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W Sebald

Publications and source records attributed to W Sebald.

At least 55 records · Page 3Linked to original sources
PubMed1998

[Biological activity of E. coli expressed BMP-4].

BMP-4 is physiologically present in low concentrations in human bone matrix. So far the protein has only been produced in small quantities by expression in mammalian cell cultures. In this study we investigated the biological activity of E. coli-expressed BMP-4. In vitro neonatal rat muscle tissue was incubated together with BMP-4 during 4 h, followed by an incubation period of 14 days on cellulose acetate membranes in BMP-free medium. The addition of 0.4 microgram BMP-4 induced cartilage formation in 1/8 samples while 4 micrograms BMP-4 showed chondroneogenesis in 2/10 samples. When the BMP-4 concentration was increased to 40 micrograms, new cartilage formation was seen in 5/7 samples. In vivo BMP-4 was implanted intramuscularly for 3 weeks in ICR mice. Amounts of 10 micrograms rhBMP-4 and more (up to 100 micrograms) constantly induced heterotopic ossicle formation. BMP-4 was also combined with a collagen carrier and implanted for 2 and 4 weeks in the abdominal muscle of SD rats. While 0.4 microgram BMP-4 showed no bone or cartilage formation, the amount of 40 micrograms BMP-4 showed new heterotopic cartilage formation, followed by endochondral ossification in almost all samples. The results prove that E. coli-expressed BMP-4 possesses the same inductive properties as mammalian-cell-expressed BMP-4.

Animals↗
PubMed1998

Inductive properties of recombinant human BMP-2 produced in a bacterial expression system.

Recombinant human BMP-2, produced in E. coli, refolded and concentrated to a purity of more than 98%, has been demonstrated to be biologically active. In vitro, amounts of 0.4 microg BMP-2 or more induced new cartilage formation in 27 out of 47 samples of a neonatal muscle tissue assay, with chondroneogenesis occurring 14 days after a four-hour contact between BMP-2 and the muscle tissue. In vivo, BMP-2 was implanted in the thigh muscle of ICR mice for a period of three weeks. Amounts of 4 microg BMP-2 and more showed heterotopic bone formation in 15 out of 17 samples. When BMP-2 was combined with a collagen carrier, amounts of 0.4 microg protein or more induced heterotopic bone formation in 30 out of 33 samples four weeks after the implantation in the abdominal wall of Sprague-Dawley rats. The results show that the E. coli-derived BMP-2 was active in different assay systems in concentrations equal to those required with mammalian cell-expressed BMP-2. It could also be demonstrated that a single morphogen (BMP-2) is enough to initiate the differentiation process associated with bone induction. The presented bacterial expression system also offers the opportunity to produce large quantities of recombinant BMP-2 for clinical applications.

Abdominal Muscles↗
PubMed1998

Radiation-induced impairment of bone healing can be overcome by recombinant human bone morphogenetic protein-2.

Radiotherapy of head and neck tumors very often results in impaired healing of craniomaxillofacial bones in the vicinity. Management of radionecrosis of bones after radiotherapy is an important clinical challenge. Bone morphogenetic proteins (BMPs) induce new bone differentiation. The aim of this study is to investigate the potential of BMPs in ameliorating radiation-induced impaired bone repair. Two 3-mm diameter defects were created in the calvaria of rats. The defects were treated with different doses of recombinant human (rh) BMP-2 using collagen type I as a carrier. Irradiation with a single dose of 1,200 rad was performed 2 or 7 days preoperatively. Unirradiated animals served as controls. New bone formation was assessed by quantitation of radiographs of the calvaria and histology on day 21 after surgery. Untreated, unirradiated defects showed a spontaneous osseous regeneration of 90 +/- 7% of the defect area within 21 days. Irradiation of the site (1,200 rad 2 days preoperatively) resulted in a profound decrease in the bone fill of the untreated defect (5 +/- 2%). Recombinant human BMP-2 in soluble collagen type I carrier delivered to the defect resulted in a significant increase of new bone formation (34 +/- 14%, P < 0.01 for 25 micrograms rhBMP-2; 77 +/- 19% for 35 micrograms rhBMP-2, P < 0.01). Type I collagen carrier alone resulted in only 7 +/- 2% healing. In conclusion, radiation-induced impairment of calvarial repair can be overcome by rhBMP-2. Thus, the concept of BMP-2-induced regeneration has potential applications in reconstructive craniomaxillofacial surgery after irradiation.

Animals↗
PubMed1997

A mixed-charge pair in human interleukin 4 dominates high-affinity interaction with the receptor alpha chain.

Human interleukin 4 (IL-4) binds to its cellular receptor with a Kd in the subnanomolar range, similar to many other 4-helix-bundle proteins interacting with members of the hematopoietin (cytokine) receptor superfamily. In the IL-4 system this interaction is predominantly determined by the extracellular domain (IL4-BP) of the receptor alpha chain (Kd approximately 150 pM). Now a high-resolution mutational and kinetic analysis has revealed that the high-affinity binding of IL-4 originates from a continuous patch of a few mostly polar or charged amino acid side chains located on helices A and C. The binding epitope comprises (i) a set of side chains determining the dissociation rate (k(off)) and (ii) a partially overlapping set determining the association rate constant (k(on)) of the IL-4/IL4-BP complex. The k(off) epitope is assembled from two juxtaposed main determinants (Glu-9 and Arg-88) surrounded by five side chains (Ile-5, Thr-13, Arg-53, Asn-89, and Trp-91) of lower importance. The cumulative increase in k(off) after alanine substitution is 10(5)-fold for the central mixed-charge pair and 3 x 10(3)-fold for the satellites. The k(on) epitope is formed by five positively charged residues on helix C (Lys-77, Arg-81, Lys-84, Arg-85, and Arg-88) and two neighboring residues on helix A (Glu-9 and Thr-13). The cumulative loss in k(on) of the alanine variants is only about 10-fold. These results provide the basis for an understanding of molecular recognition in cytokine receptor complexes and for an IL-4 antagonist design.

Alanine↗
PubMed1997

A murine interleukin-4 antagonistic mutant protein completely inhibits interleukin-4-induced cell proliferation, differentiation, and signal transduction.

We characterize here a highly efficient antagonist for interleukin-4 (IL-4) in the mouse system. In this double mutant of the murine IL-4 protein, both glutamine 116 and tyrosine 119 were substituted by aspartic acid residues. This variant (QY) bound with similar affinity to the IL-4 receptor alpha subunit as wild type IL-4 without inducing cellular responses. In contrast, QY completely inhibited in a dose-dependent manner the IL-4-induced proliferation of lipopolysaccharide-stimulated murine splenic B-cells, of the murine T cell line CTLL-2, and of the murine pre-B-cell line BA/F3. QY also inhibited the IL-4-stimulated up-regulation of CD23 expression by lipopolysaccharide-stimulated murine splenic B-cells and abolished tyrosine phosphorylation of the transcription factor Stat6 and the tyrosine kinase Jak3 in IL-4-stimulated BA/F3 cells. Selective inhibition of IL-4 may be beneficial in T-helper cell type 2-dominated diseases, like type I hypersensitivity reactions or helminthic infections. The QY mutant could be an attractive tool to study in vivo the therapeutic potential of IL-4 antagonists in mouse systems.

Animals↗
PubMed1997

IL-4 receptor complexes containing or lacking the gamma C chain are inhibited by an overlapping set of antagonistic IL-4 mutant proteins.

IL-4 signals on lymphocytes and other cells through a heterodimeric complex of two cytokine receptor proteins, IL-4R alpha and gamma C. IL-4 variants with mutations in the positions Tyr124, and (less efficiently) Arg121 and Ser125 are deficient in interaction with gamma zero and can be applied as IL-4 antagonists. Some cells respond to IL-4 without using gamma zero presumably by recruiting an alternative subunit into the receptor complex. We have investigated the effects of IL-4 mutant proteins on cells bearing this second type of IL-4 receptor complex. Mutations in the amino acid residues Arg 121 and Tyr124, but not Ser125, completely prevented cellular responses mediated by type 2 IL-4 receptors. Both receptor types are therefore affected by mutations in an overlapping but not identical site of the IL-4 molecule.

Antigens, CD↗
PubMed1997

Low-grade B cell lymphomas of mucosa-associated lymphoid tissue (MALT-type) require CD40-mediated signaling and Th2-type cytokines for in vitro growth and differentiation.

To investigate the mechanisms of T cell dependence underlying the development of extranodal mucosa-associated lymphoid tissue (MALT)-type B cell lymphomas, the activation, proliferation, and differentiation of lymphoma B cells were studied using ligand binding to the CD40 membrane receptor. The activation and proliferative response of all investigated low-grade MALT-type lymphomas (n = 6) was strongly dependent on anti-CD40-mediated signals and was complemented by cytokines produced by T helper cells of the Th2 type (interleukin-4 (IL-4) and/or IL-10). Th1 cytokines (IL-2 and/or interferon-gamma) bad little effect. Low-grade, but less so high-grade, MALT-type lymphoma B cells were induced to secrete large amounts of tumor immunoglobulin in response to IL-10. In contrast, high-grade MALT-type lymphomas (n = 5) proliferated in response to both Th2- and Th1-type cytokines and CD40 stimulation, whereas Burkitt lymphomas (n = 3) could not be rescued from apoptosis by CD40 stimulation with or without cytokines. These results suggest that CD40 signaling in combination with Th2 cytokines are essential for the development and progression of low-grade MALT-type B cell lymphoma. We conclude that T cells, which activate B cells in a CD40-dependent fashion, may contribute to lymphoma pathogenesis.

B-Lymphocyte Subsets↗
PubMed1996

Global and local determinants for the kinetics of interleukin-4/interleukin-4 receptor alpha chain interaction. A biosensor study employing recombinant interleukin-4-binding protein.

An engineered interleukin-4-binding protein (IL4-BP) representing the extracellular domain of the human interleukin-4 (IL-4) receptor alpha chain was expressed in Sf9 cells. The purified IL4-BP was immobilized via a single biotinylated SH group near the carboxyl end to a biosensor matrix and analysed in real time for interaction with IL-4 and IL-4 variants. IL-4 was bound to IL4-BP at a molar ratio of approximately 1:1. The association and dissociation at pH 7.4 and 150 mM NaCl had rate constants of 1.9 +/- 0.3 x 10(7) M-1 s-1 and 2 +/- 1 x 10(-3) s-1, respectively. Glycosylation and engineered amino acid substitutions of IL4-BP did not alter the kinetic constants as shown by a parallel analysis of IL4-BP variants produced in Escherichia coli or Chinese hamster ovary cells. The rate of association was only slightly affected in binding-deficient variants [E9Q]IL-4 and [R88Q]IL-4 and by acidic pH down to values of 4.5, but it was reduced up to fivefold at higher ionic strength. The rate of dissociation was increased 70-fold and 150-fold with the IL-4 variants and fivefold at an acidic pH of 4.5, but it was not affected by high ionic strength. Temperatures between 6 degrees C and 37 degrees C yielded similar rates of IL-4 dissociation and only a marginally reduced rate of IL-4 association at 6 degrees C. These results indicate that the high-affinity binding of IL-4 to its receptor (Kd approximately 100 pM) is mainly the result of an unusually high association rate. The IL-4/IL4-BP interaction appears to be dominated by charge effects. The exceedingly high rate of IL-4/IL4-BP association is augmented by the overall electrostatic potentials of both proteins (electrostatic steering). Localized charges and the formation of ion pairs may control the rate of complex dissociation.

Animals↗
PubMed1996

Interleukin-4 upregulates the heat shock protein Hsp90alpha and enhances transcription of a reporter gene coupled to a single heat shock element.

Transcription of the heat shock protein Hsp90alpha was strongly upregulated in human T-cells by interleukin-4 (IL-4) and to a lesser extent by IL-2, reaching peak levels after 2-3 days of stimulation. Heat shock proteins are induced within minutes under stress conditions, via heat shock factors (HSF), which activate heat shock elements (HSE). IL-4, IL-2 and IL-13 upregulated transcription of a reporter gene coupled to a single HSE site and a minimal promoter. HSE may therefore be involved in cytokine induced heat shock gene transcription in the absence of cellular stress.

Base Sequence↗
PubMed1996

Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity.

Bone morphogenetic protein 2 (BMP-2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development. A cDNA encoding mature human BMP-2 could be efficiently expressed in Escherichia coli, and after renaturation a dimeric BMP-2 protein of M(r) 26,000 was prepared with a purity greater 98%. The recombinant BMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell line (EC50 of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC50 of 15-20 nM). A peptide 1-17 representing the N-terminal basic part of BMP-2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay. These observations suggested that the N-terminai reduce the specific activity of BMP-2, probably by interacting with heparinic sites in the extracellular matrix. This conclusion was supported by a variant EHBMP-2, where the N-terminal residues 1-12 of BMP-2 had been substituted by a dummy sequence of equal length and which showed an EC50 value of around 1 nM which was affected neither by heparin nor by peptide 1-17. A physical interaction between BMP-2 and heparin could be seen in biosensor experiments, where BMP-2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin-binding of variant EHBMP-2 was negligible. These results identify the basic N-terminal domains of dimeric BMP-2 as heparin-binding sites that are not obligatory for receptor activation but modulate its biological activity.

Amino Acid Sequence↗
PubMed1996

A strict requirement of interleukin-4 for interleukin-4 induction in antigen-stimulated human memory T cells.

The role of interleukin-4 (IL-4) in the induction of IL-4 in mouse T cells is well established, but conflicting results have been reported with anti-CD3-primed human T cells and T cell clones. Therefore, IL-4 regulation was investigated in short-term cultured human T cells primed in vitro with either a superantigen or a hapten, nickel sulfate (NiSO4), for 3 days and expanded with IL-2 for another 5 days. Under these conditions, antigen-specific IL-4 producing T cells were generated in 35/40 cultures. Priming for IL-4 production was abrogated in all cultures by anti-IL-4 antibody or soluble IL-4 receptor (sIL-4R). Primed T cells that were IL-4- when cultured with IL-2 only developed an IL-4 producing phenotype when primed and expanded in the presence of exogenous IL-4. T cells primed in the presence of either endogenous or exogenous IL-4 produced 10-200-fold more IL-4 than T cells primed in the presence of anti-IL-4 antibody or sIL-4R. While IL-4 induction was absolutely dependent on IL-4, neither endogenous nor exogenous IL-4 influenced IFN-gamma synthesis. Most importantly, IL-4 induced and sIL-4R abolished priming for IL-4 production even in NiSO4-specific memory T cells from sensitized individuals. Thus, IL-4 induction in antigen-specific human memory T cell populations absolutely required IL-4. The IL-4 pathway of memory T cells retained a remarkable plasticity in sensitized individuals.

Adult↗
PubMed1995

Antagonistic mutant proteins of interleukin-4.

Interleukin-4 is a major regulator of the immune system, directing e.g. induction of a TH2 phenotype in T-cells, activation of B-cells and synthesis of IgE type antibodies, which are associated with allergic responses. Site-directed mutagenesis has revealed two sites important for receptor interaction on IL-4: site I mediates binding to the IL-4 receptor alpha subunit, and site II is involved in signal transduction through the receptor complex. Specific mutations in site II produced a series of ligands which bound to the receptor with high affinity, but had little or no agonistic activity and inhibited effects of wild type IL-4. The closely related cytokine IL-13, also a mediator of allergic processes, is antagonized as well. Antagonistic site II mutants of human IL-4 are therefore effective inhibitors with therapeutic potential for IL-4 associated diseases like type I hypersensitivity and asthma.

Amino Acid Sequence↗
PubMed1994

Design of human interleukin-4 antagonists inhibiting interleukin-4-dependent and interleukin-13-dependent responses in T-cells and B-cells with high efficiency.

Human interleukin-4 possesses two distinct sites for receptor activation. A signalling 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 alpha 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 both 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 cell line with a Ki value of approximately 100 pM. In addition, inhibition of both interleukin-4-induced and interleukin-13-induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin-4Rex, the extracellular domain of the interleukin-4 receptor alpha 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 alpha subunit in the interleukin-13 receptor system.

Antibodies, Monoclonal↗
PubMed1994

Neutralizing monoclonal antibodies define two different functional sites in human interleukin-4.

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.

Amino Acid Sequence↗
PubMed1994

Aspects of receptor binding and signalling of interleukin-4 investigated by site-directed mutagenesis and NMR spectroscopy.

Cytokines are hormones that carry information from cell to cell. This information is read from their surface upon binding to transmembrane receptors and by the subsequent initiation of receptor oligomerization. An influence on this process through mutagenesis on the hormone surface is highly desirable 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 IL-4 and the medically important IL-4 antagonists Y124D and Y124G are presented. The site around Y124 is an important epitope responsible for the ability of IL-4 to cause 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 labelled samples.

Amino Acid Sequence↗
PubMed1994

Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis.

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-alpha-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormone-receptor 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 lack 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.

Electrochemistry↗
PubMed1994

Interleukin 7 as interleukin 9 drives phytohemagglutinin-activated T cells through several cell cycles; no synergism between interleukin 7, interleukin 9 and interleukin 4.

The effects of the interleukins IL-7 and IL-9 on cell cycle progression were investigated by conventional [3H]thymidine incorporation and by the bivariate BrdU/Hoechst technique. Both IL-7 and IL-9 drive phytohemagglutinin-activated T cells through more than one cell cycle, but IL-7 was more potent 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 well, 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 suggest 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.

Cell Cycle↗