Biomedical subjects

A Herrmann

Publications and source records attributed to A Herrmann.

At least 73 records · Page 4Linked to original sources

PubMed1998

Recombination of nonreplicative RNA precursors of Sindbis virus in infected cells overexpressing murine-inducible nitric oxide synthase.

The Sindbis virus-based SINrep5 expression system is one of the most efficient vectors for gene transfer leading to fast and high expression of the gene of interest. This system was used to transfect vascular endothelial and smooth muscle cells using murine inducible nitric oxide synthase (miNOS) as a reporter gene. Infection of both cell types leads to high expression levels of miNOS. In addition, the harvested supernatant of these infected cells was used for further rounds of infections, demonstrating that recombination of the parental RNA with the helper RNA takes place and results in the production of infectious particles. As shown by RT-PCR, after recombination the miNOS gene is located in between the nonstructural and structural viral genes. This study demonstrates that despite claims in other publications, the Sindbis virus-based SINrep5 expression system leads to recombination and is thus not a safe system for in vitro and in vivo applications.

Animals↗

PubMed1998

Release of phospholipids from erythrocyte membranes by taurocholate is determined by their transbilayer orientation and hydrophobic backbone.

Bile salts mediate a specific release of phosphatidylcholine (PC) from the canalicular membrane into the bile fluid. We utilized human red blood cells (RBC) as a model system to study the release of endogenous phospholipids as well as phospholipid analogues from plasma membranes in the presence of the bile salt taurocholate (TC). Short- and long-chain fluorescent as well as spin-labeled analogues with various headgroups were chosen. RBC were labeled either on the exoplasmic or on the cytoplasmic leaflet with the analogues and incubated with various concentrations of TC. Analogues on the exoplasmic layer could be readily released by TC. Release was most efficient above the critical micellar concentration (CMC) of TC. Release was independent of the headgroup, but depended on the fatty acid chain length of the analogues; i.e., it was lower for long-chain than for short-chain labeled phospholipids. Analogues on the cytoplasmic leaflet were efficiently shielded from TC-mediated release. The preferential release of endogenous PC and sphingomyelin (SM) from the erythrocyte membrane above the CMC supports the conclusion that TC-mediated release of phospholipids occurs preferentially from the exoplasmic leaflet independent of their headgroup. However, the extent of release of endogenous phospholipids was significantly lower in comparison to that of analogues, endorsing the relevance of the hydrophobic backbone for bile salt mediated release of phospholipids. Implications for the mechanism of the release of PC from the canalicular membrane into the bile fluid are discussed.

Bile↗

PubMed1998

Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase.

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate from GTP. [1',2',3',4',5'-13C5, 2'-2H1]GTP was prepared enzymatically from [U-13C6]glucose for use as enzyme substrate. Multinuclear NMR experiments showed that the reaction catalyzed by GTP cyclohydrolase I involves the release of a proton from C-2' of GTP that is exchanged with the bulk solvent. Subsequently, a proton is reintroduced stereospecifically from the bulk solvent. This is in line with an Amadori rearrangement mechanism. The proton introduced from solvent occupies the pro-7R position in the enzyme product. The data also confirm that the reaction catalyzed by pyruvoyltetrahydropterin synthase results in the incorporation of solvent protons into positions C-6 and C-3' of the enzyme product. On the other hand, the reaction catalyzed by sepiapterin reductase does not involve any detectable incorporation of solvent protons into tetrahydrobiopterin.

Alcohol Oxidoreductases↗

PubMed1998

Acylation of the influenza hemagglutinin modulates fusion activity.

The influenza virus hemagglutinin (HA) contains three highly conserved cysteine residues at positions 551, 559, and 562 close to the carboxyl-terminus of the HA2 subunit which serve as palmitylation sites. Wild-type HA of influenza virus A/FPV/Rostock/34 (H7N1) and HA permutated by exchange of the acylated cysteine to serine residues were expressed in CV-1 cells by a SV40 vector system. Since density of immunostained HA on the cell surface measured by flow cytometric analysis did not differ between wild-type and acylation mutants, it was possible to compare acylation mutants and wild-type HA for their capacity to induce membrane fusion at low pH. The following observations were made: (1) lateral diffusion of a lipid-like fluorophore (R-18) from the erythrocyte membrane to the plasma membrane of cells expressing HA on the surface occurred equally well with mutants and wild type. (2) Diffusion of a low-molecular-weight fluorescent water-soluble probe (calcein) from erythrocytes into the cytoplasm of HA-expressing cells was not altered either. (3) However, depending on the position and the number of the deleted acylation sites, the mutants showed a reduced ability to induce syncytia. The data indicate that deacylation of the cytoplasmic tail has no measurable effect on the capacity of HA to induce membrane fusion and pore formation but that it suppresses syncytia formation.

Acylation↗

PubMed1998

Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles.

PDC-109 is the major protein of bovine seminal plasma. It binds to the bovine sperm surface at ejaculation and modulates sperm capacitation. PDC-109 displays phosphorylcholine- and heparin-binding activities which are thought to account for its sperm surface coating and glycosaminoglycan-induced sperm capacitating activities, respectively. We have characterized the interaction of isolated PDC-109 with membranes of phospholipid vesicles using a biophysical approach. Our results show that PDC-109 interacts not only with the solvent-exposed phosphorylcholine head group but also with the hydrophobic core of liposomes. Binding of PDC-109 to membranes is a very rapid, biphasic process with half times of less than one second. Maximal binding of PDC-109 to small unilamellar vesicles was achieved with a stoichiometric ratio of 10-11 phosphatidylcholine molecules/PDC-109 molecule. Incorporation of phosphatidylethanolamine or phosphatidylserine into phosphatidylcholine vesicles reduced the binding of PDC-109, suggesting that both the density of phosphorylcholine groups and the surface charge determine the interaction of the seminal plasma protein with the surface of the membrane. Electron spin resonance measurements showed that binding of PDC-109 to phosphatidylcholine vesicles caused a rigidification of the membrane. The relevance of the data for describing the role of PDC-109 in the modulation of sperm capacitation is discussed.

Animals↗

PubMed1998

Intraindividual comparison of 99mTc-labelled anti-SSEA-1 antigranulocyte antibody and 99mTc-HMPAO labelled white blood cells for the imaging of infection.

Technetium-99m labelled antigranulocyte antibodies are ready to use and are sensitive and specific in the diagnosis of infectious diseases. 99mTc-SSEA antigranulocyte antibodies have a very high affinity constant (Kd 10(-12) M) for human neutrophils (PMNs), and excellent imaging qualities with high target/background ratios. The aim of this study was to compare the diagnostic accuracy of the 99mTc-anti-SSEA-1 monoclonal antibody (Mab) with that of 99mTc-hexamethylpropylene amine oxime (HMPAO)-labelled white blood cells (WBCs). To this end, 17 patients with 23 proven infectious foci were examined with 555 MBq 99mTc-anti-SSEA-1 MAb and with 370 MBq 99mTc-HMPAO labelled autologous leucocytes within a period of 7 days. All the infections were confirmed by culture, biopsy, surgery and follow-up. Whole-body images and planar spot views with the antibody were performed at 1-h, 4-h and 24-h post injection; the biodistribution of the antibody was quantified, absorbed radiation doses were calculated and the diagnostic results were compared with the 99mTc-HMPAO WBC images. Human anti-mouse antibody (HAMA) evaluation was performed in all patients before and 3 months after antibody injection. Blood was drawn at different times after 99mTc-anti-SSEA-1 MAb injection to determine the amount of granulocyte-associated radioactivity and to calculate recovery. 99mTc-anti-SSEA-1 MAb scintigraphy detected all 23 lesions, while 21 were detected with 99mTc-HMPAO WBC scan. In this small group of patients, the sensitivity and specificity of 99mTc-anti-SSEA-1 MAb scintigraphy were 95% and 96% respectively, as compared with 91% and 82% respectively for 99mTc-HMPAO WBC scan. An increasing uptake of the injected activity in the lesion at different time points was indicative of high affinity and of specific PMN binding. There was no HAMA formation. In four of five patients investigated, a transient mild leukopenia was found at 15 min p.i.. There was increased uptake of the antibody in liver and spleen and normal uptake in kidneys and bone marrow. The estimated radiation doses for the whole body and the red bone marrow were 1.1x10(-2) cGy/37 MBq and 5.3x10(-2) cGy/37 MBq, respectively. The activity associated to the PMNs in vivo was 33.5%, 30.6%, 21.3% and 9% at 5, 15, 30 and 45 min. post-injection, respectively. It is councluded that use of 99mTc-anti-SSEA-1 antigranulocyte antibodies demonstrates promising results comparable to those obtained with 99mTc-labelled autologous WBCs. The 99mTc-labelled MAb is ready to use, has excellent image qualities and a high target/background ratio.

Adult↗

PubMed1998

[Natural coral calcium carbonate as alternative substitute in bone defects of the skull].

A biomaterial derived from natural corals with surgical applications is the calcium carbonate Biocoral. Since 1992 the author has been using this material as a bone graft substitute in maxillofacial surgery. Eighty-nine clinical implantations were done in 68 patients for different indications. The results suggested that coral grafts are well tolerated and are simultaneously partially ossified as the calcified skeleton is resorbed. Clinical cases show that use of this material has been successful.

Adult↗

PubMed1998

Immunoscintigraphy (BW 250/183) in neonates and infants with fever of unknown origin.

Fever of unknown origin is defined as a temperature above 39.0 degrees C together with a white blood cell count > or = 15,000 mm-3, the duration of fever exceeding 2 weeks and a correct diagnosis not being obtained in the first week of hospitalization. In neonates and infants with fever of unknown origin, the localization of the infectious focus is often difficult and unsatisfactory. In this retrospective study, the clinical value of 99Tcm-labelled antigranulocyte antibodies for this group of patients was investigated. Thirty-two immunoscintigrams were performed using 185-259 MBq 99Tcm-labelled antigranulocyte antibodies (BW 250/183) in 30 neonates and infants (21 boys, 9 girls, mean age 29.4 +/- 2 months), who had fever of unknown origin. Immunoscintigraphy was carried out as whole-body images (n = 7) or single planar images (n = 25) 4 h and 24 h post-injection. In children with known cardiac failure, single photon emission tomography of the thorax was performed to diagnose endocarditis (n = 2). For verification, the results of the immunoscintigrams were compared with radiology (conventional radiography = 14, MRI = 5, CT = 3), biopsy (n = 2), blood culture (n = 10) and clinical follow-up after specific therapy. In 11 of 30 children (36%), the diagnosis of an infective focus was possible with immunoscintigraphy. The sensitivity and specificity of diagnosing infective foci was 72% and 95% respectively (n = 11; colitis = 2, infection of the central permanent catheter tip = 2, middle ear infection = 1, spondylitis/discitis = 3, osteomyelitis = 2, umbilical infection = 1). In vertebral body infections, all lesions were photopenic. In 18 children (60%), no infective focus was found on immunoscintigraphy. In this group of children, the main reason (n = 5) for fever of unknown origin was chronic juvenile rheumatoid arthritis. No uptake was seen in two infants with cardiac failure and suspected endocarditis on SPET. In 3 of the 18 patients (17%), localization of an infective focus was not possible with immunoscintigraphy or on other examinations. In these patients, the fever disappeared spontaneously after a few days of antibiotic therapy. In conclusion, we have shown that 99Tcm-anti-NCA-95 scanning is a safe method with a high sensitivity and specificity for detecting infectious foci in neonates and infants with fever of unknown origin. Furthermore, this method is easy to perform, since no withdrawal of blood is necessary.

Antibodies, Monoclonal↗

PubMed1998

Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin.

The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type 1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.

Amino Acid Sequence↗

PubMed1998

[Suppressibility of thyroid autonomy in endogenous and additional exogenous TSH suppression in patients with thyroid autonomy].

AIM: The aim of this retrospective study was to examine the effect of an additional exogenous thyroid hormone suppression on the 99m Technetium Thyroid-Uptake (TcTU) in patients with thyroid autonomy and endogenous TSH suppression. METHODS: 21 patients were examined in the years 1992-1996; they had a primary endogenous suppression (end) of the TSH-b and an evaluation of the TcTU. After an additional exogenous suppression (ex) of the thyroid, the first two weeks with 75 micrograms/dl and the following 2 weeks 150 micrograms/d, or depending on the bodyweight 2 micrograms/kg levothyroxin, another evaluation of the TcTU was performed. The whole patients were called group I with a TcTUend of 1.6%-7.4%. The subgroups were selected by thresholds of the TcTUend; group Ia TcTUend > or = 1.6% and < 2.5%, group lb TcTUend > or = 2.5%, group Ic TcTUend > or = 3%. RESULTS: The intraindividual comparison showed a significant decrease of the autonomous "volume" after exogenous suppression with levothyroxin. This is shown by the following values of the TcTU: group I TcTUend = 3.47 +/- 1.65 and TcTUex = 2.91 +/- 1.38, which means a reduction of 20 rel.% (p < or = 0.001); group Ia TcTUend = 2.05 +/- 0.27, TcTUex = 1.85% +/- 0.22, which means 10 rel.% (p < or = 0.001); group Ib TcTUend = 4.18 +/- 1.59, TcTUex = 3.45% +/- 1.34, which means 20 rel.% (p < or = 0.001); group Ic TcTUend = 5.00% +/- 1.43, TcTUex = 4.05% +/- 1.32, which means 20 rel.% (p < or = 0.001). CONCLUSION: Supposing a structural variability of autonomous thyroid tissue with heterogenous TSH receptors our study shows a significant additional exogenous suppression in already endogenous suppressed TSH receptors.

Adult↗

PubMed1997

The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 A in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain alpha/beta-fold with a central four-helix bundle connecting two seven-stranded parallel beta-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1'-carbonyl group near Tyr171 OH and NADP C4'N. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1' and C2' carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.

Alcohol Oxidoreductases↗

PubMed1997

The glycosylation of rat intestinal Muc2 mucin varies between rat strains and the small and large intestine. A study of O-linked oligosaccharides by a mass spectrometric approach.

The large glycosylated domains obtained from the rat intestinal mucin Muc2 were isolated from the large and small intestine of the inbred rat strains GOT-W and GOT-BW. The expression of the rat Muc2 in the large intestine was confirmed immunochemically and by Northern blotting. Released oligosaccharides were structurally characterized by gas chromatography-mass spectrometry (neutral and sialylated species) or by tandem mass spectrometry (sulfated species), and a total of 63 structures was assigned. The large intestinal oligosaccharides were found to be identical between the strains, while the small intestinal glycosylation differed. Until now, detailed structural analysis of oligosaccharides isolated from a single mucin core or mucin domain with different origin have not been performed, and the information of different mucin glycoforms has been limited to immunochemistry. Blood group A-determinants (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-, and structures related to the blood group Sda/Cad-related epitope NeuAc/NeuGcalpha1-3(GalNAcbeta1-4)Galbeta1-, were found in GOT-BW small intestine, and also in both large intestines. Blood group H-determinants and NeuAc/NeuGcalpha1-3Galbeta1- were found in all samples. Core 1 (Galbeta1-3GalNAcalpha1-), core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-), core 3 (GlcNAcbeta1-3GalNAcalpha1-), and core 4 (GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1- were also found in all the samples. The large intestine were enriched in sulfated oligosaccharides and the small intestine contained higher amounts of sialylated species. Sulfation were found exclusively on C-6 of GlcNAc.

ABO Blood-Group System↗

PubMed1997

Mucus glycoproteins from pig gastric mucosa: identification ofdifferent mucin populations from the surface epithelium.

Pig gastric mucins were isolated from the surface epithelium of the cardiac region, corpus and antrum using density-gradient centrifugation after extraction in 6 M guanidinium chloride. In CsCl/0.5 M guanidinium chloride, mucins solubilized from the cardiac region appeared as a broad unimodal band at 1.52 g/ml whereas those from the corpus and antrum occurred as high- and low-density populations at 1.50 and 1.45 g/ml respectively. High-iron diamine reacted more strongly with the cardiac mucins and the high-density populations from corpus and antrum than with the two low-density ones. In keeping with this, approx. 60% of the oligosaccharides from the former mucins and 20% from the latter contained sulphate. All surface epithelial cells of the cardiac region stained with high-iron diamine, whereas in the corpus only the epithelium in the bottom of the pits reacted, suggesting that the high-density population from this region originates from these cells. Mucins from all regions were composed of subunits, each containing highly glycosylated domains. The mucins from the cardiac region were larger than those from the corpus and antrum, and reduced subunits as well as high-molecular-mass glycopeptides from the cardiac mucins were larger than the corresponding fragments from the other regions. Ion-exchange HPLC showed that reduced subunits from the cardiac mucins and the high-density populations from the corpus and antrum were more 'acidic' than reduced subunits from the two low-density ones. All mucins contained a 'neutral'fraction, in particular those from the antrum. Pig gastric mucus thus contains a number of distinctly different mucin populations varying in buoyant density, size, 'acidity', glycosylation, sulphation and tissue origin.

Animals↗

PubMed1997

Interaction of earthworm hemolysin with lipid membranes requires sphingolipids.

Lytic activity in the coelomic fluid of earthworm (Eisenia fetida fetida) has been ascribed to eiseniapore, a hemolytic protein of 38 kDa. Since receptors for eiseniapore on target cell membranes are not known, we used lipid vesicles of various composition to determine whether specific lipids may serve as receptors. Lytic activity of eiseniapore was probed by the relief of fluorescence dequenching from the fluorophore 8-aminonaphthalene-1,3, 6-trisulfonic acid originally incorporated into the vesicle lumen as a complex with p-xylene-bis-pyridinium bromide. Hemolysin binds to and disturbs the lipid bilayer only when distinct sphingolipids consisting of a hydrophilic head group as phosphorylcholine or galactosyl as well as the ceramide backbone, e.g. sphingomyelin, are present. Cholesterol enhances eiseniapore lytic activity toward sphingomyelin-containing vesicles probably due to interaction with sphingomyelin. Leakage of vesicles was most efficient when the lipid composition resembled that of the outer leaflet of human erythrocytes. Presumably, an oligomeric protein pore formed by six monomers is responsible for leakage of sphingomyelin-containing vesicles. The secondary structure of eiseniapore did not change upon binding to lipid membranes. The lytic activity of eiseniapore was completely abolished after its denaturation or after preincubation with polyclonal antibodies. Our results suggest that the presence of specific sphingolipids is sufficient to mediate lytic activity of eiseniapore. This action contributes to our understanding of earthworm immune responses.

Animals↗

PubMed1997

Influence of pH on phospholipid redistribution in human erythrocyte membrane.

The influence of the suspension pH (pHo) on the transmembrane mobility of spin-labeled phospholipid analogues in the human red blood cell was investigated. The passive transverse diffusion of spin-labeled phospholipid analogues was independent of pHo in the investigated range (5.8 to 8.5). However, upon acidification to pHo 5.8, a significant decrease of the rapid adenosine triphosphate (ATP)-dependent inward movement of aminophospholipids was found at physiologic ionic concentration, whereas a change of pH from 7.4 to 8.5 did not affect this transport. Evidence is given that the intracellular pH affects the active transport of aminophospholipids but not the extracellular pH. Suppression of the ATP-dependent outside-inside redistribution of aminophospholipid analogues by low pH was reversible because original transport activity was re-established upon reneutralization. pH dependence of the active phospholipid transport was not caused by the spin-labeled reporter group or by depletion of intracellular ATP. Because the same influence of pH on aminophospholipid movement could be observed for resealed ghosts, constituents of the red blood cell cytoplasm do not mediate the influence of pH on the ATP-dependent inward movement of aminophospholipids.

Adenosine Triphosphate↗

PubMed1997

Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy.

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses. At acidic pH we found a multiphasic behavior of the CD signal recorded at 283 nm. Upon lowering the pH we detected first an increase of the CD amplitude, which is associated with the formation of a fusion-competent state of HA. The initial increase was followed by a continuous decline of CD amplitude, which can be ascribed to a transformation into a fusion-inactivated conformation that is in its early phase reversible as found for A/Japan. The half-time of the different phases of the CD signal depended on the virus strain, the temperature, and the acidic pH. The results support recent hypotheses that the fusion-competent conformation is an intermediate of the fusion-inactivated structure of HA.

Circular Dichroism↗

PubMed1997

Modelling of phospholipid translocation in the erythrocyte membrane: a combined kinetic and thermodynamic approach.

A mathematical model for the dynamics of transbilayer movements of lipids in the erythrocyte plasma membrane is presented. It takes into account an active carrier which mediates the ATP-dependent translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the cytoplasmic leaflet of the membrane and passive fluxes of these lipids as well as of phosphatidylcholine, sphingomyelin and cholesterol between both layers. It is assumed that the passive fluxes are driven by concentration gradients of the lipids and by mechanical forces which result from area limitation for lipid occupation in both leaflets. Compared with a previous mathematical treatment of lipid translocation processes in the erythrocyte membrane the present model is much closer to realistic conditions, e.g. concerning the number of lipid species involved. Furthermore, the use of linear flux-force relationships as known from irreversible thermodynamics allows a simpler treatment of the passive fluxes than before and provides a relevant framework to study the coupling between the various processes. The model allows to simulate the time dependent changes of lipid concentrations which take place after activation or inhibition of ATP-dependent translocation. Using realistic parameter values it explains in quantitative terms the stationary asymmetric distribution of lipids under in vivo conditions. Using principles of metabolic control analysis we are able to quantify the role of the various active and passive processes in determining the asymmetric distribution for each lipid species.

Animals↗

PubMed1997

Targeted delivery of human neurofibromin and c-Raf-1 mutants to the cytoplasmic membrane by use of the influenza virus hemagglutinin.

Mutants of human neurofibromin and c-Raf-1 genes were fused to the 3' end of the hemagglutinin (HA) gene of influenza A virus by oligonucleotide-directed polymerase chain reaction (PCR). The two resulting chimeric genes, HA (1-534)/NF1 (1441-1518) and HA (1-534)/Raf-1 (51-132) which we designated HN and HR, respectively, were cloned in a vaccinia virus expression vector (pTMI) under the control of a T7 RNA polymerase promoter. The clones were expressed in a monkey cell line (CV-1) and the resulting chimeric proteins analysed. We found that expression levels of the chimeric proteins were similar to that of wild-type HA protein. Comparative endoglycosidase treatment revealed that the expressed chimeric proteins HN and HR were processed as wild-type HA, and FACS-analysis showed that both chimeric expression products localised in the cell membrane as the wild-type control. HN and HR expressing cells showed similar fusogenic activity as CV-1 cells transfected with wild-type HA indicating the correct topology of the fusion inducing portion (HA) of these chimera in the membrane. These findings show that the influenza virus hemagglutinin (HA) is a suitable vehicle to target foreign proteins with therapeutical potential into the cell membrane. In this respect HN and HR could potentially be used to block the abnormal signals generated by particular proteins in the cell membrane that lead to cell transformation.

Animals↗