plasma protein PDC-109 on cholesterol in the presence of phospholipids.

The interaction of the major bovine seminal plasma protein PDC-109 with cholesterol was studied by employing spin-labelled analogues. It could be shown that PDC-109 does not interact directly with cholesterol molecules. However, in the presence of phospholipids we found a strong reduction of cholesterol motion by PDC-109. The fraction of immobilized cholesterol was largest for phosphorylcholine-containing lipids. This is consistent with the preferential interaction between PDC-109 and phosphatidylcholine. It is concluded that a stronger association and interaction of PDC-109 with phosphatidylcholine leads to an enhanced fraction of immobilized cholesterol analogues, but not to a phospholipid-dependent specific interaction between the protein and cholesterol. Moreover, the interaction of PDC-109 with various spin-labelled analogues of phosphatidylcholine (lysoPC, diacylPC) was investigated. In membranes of lipid vesicles the protein caused an immobilization of the phosphatidylcholine analogues mainly in the outer membrane leaflet, with no differences between diacylPC and lysoPC. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.

Animals↗

PubMed2002

Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells.

We studied the transport of the fluorescent cholesterol analog dehydroergosterol (DHE) in polarized HepG2 human hepatoma cells. DHE delivered via methyl-beta-cyclodextrin was delivered to both the apical and basolateral membranes and became concentrated in the apical membrane within 1 min. Intracellular DHE was targeted mainly to vesicles of the subapical compartment or apical recycling compartment (SAC/ARC), where it colocalized with fluorescent transferrin and fluorescent analogs of phosphatidylcholine and sphingomyelin. In contrast, transport of DHE from the plasma membrane to the trans-Golgi network was found to be very low. Vesicles containing DHE traversed the cells in both directions, but vesicular export of DHE from the SAC/ARC to the plasma membrane domains was low. Disruption of the microtubule cytoskeleton disturbed vesicular transport of DHE but not its enrichment in the apical (canalicular) membrane. Transport of DHE to the canalicular membrane after photobleaching was very rapid (t(12) = 1.6 min) and was largely ATP-independent in contrast to enrichment of DHE in the SAC/ARC. Release of DHE from the canalicular membrane was also ATP-independent but slower than the enrichment of sterol in the biliary canaliculus (t(12) = 5.4 min). Canalicular DHE could completely redistribute to the basolateral plasma membrane but could not transfer from one cell to the other cell of an HepG2 couplet. We conclude that sterol shuttles rapidly among the plasma membrane domains and other membrane organelles and that this nonvesicular pathway includes fast transbilayer migration.

Adenosine Triphosphate↗

PubMed2002

Transbilayer movement of fluorescent phospholipid analogues in the cytoplasmic membrane of Escherichia coli.

We investigated the transmembrane movement of fluorescent labeled phospholipids in inverted inner membrane vesicles (IIMV) of Escherichia coli (E. coli) wild-type strain (MG1655), as well as in proteoliposomes reconstituted from detergent extracts of the IIMV. The transbilayer movement of 1-myristoyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoethanolamine (M-C6-NBD-PE) and -phosphocholine (M-C6-NBD-PC) was measured by a fluorescence stopped-flow back-exchange assay. Both analogues were rapidly translocated across the IIMV membrane, with half-times of <1 min (outward movement) and approximately 3 min (inward movement). No flip-flop was detected in protein-free liposomes, but in IIMV-derived proteoliposomes flip-flop of M-C6-NBD-PE occurred similarly to IIMV and could be largely eliminated by proteinase K treatment.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Lysolipids do not inhibit influenza virus fusion by interaction with hemagglutinin.

The interaction of a spin-labeled lysophosphatidylcholine analog with intact and bromelain-treated influenza viruses as well as with the bromelain-solubilized hemagglutinin ectodomain has been studied. The inhibition of fusion of influenza viruses with erythrocytes by the lysophosphatidylcholine analog was similar to that observed for non-labeled lysophosphatidylcholine. Only a weak interaction of the lysophosphatidylcholine analog with the hemagglutinin ectodomain was observed even upon triggering the conformational change of the ectodomain at a low pH. A significant interaction of spin-labeled lysophosphatidylcholine with the hemagglutinin ectodomain of intact viruses was observed neither at neutral nor at low pH, whereas a strong interaction of the lipid analog with the viral lipid bilayer was evident. We suggest that the high number of lipid binding sites of the virus bilayer and their affinity compete efficiently with binding sites of the hemagglutinin ectodomain. We conclude that the inhibition of influenza virus fusion by lysolipids is not mediated by binding to the hemagglutinin ectodomain, preventing its interaction with the target membrane. The results unambiguously argue for an inhibition mechanism based on the action of lysolipid inserted into the lipid bilayer.

Bromelains↗