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 - Velours, J. A1 - Esparza, M. A1 - Hoppe, J. A1 - Sebald, Walter A1 - Guerin, B. T1 - Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae N2 - The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the A TP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 8011/o fonnie acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 5011/o homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 genein Aspergillus nidulans. KW - Biochemie KW - ATP synthase KW - mitochondrially translated KW - proteolipid KW - sequence subunit Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62695 ER - TY - JOUR A1 - Hoppe, J. A1 - Sebald, Walter T1 - Amino acid sequence of the proteolipid subunit of the proton-translocating ATPase complex from the thermophilic bacterium PS-3 N2 - No abstract available KW - Biochemie Y1 - 1980 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62754 ER - TY - JOUR A1 - Müller, T. A1 - Sebald, Walter A1 - Oschkinat, H. T1 - Antagonist design through forced electrostatic mismatch N2 - No abstract available KW - Biochemie Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62408 ER - TY - JOUR A1 - Müller, T. A1 - Dieckmann, T. A1 - Sebald, Walter A1 - Oschkinat, H. T1 - Aspects of receptor binding and signalling of interleukin-4 investigated by site-directed mutagenesis and NMR spectroscopy N2 - Cytokines are hormones that carry information from ceJI to ceH. This information is read from their surface upon binding to transmembrane receptors and by the subsequent initiation of receptor oligomerization. An inftuence on this process through mutagenesis on the hormone surface is highly desirab)e for medical reasons. However, an understanding of hormone-receptor interactions requires insight into the structural changes introduced by the mutations. In this line structural studies on human TL-4 and the medically important IL-4 antagonists YI24D and Y124G are presented. The site a.round YI24 is an important epitope responsible for the a.bility of 11-4 t.o ca.use a signal in the target cells. It is shown that the local main-chain structure around residue 124 in the variants remains unchanged. A strategy is presented here which allows the study of these types of proteins and their variants by NMR which does not require carbon Iabeiied sa.mples. KW - Biochemie KW - Interleukin-4 KW - protein structure KW - NMR KW - signal transduction Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62444 ER - TY - JOUR A1 - von Jagow, Gerhard A1 - Sebald, Walter T1 - b-Type cytochromes N2 - No abstract available KW - Biochemie Y1 - 1980 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47383 ER - TY - JOUR A1 - Sebald, Walter T1 - Biogenesis of mitochondrial ATPase N2 - No abstract available KW - Biochemie Y1 - 1977 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47362 ER - TY - JOUR A1 - Sebald, Walter A1 - Werner, S A1 - Weiss, H T1 - Biogenesis of mitochondrial membrane proteins in Neurospora crassa N2 - no abstract available KW - Biochemie KW - Neurospora crassa Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82055 ER - TY - JOUR A1 - Gabellini, N. A1 - Harnisch, U. A1 - McCarthy, J. E. A1 - Hauska, G. A1 - Sebald, Walter T1 - Cloning and expression of the fbc operon encoding the FeS protein, cytochrome b and cytochrome c\(_1\) from the Rhodopseudomonas sphaeroides b/c\(_1\) complex N2 - The gene for the FeS protein of the Rhodopseudomonas sphaeroides b/c1 complex was identified by means of crosshybridization with a segment of the gene encoding the corresponding FeS protein of Neurospora crassa. Plasmids (pRSF1-14) containing the cross-hybridizing region, covering in total 13.5 kb of chromosomal DNA, were expressed in vitro in a homologous system. One RSF plasmid directed the synthesis of all three main polypeptides of the R. sphaeroides blc1 complex: the FeS protein, cytochrome b and cytochrome c1• The FeS protein and cytochrome c1 were apparently synthesized as precursor fonns. None of the pRSF plasmids directed the synthesis of the 10-kd polypeptide found in b/c1 complex preparations. Partial sequencing of the cloned region was performed. Several sites of strong homology between R. sphaeroides and eukaryotic polypeptides of the b/c1 complex were identified. The genes encode the three b/c1 polypeptides in the order: (5') FeS protein, cytochrome b, cytochrome c1• The three genes are transcribed to give a polycistronic mRNA of 2.9 kb. This transcriptional unit has been designated the jbc operon; its coding capacity corresponds to the size of the polycistronic mRNA assuming that only the genes for the FeS protein (jbcF), cytochrome b (jbcß) and cytochrome c1 (jbcC) are present. This could indicate that these three subunits constitute the minimal catalytic unit of the b/c1 complex from photosynthetic membranes. KW - Biochemie KW - R. sphaeroidesl KW - b/c1 complex KW - gene KW - cloning KW - in vitro expression KW - polycistronic mRNA Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62642 ER - TY - JOUR A1 - Weiss, H. A1 - Sebald, Walter A1 - Schwab, A. J. A1 - Kleinow, W. A1 - Lorenz, B. T1 - Contribution of mitochondrial and cytoplasmic protein synthesis to the formation of cytochrome b and cytochrome aa\(_3\) N2 - A cytochrome b preparation from Neurospora crassa mitochondria is found to consist of three polypeptides (apparent molecular weight 10 000, 11 000 and 32 000), a cytochrome aa3 preparation of six to seven polypeptides (apparent molecular weight 8 000, 11 000, 13 000, 18 000, 28 000 and 36 000). Selective incorporation of radioactive amino acids by eilher mitochondrial protein synthesis when the cytoplasmic one is blocked or by the cytoplasmic protein synthesis, when the mitochondrial one is blocked, indicates that one cytochrome b polypeptide (mw 32 000) and one to three cytochrome aa3 polypeptides (mw 36 000, 28 000 and 18 000) are mitochondrial translation products, the other cytochrome b and cytochrome aa3 polypeptides cytoplasmic translation products. The delayed appearance of labeling in the cytochrome b and cytochrome aa3 polypeptides compared to the average cell protein after a pulse of <~H leueine revealed that these polypeptides are derived from separate pools of precursor polypeptides. The pool sizes range from 2 p. cent to 25 p. cent of the amount of the corresponding polypeptide present in the cytochromes. The 32 000 molecular weight polypeptide of cytochrome band at least the 18 000 molecular weight polypeptide of cytochrome aa\(_3\) are mitochondrial translation products as well in the fungus Neurospora crassa as in the insect Locusta migratoria. So, despite the fact that the size of mitochondrial DNA and mitochondrial ribosomes is reduced in insects, the products have maintained their characteristics. KW - Biochemie Y1 - 1973 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62835 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 - Sebald, Walter A1 - Schwab, A. J. A1 - Bücher, T. T1 - Cycloheximide resistant amino acid incorporation into mitochondrial protein from Neurospora crassa in vivo N2 - No abstract available KW - Biochemie Y1 - 1969 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62900 ER - TY - JOUR A1 - Weiss, H. A1 - Sebald, Walter A1 - Bücher, T. T1 - Cycloheximide resistant incorporation of amino acids into a polypeptide of the cytochrome oxidase of Neurospora crassa N2 - Radioaetive leueine was ineorporated by N eurospora crassa mitoehondria in vivo in the presence of cyeloheximide. When the membrane protein of these mitochondria was ehromatographieally separated on oleyl polymethaerylie aeid resin, & nurober of fraetions were obtained whieh differ with respeet to their eontents of radioaetivity and eytoehromes. The highest speeifie radioaetivity was found in the fraction eontaining eytoehrome aa3• This fraetion proved to be a pure and enzymatically aetive cytoehrome oxidase. Its ratio of absorbanee at 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodeeylsulfate gel-electrophoresis, this enzymewas separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioaetivity. KW - Biochemie Y1 - 1971 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62866 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 - 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 - Tony, H. P. A1 - Shen, B. J. A1 - Reusch, P. A1 - Sebald, Walter T1 - Design of human interleukin-4 antagonists inhibiting interleukin-4-dependent and interleukin-13-dependent responses in T-cells and B-cells with high efficiency N2 - Human interleukin-4 possesses two distinct sites for receptor activation. A signaHing site, comprising residues near the C-terminus on helix D, determines the efficacy of interleukin-4 signal transduction without affecting the binding to the interleukin-4 receptor a subunit. A complete antagonist and a series of low-efficacy agonist variants of human interleukin-4 could be generated by introducing combinations of two or three negatively charged aspartic acid residues in this site at positions 121, 124, and 125. One of the double variants, designated [R121D,Y124D]interleukin-4, with replacements of böth Arg121 and Tyr124 by aspartic acid residues was completely inactive in all analysed cellular responses. The loss of efficacy in [R121D,Y124D]interleukin-4 is estimated to be larger than 2000-fold. Variant [R121D,Y124D]interleukin-4 was also a perfect antagonist for inhibition of interleukin-13-dependent responses in B-cells and the TF-1 cellline with a K\(_i\) value of approximately 100 pM. In addition, inhibition of both interleukin-4-induced and interleuk.in-13- induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin-4Rm the extracellular domain of the interleuk.in-4 receptor a subunit. These results indicate that efficient interleukin-4 antagonists can be designed on the basis of a sequential two-step activation model. In addition, the experiments indicate the functional participation of the interleukin-4 receptor a subunit in the interleukin-13 receptor system. KW - Biochemie Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62394 ER - TY - JOUR A1 - Schwab, A. J. A1 - Sebald, Walter A1 - Weiss, H. T1 - Different pool sizes of the precursor polypeptides of cytochrome oxidase from Neurospora crassa. N2 - Pulse-labelling experiments with growing Neurospora crassa revealed that the polypeptides composing the protein moiety of a cytochrome oxidase preparation are derived from at least four independent pools of precursor polypeptides. The pool sizes range from 2 ° f 0 to 25 °/0 of the amount of the corresponding polypeptide present in cytochrome oxidase. The smallest pool is assigned to a polypeptide of mitochondrial origm. Serial pools were found for one of the polypeptides. KW - Biochemie Y1 - 1972 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62841 ER - TY - JOUR A1 - Sebald, Walter A1 - Bücher, T. A1 - Olbrich, B. A1 - Kaudewitz, F. T1 - Electrophoretic pattern of and amino acid incorporation in vitro into the insoluble mitochondrial protein of neurospora crassa wild type and mi-1 mutant N2 - No abstract available KW - Biochemie Y1 - 1968 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62926 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 - Klammert, Uwe A1 - Müller, Thomas D. A1 - Hellmann, Tina V. A1 - Wuerzler, Kristian K. A1 - Kotzsch, Alexander A1 - Schliermann, Anna A1 - Schmitz, Werner A1 - Kuebler, Alexander C. A1 - Sebald, Walter A1 - Nickel, Joachim T1 - GDF-5 can act as a context-dependent BMP-2 antagonist JF - BMC Biology N2 - Background Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor. Results Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites. Conclusions Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs. KW - growth and differentiation factor 5 KW - ligand-receptor complex KW - crystal structure KW - antagonist Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125550 VL - 13 IS - 77 ER -