@phdthesis{Delto2015,
  author    = {Delto, Carolyn Francesca},
  title     = {Structural and Biochemical Characterization of the GABA(A) Receptor Interacting Protein Muskelin},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115922},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {In a study from 2011, the protein muskelin was described as a central coordinator of the retrograde transport of GABA(A) receptors in neurons. As muskelin governs the transport along actin filaments as well as microtubules, it might be the first representative of a novel class of regulators, which coordinate cargo transport across the borders of these two independent systems of transport paths and their associated motorproteins. To establish a basis for understanding the mode of operation of muskelin, the aim of this thesis was an in-depth biochemical and structural characterization of muskelin and its interaction with the GABA(A) receptor. One focus of the work was the analysis of the oligomerization of muskelin. As could be demonstrated, the oligomerization is based on two independent interactions mediated by different domains of the protein: a known interaction of the N-terminal discoidin domain with the C-terminal portion, termed head-to-tail interaction, and a dimerization of the LisH motif in muskelin that was so far neglected in the literature. For the detailed studies of both binding events, the solution of a crystal structure of a fragment of muskelin, comprising the Discoidin domain and the LisH motif, was an important basis. The fragment crystallized as a dimer, with dimerization being mediated solely by the LisH motif. Biochemical analysis corroborated that the LisH motif in muskelin serves as a dimerization element, and, moreover, showed that the C-terminal domain of the protein substantially stabilizes this dimerization. In addition, the crystal structure revealed the molecular composition of the surface of the head in the head-to-tail interaction, namely the discoidin domain. This information enabled to map the amino acids contributing to binding, which showed that the binding site of the head-to-tail interaction coincides with the generic ligand binding site of the discoidin domain. As part of the analyses, residues that are critical for LisH-dimerization and the head-to-tail binding, respectively, were identified, whose mutation specifically interfered with each of the interactions separately. These mutations allowed to investigate the interplay of these interactions during oligomerization. It could be shown that recombinant muskelin assembles into a tetramer to which both interactions, the LisH-dimerization and the head-to-tail binding, contribute independently. When one of the two interactions was disturbed, only a dimer mediated via the respective other interaction could be formed; when both interactions were disturbed, the protein was present as monomer. Furthermore, Frank Heisler in the group of Matthias Kneussel was able to show the drastic impact of an impaired LisH-dimerization on muskelin in cells using these mutations. Disturbing the LisH-dimerization led to a complete redistribution of the originally cytoplasmic muskelin to the nucleus which was accompanied by a severe impairment of its function during GABA(A) receptor transport. Following up on these results in an analysis of muskelin variants, for which alterations of the subcellular localization had been published earlier, the crucial influence of LisH-dimerization to the subcellular localization and thereby the role of muskelin in the cell was confirmed. The biochemical studies of the interaction of muskelin and the alpha1 subunit of the GABA(A) receptor demonstrated a direct binding with an affinity in the low micromolar range, which is mediated primarily by the kelch repeat domain in muskelin. For the binding site on the GABA(A) receptor, it was confirmed that the thirteen most C-terminal residues of the intracellular domain are critical for the binding of muskelin. In accordance with the strong conservation of these residues among the alpha subunits of the GABA(A) receptor, it could be shown that an interaction with muskelin in vitro is also possible for the alpha2 and alpha5 subunits. Based on the comparison of the binding sites between the homologous subunits, tentative conclusions can be drawn about the details of the binding, which may serve as a starting point for follow-up studies. This thesis thereby makes valuable contributions to the understanding of muskelin, in particular the significance of its oligomerization. It furthermore provides an experimental framework for future studies that address related topics, such as the characterization of other muskelin interaction partners, or the questions raised in this work.},
  subject      = {Oligomerisation},
  language  = {en}
}
@phdthesis{Kober2012,
  author    = {Kober, Franz-Xaver Wilhelm},
  title     = {Molecular insights into the protein disulfide isomerase family},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72144},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Upon synthesis, nascent polypeptide chains are subject to major rearrangements of their side chains to obtain an energetically more favorable conformation in a process called folding. About one third of all cellular proteins pass through the secretory pathway and undergo oxidative folding in the endoplasmic reticulum (ER). During oxidative folding, the conformational rearrangements are accompanied by the formation of disulfide bonds - covalent bonds between cysteine side chains that form upon oxidation. Protein disulfide isomerase (PDI) assists in the folding of substrates by catalyzing the oxidation of pairs of cysteine residues and the isomerization of disulfide bonds as well as by acting as chaperones. In addition to PDI itself, a family of related ER-resident proteins has formed. All PDI family members share the thioredoxin fold in at least one of their domains and exhibit a subset of the PDI activities. Despite many studies, the role of most PDI family members remains unclear. The project presented in this thesis was aimed to establish tools for the biochemical characterization of single members of the PDI family and their role in the folding process. A combination of fluorescence based assays was developed to selectively study single functions of PDI family members and relate their properties of either catalysis of oxidation or catalysis of isomerization or chaperone activity to the rest of the protein family. A binding assay using isothermal titration calorimetry (ITC) was established to complement the activity assays. Using ITC we could show for the first time that members of the PDI family can distinguish between folded and unfolded proteins selectively binding the latter. The unique information provided by this method also revealed a two-site binding of unfolded proteins by PDI itself. In addition to the functional characterization, experiments were conducted to further investigate the oligomeric state of PDI. We could show that the equilibrium between structurally different states of PDI is heavily influenced by the redox state of the protein and its environment. This new data could help to further our understanding of the interplay between oxidases like PDI and their regenerative enzymes like Ero1, which may be governed by structural changes in response to the change in redox status. Another structural approach was the screening of all investigated PDI family members for suitable crystallization conditions. As a result of this screening we could obtain protein crystals of human ERp27 and were able to solve the structure of this protein with X-ray crystallography. The structure gives insight into the mechanisms of substrate binding domains within the PDI family and helps to understand the interaction of ERp27 with the redox active ERp57. In collaboration with the group of Heike Hermanns we could further show the physiological importance of this interaction under oxidative stress. In conclusion, the project presented in this thesis provides novel tools for an extensive analysis of the activities of single PDI family members as well as a useful set of methods to characterize novel oxidoreductases and chaperones. The initial results obtained with the our novel methods are very promising. At the same time, the structural approach of this project could successfully solve the structure of a PDI family member and give information about the interplay within the PDI family.},
  subject      = {Biochemie},
  language  = {en}
}
@article{SchaeferWeibelDonatetal.2012,
  author    = {Sch{\"a}fer, Simon and Weibel, Stephanie and Donat, Ulrike and Zhang, Qian and Aguilar, Richard J. and Chen, Nanhai G. and Szalay, Aladar A.},
  title     = {Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78220},
  year      = {2012},
  abstract  = {Background: Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods: For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results: GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions: Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.},
  subject      = {Biochemie},
  language  = {en}
}
@phdthesis{Zanucco2011,
  author    = {Zanucco, Emanuele},
  title     = {Role of oncogenic and wild type B-RAF in mouse lung tumor models},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69603},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Von Wachstumsfaktoren regulierte Signalkaskaden sind Schl{\"u}sselelemente in der Gewebeentwicklung und Geweberegeneration. Eine Deregulation dieser Kaskaden f{\"u}hrt zu Entwicklungsst{\"o}rungen und neoplastischen Krankheiten. F{\"u}r viele humane Krebsformen sind aktivierende Mutationen der Kinasen der RAF Familie verantwortlich. Das erste Projekt dieser Doktorarbeit fokussiert auf der Rolle des B-RAF V600E, welches als eine der am h{\"a}ufigsten vorkommenden Mutantionen in humanen Krebszellen identifiziert worden ist. Um die onkogene Funktion des B-RAF V600E zu untersuchen, haben wir transgene Mauslinien hergestellt, welche das aktivierte Onkogen spezifisch in alveolaren Lungenepithelzellen des Typ II exprimieren. Konstitutive Expression des B-RAF V600E f{\"u}hrte zu einer abnormen alveolaren Epithelzellbildung und zu Emphysem-{\"a}hnlichen L{\"a}sionen. Diese L{\"a}sionen wiesen Zeichen einer Gewebsumstrukturierung auf, oft in Assoziation mit chronischer Inflammation und geringer Inzidenz von Lungentumoren. Die Infiltration der entz{\"u}ndlichen Zellen erfolgte erst nach der Entstehung von Emphysem-{\"a}hnlichen L{\"a}sionen und k{\"o}nnte zur sp{\"a}teren Tumorbildung beigetragen haben. Diese Ergebnisse unterst{\"u}tzen ein Modell, in welchem der kontinuierliche regenerative Prozess eine tumorf{\"o}rdernde Umgebung schafft. Dabei induziert die Aktivit{\"a}t des onkogenen B-RAF eine alveolare St{\"o}rung, welche urs{\"a}chlich verantwortlich ist f{\"u}r den kontinuierlichen regenerativen Prozess. Das zweite Projekt fokussiert auf die Rolle von endogenem (wildtypischen) B-RAF in einem durch onkogenes C-RAF induzierten Maus Lungentumormodell. F{\"u}r unsere Untersuchungen haben wir eine Mauslinie geschaffen, in welcher B-RAF in den C-RAF Lungentumoren konditionell eliminiert werden kann. Eine konditionelle Eliminierung des B-RAF hat die Entstehung von Lungentumoren nicht blockiert, aber zu reduziertem Tumorwachstum gef{\"u}hrt. Dieses reduzierte Tumorwachstum konnte auf eine reduzierte Zellproliferation zur{\"u}ckgef{\"u}hrt werden. Außerdem konnten wir durch die B-RAF Elimination eine Reduktion der Intensit{\"a}t der mitogenen Signalkaskade beobachten. Insgesamt deuten die Ergebnisse darauf hin, dass das onkogene Potential von C-RAF in vivo unabh{\"a}ngig von B-RAF ist und eine Kooperation von B-RAF und C-RAF jedoch f{\"u}r die vollst{\"a}ndige Aktivierung der mitogenen Signalkaskade wichtig ist.},
  subject      = {Lungenkrebs},
  language  = {en}
}
@phdthesis{Fischer2010,
  author    = {Fischer, Andreas},
  title     = {The Role of Protein-Protein Interactions in the Activation Cycle of RAF Kinases},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48139},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Members of the RAF protein kinase family are key regulators of diverse cellular processes. The need for isoform-specific regulation is reflected by the fact that all RAFs not only display a different degree of activity but also perform isoform-specific functions at diverse cellular compartments. Protein-protein-interactions and phosphorylation events are essential for the signal propagation along the Ras-RAF-MEK-ERK cascade. More than 40 interaction partners of RAF kinases have been described so far. Two of the most important regulators of RAF activity, namely Ras and 14-3-3 proteins, are subject of this work. So far, coupling of RAF with its upstream modulator protein Ras has only been investigated using truncated versions of RAF and regardless of the lipidation status of Ras. We quantitatively analyzed the binding properties of full-length B- and C-RAF to farnesylated H-Ras in presence and absence of membrane lipids. While the isolated Ras-binding domain of RAF exhibit a high binding affinity to both, farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases demonstrate crucial differences in their affinity to Ras. In contrast to C-RAF that requires carboxyterminal farnesylated H-Ras for interaction at the plasma membrane, B-RAF also binds to nonfarnesylated H-Ras in the cytosol. For identification of the potential farnesyl binding site we used several fragments of the regulatory domain of C-RAF and found that the binding of farnesylated H-Ras is considerably increased in the presence of the cysteine-rich domain of RAF. In B-RAF a sequence of 98 amino acids at the extreme N terminus enables binding of Ras independent of its farnesylation status. The deletion of this region altered Ras binding as well as kinase properties of B-RAF to resemble C-RAF. Immunofluorescence studies in mammalian cells revealed essential differences between B- and C-RAF regarding the colocalization with Ras. In conclusion, our data suggest that that B-RAF, in contrast to C-RAF, is also accessible for nonfarnesylated Ras in the cytosolic environment due to its prolonged N terminus. Therefore, the activation of B-RAF may take place both at the plasma membrane and in the cytosolic environment. Furthermore, the interaction of RAF isoforms with Ras at different subcellular sites may also be governed by the complex formation with 14-3-3 proteins. 14-3-3 adapter proteins play a crucial role in the activation of RAF kinases, but so far no information about the selectivity of the seven mammalian isoforms concerning RAF association and activation is available. We analyzed the composition of in vivo RAF/14-3-3 complexes isolated from mammalian cells with mass spectrometry and found that B-RAF associates with a greater variety of 14-3-3 proteins than C- and A-RAF. In vitro binding assays with purified proteins supported this observation since B-RAF showed highest affinity to all seven 14-3-3 isoforms, whereas C-RAF exhibited reduced affinity to some and A-RAF did not bind to the 14-3-3 isoforms epsilon, sigma, and tau. To further examine this isoform specificity we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. By deleting one of the two 14-3-3 isoforms in Saccharomyces cerevisiae we were able to show that homodimeric 14-3-3 proteins are sufficient for functional activation of B- and C-RAF. In this context, the diverging effect of the internal, inhibiting and the activating C-terminal 14-3-3 binding domain in RAF could be demonstrated. Furthermore, we unveil that prohibitin stimulates C-RAF activity by interfering with 14-3-3 at the internal binding site. This region of C-RAF is also target of phosphorylation as part of a negative feedback loop. Using tandem MS we were able to identify so far unknown phosphorylation sites at serines 296 and 301. Phosphorylation of these sites in vivo, mediated by activated ERK, leads to inhibition of C-RAF kinase activity. The relationship of prohibitin interference with 14-3-3 binding and phosphorylation of adjacent sites has to be further elucidated. Taken together, our results provide important new information on the isoform-specific regulation of RAF kinases by differential interaction with Ras and 14-3-3 proteins and shed more light on the complex mechanism of RAF kinase activation.},
  subject      = {Signaltransduktion},
  language  = {en}
}
@article{SchwarzHameisterGessleretal.1994,
  author    = {Schwarz, Klaus and Hameister, Horst and Gessler, Manfred and Grzeschik, Karl-Heinz and Hansen-Hagge, Thomas E. and Bartram, Claus R.},
  title     = {Confirmation of the localization of the human recombination activating gene 1 (RAG1) to chromosome 11p13},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59136},
  year      = {1994},
  abstract  = {The human recombination activating gene 1 (RAGl) has previously been mapped to chromosomes 14q and 11 p. Here we confirm the chromosome 11 assignment by two independent approaches: autoradiographic and fluorescence in situ hybridization to metaphase spreads and analysis of human-hamster somatic cell hybrid DNA by the polymerase chain reaction (PCR) and Southern blotting. Our results unequivocally localize RAG1 to llp13.},
  subject      = {Biochemie},
  language  = {en}
}
@article{SchwartzNeveEisenmanetal.1994,
  author    = {Schwartz, Faina and Neve, Rachel and Eisenman, Robert and Gessler, Manfred and Bruns, Gail},
  title     = {A WAGR region gene between PAX-6 and FSHB expressed in fetal brain},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59125},
  year      = {1994},
  abstract  = {Developmental delay or mental retardation is a frequent component of multi-system anomaly syndromes associated with chromosomal deletions. Isolation of genes involved in the mental dysfunction in these disorders should define loci important in brain formation or function. We have identified a highly conserved locus in the distal part of 11 p 13 that is prominently expressed in fetal brain. Minimal expression is observed in a number of other fetal tissues. The gene maps distal to PAX-6 but proximal to the loci for brain-derived neurotrophic factor (BDNF) and the beta subunit of follicle stimulating hormone (FSHB), within a region previously implicated in the mental retardation component of some WAGR syndrome patients. Within fetal brain, the corresponding transcript is prominent in frontal, motor and primary visual cortex as weil as in the caudate-putamen. The characteristics of this gene, including the striking evolutionary conservation at the locus, suggest that the encoded protein may function in brain development.},
  subject      = {Biochemie},
  language  = {en}
}
@article{MuellerDieckmannSebaldetal.1994,
  author    = {M{\"u}ller, T. and Dieckmann, T. and Sebald, Walter and Oschkinat, H.},
  title     = {Aspects of receptor binding and signalling of interleukin-4 investigated by site-directed mutagenesis and NMR spectroscopy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62444},
  year      = {1994},
  abstract  = {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.},
  subject      = {Biochemie},
  language  = {en}
}
@article{MuellerSebaldOschkinat1994,
  author    = {M{\"u}ller, T. and Sebald, Walter and Oschkinat, H.},
  title     = {Antagonist design through forced electrostatic mismatch},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62408},
  year      = {1994},
  abstract  = {No abstract available},
  subject      = {Biochemie},
  language  = {en}
}
@article{ReuschArnoldHeusseretal.1994,
  author    = {Reusch, P. and Arnold, S. and Heusser, C. and Wagner, K. and Weston, B. and Sebald, Walter},
  title     = {Neutralizing monoclonal antibodies define two different functional sites in human interleukin-4},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62418},
  year      = {1994},
  abstract  = {Human interleukin-4 (IL-4) is a small four-helix-bundle protein which is essential for organizing defense reactions against macroparasites, in particular helminths. Human IL-4 also appears to exert a pathophysiological role during various IgE-mediated allergic diseases. Seven different monoclonal antibodies neutralizing the activity of human IL-4 were studied in order to identify functionally important epitopes. A collection of 41 purified IL-4 variants was used to analyse how defined amino acid replacements affect binding affinity for each individual mAb. Specific amino acid positions could be assigned to four different epitopes. mAbs recognizing epitopes on helix A and/or C interfered with IL-4 receptor binding and thus inhibited IL-4 function. However, other mAbs also inhibiting IL-4 function recognized an epitope on helix D of IL-4 and did not inhibit IL-4 binding to the receptor protein. One mAb, recognizing N-terminal and C-terminal residues, partially competed for binding to the receptor. The results of these mAb epitope analyses confirm and extend previous data on the functional consequences of the amino acid replacements which showed that amino acid residues in helices A and C of IL-4 provide a binding site for the cloned IL-4 receptor and that a signalling site in helix D interacts with a further receptor protein.},
  subject      = {Biochemie},
  language  = {en}
}
@article{DemchukMuellerOschkinatetal.1994,
  author    = {Demchuk, E. and Mueller, T. and Oschkinat, H. and Sebald, Walter and Wade, R. C.},
  title     = {Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62424},
  year      = {1994},
  abstract  = {Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-a-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormonereceptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 Iack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors.},
  subject      = {Biochemie},
  language  = {en}
}
@article{LehrnbecherPootOrschescheketal.1994,
  author    = {Lehrnbecher, T. and Poot, M. and Orscheschek, K. and Sebald, Walter and Feller, A. C. and Merz, H.},
  title     = {Interleukin 7 as interleukin 9 drives phytohemagglutinin-activated T cells through several cell cycles; no synergism between interleukin 7, interleukin 9 and interleukin 4},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62438},
  year      = {1994},
  abstract  = {The effects of the interlenkins IL-7 and IL-9 on cell cycle progression were investigated by conventional [3H]thymidine incorporation and by the bivariate BrdU/Hoechst technique. 8oth IL· 7 and IL-9 drive phytohemagglutinin-activated T cells through more than one cell cycle, but IL-7 wasmorepotent on cell cycle progression than IL-9. Neither synergistic nor inhibitory effects were seen between various combinations of the lymphokines IL-7, IL-9 and IL-4 compared to each lymphokine alone. When T cells are activated with phytohemagglutinin for 3 days, all or most IL-4 responsive cells respond to IL-7 as weil, whereas only a part of IL-7 responders are IL-4 responders. In contrast, when T cells are activated with phytohemagglutinin for 7 days, the quantitative data of the cell cycle distribution soggest that the population of IL-7 responders is at least an overlapping, if not a real subset of the population of the IL-4 responders.},
  subject      = {Biochemie},
  language  = {en}
}
@article{KueblerReutherKirchneretal.1994,
  author    = {K{\"u}bler, N. and Reuther, J. and Kirchner, T. and Pfaff, M. and M{\"u}ller-Hermelink, H. K. and Albert, R. and Sebald, Walter},
  title     = {IgG monoclonal antibodies that inhibit osteoinductivity of human bone matrix-derived proteins (hBMP/NCP)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62388},
  year      = {1994},
  abstract  = {Monoclonal hBMP/NCP (human bone morphogenetic protein anrl associaterl noncollagenous proteins) antiborlies of the lgG class were prorlucerl. In vitro, 12 of 19 hBMP/NCP antiborlies showerl functional inhibition of hBMP/ NCP-induced chondroneogenesis in a neonatal muscle tissue assay. Inducing factors were characterized by their inhibiting antibodies with immunoblotting. Several peptide factors seem to be involved in the cascade of inducerl chondro- and osteogenesis.},
  subject      = {Biochemie},
  language  = {en}
}
@article{TonyShenReuschetal.1994,
  author    = {Tony, H. P. and Shen, B. J. and Reusch, P. and Sebald, Walter},
  title     = {Design of human interleukin-4 antagonists inhibiting interleukin-4-dependent and interleukin-13-dependent responses in T-cells and B-cells with high efficiency},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62394},
  year      = {1994},
  abstract  = {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 b{\"o}th 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.},
  subject      = {Biochemie},
  language  = {en}
}
@article{GesslerKonigMooreetal.1993,
  author    = {Gessler, Manfred and Konig, Anja and Moore, Jay and Qualman, Steven and Arden, Karen and Cavenee, Webster and Bruns, Gail},
  title     = {Homozygous inactivation of WTI in a Wilms' tumor associated with the WAGR syndrome},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59146},
  year      = {1993},
  abstract  = {Wilms' tumor is a childhood nephroblastoma that is postulated to arise through the inactivation of a tumor suppressor gene by a two-hit mechanism. A candidate II p 13 Wilms' tumor gene, WTI, has been cloned and shown to encode a zinc finger protein. Patients with the WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation) have a high risk of developing Wilms' tumor and they carry constitutional deletions of one chromosome II allele encompassing the WTI gene. Analysis of the remaining WTI allele in a Wilms' tumor from a WAGR patient revealed the deletion of a single nucleotide in exon 7. This mutation likely played a key role in tumor formation, as it prevents translation of the DNA-binding zinc finger domain that is essential for the function of the WTI polypeptide as a transcriptional regulator.},
  subject      = {Biochemie},
  language  = {en}
}
@article{HenryHooversBarichardetal.1993,
  author    = {Henry, Isabelle and Hoovers, Jan and Barichard, Fernande and Berth{\´e}as, Marie-Francoise and Puech, Anne and Prieur, Fabienne and Gessler, Manfred and Bruns, Gail and Mannens, Marcel and Junien, Claudine},
  title     = {Pericentric intrachromosomal insertion responsible for recurrence of del(11)(p13p14) in a family},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59157},
  year      = {1993},
  abstract  = {The combined use of qualitative and quantitative analysis of I I p I 3 polymorphic markers tagether with chromosomal in situ suppression hybridization (CISS) with biotin labeled probes mapping to I I p allowed us to characterize a complex rearrangement segregating in a family. We detected a pericentric intrachromosomal insertion responsible (or recurrence of del( I I )(p 13p 14) in the family: an insertion of band I I p 13-p 14 carrying the genes for predisposition to Wilms' tumor, WT I, and for aniridia, AN2, into the long arm of chromosome I I in II q 13-q 1<4. Asymptomatic balanced carriers were observed over three generations. Classical cytogenetics had failed to detect this anomaly in the balanced carriers, who were first considered to be somatic mosaics for del( II )(p 13). Two of these women gave birth to children carrying a deleted chromosome II. most likely resulting from the loss of the I I p 13 band inserted in I I q. Although in both cases the deletion encompassed exactly the same maternally inherited markers, there was a wide Variation in clinical expression. One child, with the karyotype 46,XY,del(ll)(pllpl4), presented the full-blown WAGR syndrome with anlridia, mental retardation, Wilms' tumor, and pseudohermaphroditism, but also had proteinuria and glomerular sclerosis reminiscent of Drash syndrome. In contrast, the other one, a girl with the karyotype 46,XX,del( I I )(p I 3), only had aniridia. Although a specific set of mutational sites has been observed in Drash patients, these findings suggest that the loss of one copy of the WTI gene can result in similar genital and kidney abnormalities.},
  subject      = {Biochemie},
  language  = {en}
}
@article{KonigJakubiczkaWieackeretal.1993,
  author    = {Konig, Anja and Jakubiczka, Sybille and Wieacker, Peter and Schl{\"o}sser, Hans W. and Gessler, Manfred},
  title     = {Further evidence that imbalance of WT1 isoforms may be involved in Denys-Drash syndrome},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59167},
  year      = {1993},
  abstract  = {No abstract available},
  subject      = {Biochemie},
  language  = {en}
}
@article{KruseShenArnoldetal.1993,
  author    = {Kruse, N. and Shen, B. J. and Arnold, S. and Tony, H. P. and M{\"u}ller, T. and Sebald, Walter},
  title     = {Two distinct functional sites of human interleukin 4 are identified by variants impaired in either receptor binding or receptor activation},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62451},
  year      = {1993},
  abstract  = {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.},
  subject      = {Biochemie},
  language  = {en}
}
@article{PoulatMorinKonigetal.1993,
  author    = {Poulat, F. and Morin, D. and Konig, A. and Brun, P. and Giltay, J. and Sultan, C. and Dumas, R. and Gessler, Manfred and Berta, P.},
  title     = {Distinct molecular origins for Denys-Drash and Frasier syndromes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59172},
  year      = {1993},
  abstract  = {The direct involvment of the Wilm's tumor suppressor gene (WTl) in Denys-Drash syndrome through mutations within exons 8 or 9 has recently been established. The absence of such alterations in three patients with Frasier syndrome provides a molecular basis for distinguishing these two syndromes that are associated with streak gonads, pseudohermaphroditism and renal failure.},
  subject      = {Biochemie},
  language  = {en}
}
@article{KruseTonySebald1992,
  author    = {Kruse, N. and Tony, H. P. and Sebald, Walter},
  title     = {Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62469},
  year      = {1992},
  abstract  = {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.},
  subject      = {Biochemie},
  language  = {en}
}