TY - JOUR A1 - McCarthy, J. E. A1 - Schairer, H. U. A1 - Sebald, Walter T1 - Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation N2 - The c, b and ö subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro andin vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating A TP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit ö. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching > 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage A is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of A TP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons. KW - Biochemie KW - E. coli atp operon KW - subunit stoichiometry KW - in vitro and in vivo expression KW - translational initiation Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62657 ER - TY - JOUR A1 - Lindenmaier, W. A1 - Dittmar, K. E. A1 - Hauser, H. A1 - Necker, A. A1 - Sebald, Walter T1 - Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo N2 - A method has been developed that allows the isolation of genomic clones from a cosmid library by homologaus recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into A. phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologaus plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 genewas restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively. KW - Biochemie KW - Recombinant DNA KW - DNA mediated gene transfer KW - expression plasmid KW - screening KW - packaging KW - bacteriophage lambda Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62662 ER - TY - JOUR A1 - Weich, H. A. A1 - Sebald, Walter A1 - Schairer, H. U. A1 - Hoppe, J. T1 - The human osteosarcoma cell line U-2 OS expresses a 3.8 kilobase mRNA which codes for the sequence of the PDGF-B chain N2 - A cDNA clone of about 2500 basepairswas prepared from the human osteosarcoma cellline U-2 OS by hybridizing with a v-sis probe. Sequence analysis showed that this cDNA contains the coding region for the PDGF-B chain. Here we report that the mitogen secreted by these osteosarcoma cells contains the PDGF-B chain and is probably a homodimer of two B-chains. KW - Biochemie KW - Platelet-derived growthfactor KW - cDNA KW - Oncogene KW - Tumor cell Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62588 ER - TY - JOUR A1 - Hoppe, J. A1 - Gatti, D. A1 - Weber, H. A1 - Sebald, Walter T1 - Labeling of individual amino acid residues in the membrane-embedded F\(_0\) part of the F\(_1\) F\(_0\) ATP synthase from Neurospora crassa. Influence of oligomycin and dicyclohexylcarbodiimide N2 - Three F0 subunits and the F\(_1\) subunit P of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[\(^{125}\)I]iodophenyl)diazirine ([\(^{125}\)I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confmed to five residues at the NH2-terminus and five residues at the C-terminus ofthe protein. Labeling occurred at similar positions compared with the homologaus protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent oflabeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodümide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F\(_0\) subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F\(_0\) subunits. KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62598 ER - TY - JOUR A1 - Hoppe, J. A1 - Sebald, Walter T1 - Topological studies suggest that the pathway of the protons through F\(_0\) is provided by amino acid residues accessible from the lipid phase N2 - The structure of the F0 part of ATP synthases from E. coli and Neurospora crassa was analyzed by hydrophobic surface labeling with [125I]TID. In the E. co/i F0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. Labeted amino acid residues were identified by Edman degradation of the dicyclohexylcarbodiimide binding (DCCD) proteins from E. coli and Neurospora crassa. The very similar patterns obtained with the two homologaus proteins suggested the existence of tightly packed cx-helices. The oligomeric structure of the DCCD binding protein appeared to be very rigid since little, if any, change in the labeling patternwas observed upon addition of oligomycin or DCCD to membranes from Neurospora crassa. When membrancs were pretrcated with DCCD prior to the reaction with [125I]TID an additionally labeled amino acid appeared at the position of Glu·65 which binds DCCD covalently, indicating the Jocation of this inhibitor on the outside of the oligomer. It is suggested that proton conduction occurs at the surface of the oligomer of the DCCD binding protein. Possibly this oligomer rotates against the subunit a or b and thus enables proton translocation. Conserved residues in subunit a, probably located in the Iipid bilayer, might participate in the pro· ton translocation mechanism. N2 - La structure de la partie F0 de l'ATP synthase a ete analysee au moyen de marquage par /e reactif hydrophobe TJD[125 I]. Les trois sous-unites de E. coli F0 sont accessibles au reactif ce qui semble indiquer que ces sous-unites sont integrees dans Ia membrane defaron independante. Les amino-acides marques ont ete identifies par Ia degradation d'Edman des profeines d'E. coli et de Neurospora associees au dicyclohexylcarbodiimide (DCCD). L 'analogie des courbes obtenues pour /es deux proteines homologues suggere l'existence d'a-helices rangees de faron serree. La structure oligomerique de Ia proreine associee au DCCD semble etre tres rigide puisque pratiquement aucun changement dans /e marquage n 'a ete observe par addition d'oligomycine ou de DCCD aux membranes de Neurospora crassa. Quand /es membranes sont traitees avec /e DCCD avant Ia reaction avec TJD[125 I], un amino-acide additionnellement marque apparait a Ia position Glu·65 et forme avec le DCCD une Iiaison covalente. Ce dernier resu/tat indique Ia localisation de cet inhibiteur a /'exterieur de J'oligo· mere. II semble donc que Ia conduction des protons ait lieu a Ia surface de /'oligomere de Ia proteine associee au DCCD. II serait possib/e que l'o/igomere se retourne contre Ia sous-unite a ou b, permettunt de ce fait Ia translocation des protons. Les residus conserves de Ia sous-unite a, probab/ement Jocalises dans Ia double couche lipidique, pourraient participer au mecanisme de translocation des protons. KW - Biochemie KW - conduction de protons KW - proteines membranaires KW - carbenes KW - proton conduction KW - membrane proteins KW - carbenes Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62602 ER - TY - JOUR A1 - Gabellini, N. A1 - Sebald, Walter T1 - Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides. Evaluation of the deduced amino acid sequences of the FeS protein, cytochrome b and cytochrome c\(_1\) N2 - No abstract available KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62615 ER - TY - JOUR A1 - McCarthy, J. E. A1 - Sebald, Walter A1 - Gross, G. A1 - Lammers, R. T1 - Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes N2 - No abstract available KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62626 ER - TY - JOUR A1 - Kleene, R. A1 - Pfanner, N. A1 - Pfaller, R. A1 - Link, T. A. A1 - Sebald, Walter A1 - Neupert, W. A1 - Tropschug, M. T1 - Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria N2 - No abstract available KW - Biochemie Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62566 ER - TY - JOUR A1 - Römisch, J. A1 - Tropschug, M. A1 - Sebald, Walter A1 - Weiss, H. T1 - The primary structure of cytochrome c\(_1\) from Neurospora crassa N2 - No abstract available KW - Biochemie Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62578 ER - TY - JOUR A1 - Weigel, U. A1 - Meyer, M. A1 - Sebald, Walter T1 - Mutant proteins of human interleukin 2. Renaturation yield, proliferative activity and receptor binding N2 - No abstract available KW - Biochemie Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62543 ER - TY - JOUR A1 - Flügge, U. I. A1 - Fischer, K. A1 - Gross, A. A1 - Sebald, Walter A1 - Lottspeich, F. A1 - Eckerskorn, C. T1 - The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts N2 - No abstract available KW - Biochemie Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62559 ER - TY - JOUR A1 - Merz, H. A1 - Fliedner, A. A1 - Lehrnbecher, T. A1 - Sebald, Walter A1 - Müller-Hermelink, H. K. A1 - Feller, A. C. T1 - Cytokine expression in B-cell non-Hodgkin lymphomas N2 - No abstract available KW - Biochemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62539 ER - TY - JOUR A1 - Lehrnbecher, T. A1 - Merz, H. A1 - Sebald, Walter A1 - Poot, M. T1 - Interleukin 4 drives phytohemagglutinin-activated T cells through several cell cycles: no synergism between interleukin 2 and interleukin 4 N2 - Cell kinetic studies of T cells stimulated with the interleukin 2 (11-2), D-4, or both lymphokines were performed with conventional [3H] thymidine incorporation and with the bivariate BrdU/Hoechst technique. 11-2 and 11-4 are able to drive phytohemagglutininactivated T cells through more than one cell cycle. Neither synergistic nor inhibitory efl'ect on T -cell proliferationwas seen for the stimulation with both 11-2 and 11-4 as compared with the effect ofll-2 alone. The quantitative data ofthe cell cycle distribution ofphytohemagglutininactivated T cells suggestthat the population ofll-4-responsive cells is at least an overlapping population, if not a real subset of the ·population of the 11-2-responsive cells. KW - Biochemie KW - BrdU-Hoechst KW - cell cycle KW - flow cytometry KW - interleukin 2 KW - interleukin 4 Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62491 ER - TY - JOUR A1 - Merz, H. A1 - Fliedner, A. A1 - Orscheschek, K. A1 - Binder, T. A1 - Sebald, Walter A1 - Müller-Hermelink, H. K. A1 - Feller, A. C. T1 - Cytokine expression in T-cell lymphomas and Hodgkin's disease. Its possible implication in autocrine or paracrine production as a potential basis for neoplastic growth N2 - No abstract available KW - Biochemie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62483 ER - TY - JOUR A1 - Kruse, N. A1 - Lehrnbecher, T. A1 - Sebald, Walter T1 - Site-directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human interleukin-4 N2 - Mutant proteins (muteins) of human lnterleukin-4 (llA) were constructed by means of in vitro mutagenesis. The muteins were expressed in E. co/1, submitted to a renaturation and purification protocol and analysed for biological activity. Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted. in a nearly co•mplete loss · of biological actiyity and an unstable protein. The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity. Exchange of the other four cysteines or of · the single tryptophane had smaller etTects. KW - Biochemie KW - Interleukin 4 (human) KW - Recombinant KW - ln vitro mutagenesis KW - Structure-function Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62505 ER - TY - JOUR A1 - Kruse, N. A1 - Tony, H. P. A1 - Sebald, Walter T1 - Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement N2 - lnterleukin-4 (IL-4) represents a prototypic lymphokine (for a recent review see Paul, 1991). It promotes differentiation of B-cells and the proliferation of T- and B-cell, and other cell types of the lymphoid system. An antagonist of human IL-4 was discovered during the studies presented here after Tyr124 of the recombinant proteinbad been substituted by an aspartic acid residue. This IL-4 variant, Y124D, bound with high affinity to the IL-4 receptor (K\(_D\) = 310 pM), but retained no detectable proliferative activity for T -<:ells and inhibited IL-4-dependent T -cell proliferation competitively (K\(_i\) = 620 pM). The loss of efficacy in variant Y124D was estimated to be > 100-fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B-cells. The subsitution of Tyr124 by either phenylalanine, histidine, asparagine, Iysine or glycine resulted in partial agonist variants with unaltered receptor binding atTmity and relatively small deficiencies in efficacy. These results demoostrate that high affinity binding and signal generation can be uncoupled efticiently in a Iigand of a receptor betonging to the recently identified hematopoietin receptor family. In addition we show for the first time, that a powerful antagonist acting on the IL-4 receptor system can be derived from the IL-4 protein. KW - Biochemie KW - drug design KW - partial agonists KW - receptor signalling Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62469 ER - TY - JOUR A1 - Dummer, R. A1 - Posseckert, G. A1 - Nestle, F. A1 - Witzgall, R. A1 - Burger, M. A1 - Becker, J. C. A1 - Schäfer, E. A1 - Wiede, J. A1 - Sebald, Walter A1 - Burg, G. T1 - Soluble interleukin-2 receptors inhibit interleukin 2-dependent proliferation and cytotoxicity: explanation for diminished natural killer cell activity in cutaneous T-cell lymphomas in vivo? N2 - No abstract available KW - Biochemie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62473 ER - TY - JOUR A1 - Duschl, Albert A1 - Jahn, Ute A1 - Bertling, Claudia A1 - Sebald, Walter T1 - A comparison of assays for the response of primary human T-cells upon stimulation with interleukin-2, interleukin-4 and interleukin-7 N2 - The most commonly used assay to quantitate the response of peripheral T~cells upon stimulation with growth factors is determination of incorporated (JH]TdR. We compared thls test to three other methods: 1. direct countlog of cells with a Coulter type counter as reference assay, 2. a colorimetric assay using the tetrazolium dye 3-[ 4,S-dimethylthiazol-l-yl]-2,5diphenyl tetrazolium (MTT), which is a cheap and increasingly popular non-radioactive method and 3. incorporation of the thymidine analog 5-bromo-2'-deoxyuridine detection with a monoclonal antibody on cytospins. Primary human PHA-blasts from >30 healthy individuals were stimulated with IL-2, IL-4 aod IL-7 and assayed with up to four different methods. We discuss the advantages and disadvantages of the assays used and tbe effects of differences between cell preparations. We observed no significant variations between individuals for the dose dependence, but the relative emctency of IL4 compared to IL-2 and IL-7 was variable. This was probably due to the slower response observed upon stimulation with this factor. KW - T-Lymphozyt KW - Interleukin 2 KW - Interleukin 4 KW - Interleukin 7 KW - T-cells KW - proliferation assays KW - IL-2 KW - IL-4 KW - IL-7 Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86750 ER - TY - JOUR A1 - Kruse, N. A1 - Shen, B. J. A1 - Arnold, S. A1 - Tony, H. P. A1 - Müller, T. A1 - Sebald, Walter T1 - Two distinct functional sites of human interleukin 4 are identified by variants impaired in either receptor binding or receptor activation N2 - Interleukin 4 (IL-4) exerts a decisive role in the coord.ination of proteelive immune responses against parasites, particularly helminths. A disregulation of ll.r4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human ll.r4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4-helix-bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL-4 variants deficient in binding to the extracellular domain of the ll.r4 receptor (IL-4ReJ. In parallel, up to 1000-fold increased concentrations of this type of variant were required to induce T -cell proliferation and B-eeil CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild-type).ß.A variants affected at site 2 exhibited partial agonist activity during T -cell proliferation; however, they still bound with high affinity to IL-4Rex. [The generation of an IL-4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO }., 11, 3237-3244)]. These findings indicate that IL-4 functions by bind.ing IL-4Rex via site 1 which is constituted by residues on helices A and C. They further suggest that the association of a second, still undetined receptor protein with site 2 in helix D activates the receptor system and generates a transmembrane signal. KW - Biochemie KW - drug design/partial agonists KW - receptor signalling Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62451 ER - TY - JOUR A1 - Kübler, Norbert A1 - Reuther, Jürgen A1 - Kirchner, Thomas A1 - Priessnitz, Bernd A1 - Sebald, Walter T1 - Osteoinductive, morphologic, and biomechanical properties of autolyzed, antigen-extracted, allogeneic human bone N2 - Autolyzed, antigen-extracted, allogeneic (AAA) bone was prepared from human cortical bone and its morphologic, biomechanical, and osteoinductive properlies were compared with untreated (frozen) as well as lyophilized human bone. Scanning electron microscopy revealed removal of inprganic calcium phosphates and persistence of shrunken collagen fibrils on the surface of AAA bone matrix. Biomechanical testing of differently prepared bone samples showed that lyophilization increased both the modulus of elasticity (P < .00001) and the compressive strength (P < .00001 ). Depending on the depth of decalcification in the preparation of AAA bone, both measured values decreased in rehydrated AAA bone compared with untreated bone {P < .00001 ). Completely demineralized and rehydrated AAA bone was soft, flexible, and showed very little compressive strength. Differences in biomechanical behavior between samples drilled longitudinally or perpendicularly to the diaphyseal bone axis were observed. Xenogeneic human bone samples were implanted in muscle pouches of Sprague-Dawley rats for 6 weeks. AAA bone implants showed chondrogenesis and osteogenesis in 50% of the cases, while untreated or lyophilized bone implants induced no new cartilage or bone formation. As decalcification exposed xenogeneic organic matrix components, AAA bone implants provoked the highest inflammatory reaction. When AAA bone samples were implanted in immunosuppressed rats, the inflammatory reaction was suppressed and 94o/o of the implants showed endochondral bone formation. The chondroinductivity of the bone samples also was tested in vitro using neonatal rat muscle tissue to avoid interference with inflammatory cells and secreted cytokines. In this assay, 68°/o of AAA bone samples induced chondroneogenesis, while untreated as weil as lyophilized bone samples failed to induce any cartilage formation. The results clearly dernonstrafe that AAA bone has osteoinductive properties. Biomechanical stability of AAA bone implants depends on the degree of demineralization. Thus, they can be prepared in an appropriate manner for different indications in oral and maxillofacial surgery. KW - Mund-Kiefer-Gesichts-Chirurgie KW - Oralchirurgie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86715 ER -