04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Extraordinary biological membrane structures resulting from different local membrane curvatures(1992) Meyer, Helmut W.; Hülser, Dieter F.The bilayer arrangement of amphiphilic molecules is not only the basic structure of rather flat biological membranes, but also of regularly curved bilayers in most cubic phase structures. The basis of these cubic phase structures are infinite periodical minimal surfaces (IPMS). Extraordinary biological membrane structures resembling such IPMS were found as periodically curved bilayers in areas of the plasma membrane in a Streptomyces strain and in liposomes prepared from its extracted lipids. This structure consists of a transition of convex to concave curvatures and vice versa. A structure with curvatures in one direction only was observed in vacuolar membranes of yeast cells with a genetic defect. Our electron microscopical analysis of freeze fractured membranes of these cells revealed not only fully invaginated but also flat particle-free areas which were mainly circularly shaped, some elongated areas, however, were also present. In addition, sometimes periodical arrangements were detected which obviously are not related to IPMS structures. Both structures, however, indicate a high proportion of wedge-shaped lipid molecules in the bilayer.Item Open Access Transduction of chemical signals in dictyostelium cells(1984) Gerisch, Günther; Tsiomenko, Arnold; Stadler, Joachim; Claviez, Michael; Hülser, Dieter F.; Rossier, ClaudeThree different functions of cyclic AMP in D discoideum are known: (1) cAMP acts as a chemoattractant during cell aggregation, (2) it controls cell development, particularly the acquisition of aggregation competence, and (3) it is involved in terminal cell differentiation. In this report we will concentrate on the functions 1 and 2 of cAMP. Chemotaxis requires the recognition of concentration gradients in the environment by attractant binding to cell surface receptors, the processing of signals from the receptors to the contractile system of the cells, extension of pseudopods at one part, and contraction at other parts of the cells in accord with the external gradient. One pathway of signal processing from the receptors to the contractile system involves the regulation of a myosin kinase. The control of development up to aggregation competence is largely dependent on the temporal pattern of cAMP application: Only repetitive pulses enhance development. This effect has been studied using the expression of a membrane glycoprotein called contact site A as a differentiation marker.Item Open Access A systems biology approach to dynamic modeling and inter-subject variability of statin pharmacokinetics in human hepatocytes(2011) Bucher, Joachim; Riedmaier, Stephan; Schnabel, Anke; Marcus, Katrin; Vacun, Gabriele; Weiss, Thomas S.; Thasler, Wolfgang E.; Nüssler, Andreas K.; Zanger, Ulrich M.; Reuss, MatthiasBackground The individual character of pharmacokinetics is of great importance in the risk assessment of new drug leads in pharmacological research. Amongst others, it is severely influenced by the properties and inter-individual variability of the enzymes and transporters of the drug detoxification system of the liver. Predicting individual drug biotransformation capacity requires quantitative and detailed models. Results In this contribution we present the de novo deterministic modeling of atorvastatin biotransformation based on comprehensive published knowledge on involved metabolic and transport pathways as well as physicochemical properties. The model was evaluated on primary human hepatocytes and parameter identifiability analysis was performed under multiple experimental constraints. Dynamic simulations of atorvastatin biotransformation considering the inter-individual variability of the two major involved enzymes CYP3A4 and UGT1A3 based on quantitative protein expression data in a large human liver bank (n = 150) highlighted the variability in the individual biotransformation profiles and therefore also points to the individuality of pharmacokinetics. Conclusions A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology.Item Open Access Enrichment of dibenzofuran utilizing bacteria with high co-metabolic potential towards dibenzodioxin and other anellated aromatics(1989) Strubel, Volker; Rast, Hans G.; Fietz, Walter H.; Knackmuss, Hans-Joachim; Engesser, Karl-HeinrichDibenzofuran degrading bacteria were enriched from various environmental sources. A mutualistic mixed culture of strain DPO 220 and strain DPO 230 was characterized. Strain DPO 220 alone showed limited growth with dibenzofuran as sole source of carbon and energy (td ≥ 4.5 h). A labile degradation product, C12H10O5, and salicylate were isolated from the culture fluid. Salicylate was found to be a central intermediate of DBF-degradation.Strain DPO 220 co-metabolized a wide range of anellated aromatics as well as heteroaromatics. High rates of co-oxidation of dibenzodioxin demonstrate analogue-enrichment to be a powerful technique for selecting enzymatic activities for otherwise non-degradable substrates.Item Open Access Patch-clamp measurements of gap-junction channels in cultured cells(1992) Hülser, Dieter F.; Eckert, Reiner; Zempel, Günther; Paschke, Dietmar; Dunina-Barkovskaja, AntoninaDirect intercellular communication in most tissues is made possible by proteinaceous pores called gap-junction channels. These channels bridge the extracellular gap between apposed cells and connect their intracellular compartments both electrically and metabolically. The extracellular parts of two hemichannels - the connexons - are linked thus forming a communicating gap-junction channel. A connexon is a hexamer of protein subunits which are members of the connexin family. Since connexin 32 (Cx32) was the first gap-junction channel protein to be sequenced from hepatocytes, it serves as a reference to which all other gap-junction proteins are compared. The individual channel conductance may vary between 25 and 150 pS. Gap-junction channels of some tissues are more voltage sensitive (e.g. liver) than others (e.g. heart). The question whether these differences in electrical properties may be attributed to the different connexins being expressed in these tissues is still unanswered. Several approaches to resolve this problem will be discussed in this contribution, all are based on double whole-cell patch-clamp measurements using isolated cell pairs, as follows: (1) Cells with two different channel conductances perfused with anti connexin antibodies to specifically block one channel species; (2) Cells with only one connexin species selected by immunological characterization; (3) Weakly coupled HeLa cells transfected with specific connexin genes, a method which resulted in better correlations between connexin type and single channel properties.Item Open Access Timing the early events during sea urchin fertilization(1983) Schatten, Gerald; Hülser, Dieter F.To determine precisely the timing, duration, and sequences of the earliest events during sea urchin (Lytechinus variegatus) fertilization, the bioelectric recordings of microelectrode-impaled eggs were electronically superimposed, by video mixing, over the microscopic differential interference contrast image of the same egg at insemination. Videotape analysis, utilizing a slow-motion analyzer, demonstrates that the successful sperm triggers the bioelectric membrane potential reversal within 3.36 ± 3.02 sec (0.72-9.76 sec range; Σ = 23 eggs) of sperm-egg attachment. This sperm, actively gyrating about its attachment site, is indistinguishable from the other, unsuccessful sperm until 12.66 ± 2.72 sec (6.72-16.60 sec range; Σ = 15) later when the sperm tail ceases its beating and sperm incorporation ensues. The cortical granules begin to discharge, and the fertilization coat starts to elevate at the fusion site at 20.79 ± 3.18 sec (13.62-26.08 sec range; Σ = 12) after the onset of the fertilization potential, i.e., an average of about 8 sec after the cessation of sperm-tail motility during incorporation. In most cases, the bioelectric responses starts within 7 sec of sperm adhesions; if the data are analyzed excluding the few slow cases, the fertilization potential is found to start 1.93 sec (±1.28 sec) after sperm attachment. These results indicate that the first successful sperm triggers the fast block to polyspermy within 3.4 sec, perhaps as quickly as 1.9 sec, of sperm-egg adhesion, about 13 sec before the first morphological indication of fertilization, and about 21 sec before the characteristic elevation of the fertilization coat responsible for the late block to polyspermy.Item Open Access Effects of intrastriatal blockade of glutamatergic transmission on the acquisition of T-maze and radial maze tasks(1989) Hauber, Wolfgang; Schmidt, Werner J.Prefrontal cortex and neostriatum constituting the prefrontal system are connected by glutamatergic neurones. The involvement of this corticostriatal projection in control of maze performance of rats was investigated. Glutamatergic transmission mediated by N-methyl-D-aspartate (NMDA) receptors was blocked by intrastriatal injections of dl-2-amino-5-phosphonovaleric acid (AP-5) (50 nmole in 0.5 Mgrl). In experiment 1, intrastriatal AP-5 was found to increase the number of errors during acquisition of a delayed alternation task in a T-maze. In experiment 2, the effect of intrastriatal AP-5 on acquisition of different 8 arm maze tasks was investigated. AP-5 did not affect the number of reentries on spontaneous and reinforced alternation; pre- and postdelay errors on delayed alternation were not altered. Therefore, intrastriatal NMDA receptor blockade impairs acquisition of a delayed alternation in a T-maze, while intrastriatal blockade of NMDA receptors does not affect acquisition of different 8 arm maze tasks.Item Open Access Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions(1983) Willecke, Klaus; Müller, Dagmar; Drüge, Petra Maria; Frixen, Uwe; Schäfer, Reinhold; Dermietzel, Rolf; Hülser, Dieter F.Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec−). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec− phenotype most stringently showed the following characteristics: 1. 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec− phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. 4. The mec− defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec−, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec− defect.Item Open Access 6-Hydroxydopamine lesion of the rat prefrontal cortex impairs motor initiation but not motor execution(1994) Hauber, Wolfgang; Bubser, Michael; Schmidt, Werner J.We examined the effects of bilateral 6-hydroxydopamine (6-OHDA) lesions of the medial prefrontal cortex (PFC) in rats on motor initiation and execution in a simple reaction time task. Reaction times (RT) and movement times (MT) were measured in trained rats on four preand postoperative days. Animals with 6-OHDA lesions were selectively impaired on motor initiation as measured by a significant increase in RT on each postoperative day. Motor execution was intact postoperatively, since MT was not altered. Neurochemical analysis revealed a significant depletion of prefrontal dopamine (DA) and noradrenaline (NA) in lesioned animals. It was concluded that DA and, to a lesser extent, NA in the rat PFC were involved in monitoring RT performance.Item Open Access Model-based analysis of an adaptive evolution experiment with Escherichia coli in a pyruvate limited continuous culture with glycerol(2012) Feuer, Ronny; Gottlieb, Katrin; Viertel, Gero; Klotz, Johannes; Schober, Steffen; Bossert, Martin; Sawodny, Oliver; Sprenger, Georg; Ederer, MichaelBacterial strains that were genetically blocked in important metabolic pathways and grown under selective conditions underwent a process of adaptive evolution: certain pathways may have been deregulated and therefore allowed for the circumvention of the given block. A block of endogenous pyruvate synthesis from glycerol was realized by a knockout of pyruvate kinase and phosphoenolpyruvate carboxylase in E. coli. The resulting mutant strain was able to grow on a medium containing glycerol and lactate, which served as an exogenous pyruvate source. Heterologous expression of a pyruvate carboxylase gene from Corynebacterium glutamicum was used for anaplerosis of the TCA cycle. Selective conditions were controlled in a continuous culture with limited lactate feed and an excess of glycerol feed. After 200–300 generations pyruvate-prototrophic mutants were isolated. The genomic analysis of an evolved strain revealed that the genotypic basis for the regained pyruvate-prototrophy was not obvious. A constraint-based model of the metabolism was employed to compute all possible detours around the given metabolic block by solving a hierarchy of linear programming problems. The regulatory network was expected to be responsible for the adaptation process. Hence, a Boolean model of the transcription factor network was connected to the metabolic model. Our model analysis only showed a marginal impact of transcriptional control on the biomass yield on substrate which is a key variable in the selection process. In our experiment, microarray analysis confirmed that transcriptional control probably played a minor role in the deregulation of the alternative pathways for the circumvention of the block.