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H Baumann

Publications and source records attributed to H Baumann.

At least 91 records · Page 5Linked to original sources
PubMed1995

Interleukin-6-type cytokines and their receptors for gene therapy of melanoma.

B-78-H1 melanoma cells were stably transfected with cDNAs encoding human IL-6, human LIF, murine sIL-6R and murine sLIFR. The mock transfected and transfected cells demonstrated no detectable H-2Kb molecules. B-78 transfected cells were subcutaneously (s.c.) and intravenously (i.v.) injected to B57BL/6 x C3H mice. Control B-78 cells formed tumors and lung metastases in injected animals. Cells transfected with IL-6, LIF and sIL-6R showed greatly reduced tumor and metastases formation. Transfection of IL-6, sIL-6R or LIF had similar protective effects while the combination of IL-6 and sIL-6R was most effective. In contrast, cells transfected with sLIFR showed reduced metastasis formation but increased tumor growth compared to mock transfected cells. Kinetic analysis demonstrated a 3 weeks lag period between the formation of tumors by B-78 cells and the combination of B-78 cells transfected with IL-6 and sIL-6R. No such lag phase was seen when B-78-IL-6 or B-78-sIL-6R cells were injected alone. Mice primarily injected s.c. with a mixture of IL-6 and sIL-6R transfected cells and rechallenged after 2 weeks with parental B-78 cells demonstrated long-lasting antitumor immunity. IL-6 and sIL-6 transfected cells used alone for immunization had only limited effect. Injection of transfected cells into SCID mice which are characterized by greatly reduced number of T and B cells, showed a protective effect of sIL-6R on metastasis formation by B-78 cells. beta 2m knockout mice lacking CD8+ T cells, injected with B-78 cells developed tumors and died after 2 weeks. However, B-78 cells transfected with IL-6 developed tumors in only 50% of animals. Mice without tumors rechallenged with B-78 cells demonstrated required immunity against parental melanoma cells. The results obtained indicate that studied IL-6-type cytokines and their respective soluble receptors affect murine melanoma growth and metastasis formation. The major finding of these studies is that IL-6 complexed with sIL-6R demonstrated qualitatively different biological activity than IL-6 alone especially in stimulating long lasting anti-melanoma immunity. The proposed mechanism of action of such complexes beside activation of cytotoxic T lymphocytes is activation of NK cells.

Animals↗
PubMed1995

Separate signaling mechanisms are involved in the control of STAT protein activation and gene regulation via the interleukin 6 response element by the box 3 motif of gp130.

The cytoplasmic receptor sequences required for the transcriptional control via the IL-6 response element (IL-6RE) and the hematopoietin receptor response element (HRRE) in hepatoma cells were defined by transient expression of wild-type and mutant granulocyte-colony stimulating factor receptor-gp130 chimeric receptors. gp130 generated two separate transcriptional signals, one of which was directed to IL-6RE and required an intact box 3 motif, and another, which was directed to HRRE and was box 3-independent. The activation of DNA-binding of STAT3 required the same gp130 domains as the IL-6RE response. A box 3-independent activation of STAT proteins was achieved by overexpression of the kinases JAK2 or TYK2. The increase in the DNA-binding activity of STAT proteins, however, did not result in a corresponding increase in transcription via either IL-6RE or HRRE. The data indicate that activation of the DNA-binding potential of STAT proteins via gp130 is not sufficient to achieve transcriptional up-regulation of specific target genes.

Animals↗
PubMed1995

Structural characterization of a short peptide fragment that mediates estrogen-receptor dimerization.

In a recent study it is suggested that a short peptide fragment within the estrogen receptor ligand-binding and dimerization domain might act as a constitutively active dimerization motif [Lees, J. A., Fawell, S. E., White, R. & Parker, M. G. (1990) Mol. Cell. Biol. 10, 5529-5531]. We used NMR and CD spectroscopies to characterize the structure and biophysical properties of a synthetic peptide comprising residues Thr500-His528 of the mouse estrogen receptor, including the putative dimerization motif. We found that residues Leu501-Asn523 form a nascent helix in water solution, whereas the C-terminal (Lys524-His528) has no propensity for alpha-helical conformation. We found no evidence for a strong homodimerization activity of the peptide. However, we observed concentration-dependent NMR chemical shifts of several residues that would be located on the same face of an alpha-helix. This observation suggests a weak, but specific dimerization/oligomerization of the peptide at millimolar concentrations.

Amino Acid Sequence↗
PubMed1995

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

3T3 Cells↗
PubMed1995

DNA-binding surface of the Sso7d protein from Sulfolobus solfataricus.

We have used nuclear magnetic resonance (n.m.r.) spectroscopy to identify the DNA-binding surface of the abundant, small and basic protein Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus. The Sso7d protein was previously found to bind strongly to double-stranded DNA sequences and to protect DNA from thermal denaturation, indicating that it might assume a similar function in vivo. Several amide resonances in two-dimensional n.m.r. 1H, 15N correlation spectra of 15N-enriched Sso7d are shifted and broadened upon addition of small amounts of ten base-pair or 19 base-pair duplex DNA oligomers under conditions where Sso7d-DNA complexes exchange rapidly on the n.m.r. time scale. The locations of the corresponding amides in the Sso7d structure define the surface that interacts with DNA. This surface coincides with a continuous region of strong positive electrostatic potential, which was calculated by means of numerical solution of the Poisson-Boltzmann equation. A model of the non-specific Sso7d-DNA complex is suggested based on the present data and previously obtained evidence that Sso7d interacts with the DNA major groove. The protein-DNA interface consists of a triple-stranded beta-sheet, which interacts with the DNA major groove and a reverse turn connecting the two strands of a double-stranded beta-sheet, which interacts with the minor groove. We note that the five (of 14) lysine side-chains that are specifically subjected to N zeta-monomethylation in the cell are located on surfaces of Sso7d that are exposed to the solvent in the proposed Sso7d-DNA complex.

Archaeal Proteins↗
PubMed1995

Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor.

A 58-amino acid polypeptide containing the functional core region, the tau 1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the tau 1 core peptide fragment. Circular dichroism spectroscopy shows that the tau 1 core and a larger fragment encompassing the intact tau 1 domain are largely unstructured in water solution under a variety of pH conditions. The tau 1 core, however, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for alpha-helical conformation. Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character. Potentially helix-breaking proline substitutions, in proposed alpha-helical regions, lead to reduced activity, suggesting that alpha-helices are important for transactivation in vivo.

Amino Acid Sequence↗
PubMed1995

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor gamma chain is required for IL-7 signal transduction.

The interleukin (IL)-2 receptor gamma chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor gamma chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.

Amino Acid Sequence↗
PubMed1995

Combined NRA, channeling-RBS and FTIR ellipsometry analyses for the determination of the interface and bonding state of thin SiO(x) and SiN(x)O(y) layers.

The molecular ions O(+)(2) and NO(+) are im- planted at room temperature into single-crystal silicon with an energy of E=6 keV/atom at fluences ranging from 2.5x10(16) to 3.5x10(17) at/cm(2). The samples are processed by electron beam rapid thermal annealing at 1100 ( degrees )C for 15 s. The depth distributions of the implanted specimens ((18)O) are determined by nuclear reaction analyses using the reaction (18)O(p,alpha)(15)N. Channeling-RBS measurements are performed to obtain the interface structure between the implanted layer and the single-crystal Si substrate. The chemical bonding state of as-implanted and implanted-annealed specimens is observed by FTIR ellipsometry measurements.

Journal Article↗
PubMed1995

A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates.

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.

Carbon Isotopes↗
PubMed1994

Signaling by the cytoplasmic domain of hematopoietin receptors involves two distinguishable mechanisms in hepatic cells.

The receptor for granulocyte colony stimulating factor (G-CSFR) and chimeric receptors consisting of the extracellular domain of G-CSFR and the transmembrane and cytoplasmic domain of the leukemia inhibitory factor receptor, gp130, or c-mpl function as homodimeric complexes. These receptors mediate a similar stimulation of gene transcription via separate regulatory elements of acute phase plasma protein genes. To identify the receptor regions within the cytoplasmic domains necessary for transcriptional regulation, the receptors were transiently expressed in rat hepatoma cells. Each receptor form reconstituted G-CSF-induced expression of a chloramphenicol acetyltransferase gene construct containing the cytokine response element of the rat alpha 1-acid glycoprotein gene. This regulation required the presence of two conserved sequence motifs (referred to as box 1 and box 2) in the cytoplasmic domains of each receptor. With the exception of G-CSFR-MPL chimera, the receptors also mediated a similarly high stimulation via the IL-6 response element of the rat beta-fibrinogen and hemopexin genes. Regulation of the IL-6 response element required, however, in addition to boxes 1 and 2, a third sequence motif (box 3). This motif is absent in the cytoplasmic domain of c-mpl, possibly explaining its inability to activate the IL-6 response element. When cells which express receptor forms with prominent box 3 function were treated with suramin, a ligand-independent gene stimulation via the IL-6 response element was observed. The suramin effect probably involves a receptor dimerization mediated by the extracellular G-CSFR domain and by the intracellular regions that include box 3.

Acute-Phase Proteins↗
PubMed1994

Solution structure of a trisaccharide-antibody complex: comparison of NMR measurements with a crystal structure.

NMR and crystallography have been used to study antigen conformational changes that occur in a trisaccharide-Fab complex in solution and in the solid state. NOE buildup rates from transferred NOE experiments show that the antigenic determinant of a Salmonella lipopolysaccharide, represented by the trisaccharide methyl glycoside alpha-D-Galp(1-->2 [alpha-D-Abep(1-->3)]- alpha-D-Manp1-->OMe (1), undergoes a protein-induced conformational shift about the Gal-->Man glycosidic linkage when it is bound by a monoclonal antibody in aqueous solution. The same trisaccharide was crystallized with Fab, and a solved structure at 2.1-A resolution revealed that the conformation of the trisaccharide ligand was similar to that seen in a dodesaccharide-Fab complex [Cygler et al. (1991) Science 253, 442-445), where the Gal-Man linkage also experienced a similar conformational shift. Distance constraints derived from the TRNOE buildup curves are consistent with two bound trisaccharide conformations, one of which correlates with the ligand conformation of the crystalline Fab-trisaccharide complex. In this bound conformation, short interatomic distances between Abe O-2 and Gal O-2 permit an oligosaccharide intramolecular hydrogen bond. Despite its relatively low energy, a preponderance of this conformer could not be detected in aqueous or DMSO solutions of free trisaccharide by either 1H or 13C NMR experiments. In DMSO, a different intramolecular hydrogen bond between Abe O-2 and Man O-4 was observed due to a solvent-induced shift in the conformational equilibria (relative to aqueous solution). Molecular modeling of the trisaccharide in the binding site and as the free ligand suggested that the protein imposes an induced fit on the antigen, primarily resulting in a shift of the Gal-Man phi torsional angle. This reduces the interproton separation between Abe H-3 and Gal H-1 with a marked increase in the intensity of the previously weak NOEs between the protons of the noncovalently linked galactose and abequose residues. The impact of the conformational shift on gross trisaccharide topology is sufficiently small that binding modes inferred from functional group replacements are not impaired.

Antibodies, Monoclonal↗
PubMed1994

The rat and human hemopexin genes contain an identical interleukin-6 response element that is not a target of CAAT enhancer-binding protein isoforms.

Hemopexin (Hx) is an abundant acute-phase protein (APP) that binds heme with high affinity. In rat hepatic cells, the transcription rate of the Hx gene is increased by interleukin (IL)-1 and IL-6. To investigate the cis-acting regulatory elements (REs) responsive to these hormones, chloramphenicol acetyltransferase constructs of rat and human Hx gene sequences were tested in transiently transfected hepatoma cells. An IL-6-RE was identified in the promoter of both rat and human Hx genes, the function of which was dependent on the core sequence (CCGGGAA) common in other APP genes. The previously characterized Hx A element mediated a relatively minor cytokine response as compared with the Hx IL-6-RE. The human Hx A element, in contrast to the rat and human Hx IL-6-REs, was strongly trans-activated by cotransfected CAAT enhancer-binding proteins (C/EBP)-beta and -delta. The rat gene homolog of the human Hx A element was inactive as a cytokine RE and was minimally trans-activated by C/EBP isoforms. Results of electrophoretic mobility shift assays indicated that the Hx IL-6-RE is a binding site for the IL-6-inducible nuclear protein IL-6 RE-BP, which also binds to the conserved IL-6-REs of other APP genes and is distinct from C/EBP beta.

Animals↗
PubMed1994

Impaired immune and acute-phase responses in interleukin-6-deficient mice.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates various aspects of the immune response, acute-phase reaction and haematopoiesis (for reviews see refs 1, 2). In vitro, leukaemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and interleukin-11 display overlapping activities with IL-6. This functional redundancy may be explained by the interactions of specific binding receptors with a common signal-transducing receptor (gp130) (for reviews see refs 3, 4). To elucidate the unique function of IL-6 in vivo, we have disrupted the IL-6 gene by homologous recombination. IL-6-deficient mice develop normally. They fail to control efficiently vaccinia virus and infection with Listeria monocytogenes, a facultative intracellular bacterium. The T-cell-dependent antibody response against vesicular stomatitis virus is impaired. Further, the inflammatory acute-phase response after tissue damage or infection is severely compromised, whereas it is only moderately affected after challenge with lipopolysaccharide. We conclude that IL-6 production induced by injury or infection is an important in vivo SOS signal which coordinates activities of liver cells, macrophages and lymphocytes.

Acute-Phase Reaction↗
PubMed1994

Species-specific changes in regulatory elements of mouse haptoglobin genes.

Although expression of the haptoglobin (HP) as an acute phase reactant is evolutionarily conserved among mammals, there are differences among species with regard to the hormones required for stimulation. Using primary hepatocyte cultures, we show that in Mus caroli, as in rat, IL-1 and IL-6 are stimulatory, whereas in M. domesticus, as in humans, IL-1 response is diminished. In vivo, an acute inflammatory process increases hepatic HP expression in both mouse species up to 30-fold but minimally affects the low level HP expression in the lung. To define the species-specific differences in regulation, we isolated the hormone-responsive elements of the HP gene from the Mus species, M. domesticus, M. caroli, and M. saxicola. Functional studies in transfected hepatoma cells revealed an exceptionally strong dexamethasone response for all three murine HP gene elements. The IL-6 response was less prominent than in rat or human. A modest response to IL-1 was observed in M. caroli and M. saxicola. A mouse-specific insertion of a polypurine sequence led to a binding site for the PEA3 transcription factor in the HP gene promoter of M. domesticus and M. saxicola, but not M. caroli. The specific regulatory effects of glucocorticoid receptor, C/EBP beta, and Ets proteins were documented by co-transfection.

Animals↗
PubMed1994

Evolution of murine alpha 1-proteinase inhibitors: gene amplification and reactive center divergence.

The organization and sequence of genes encoding the alpha 1-proteinase inhibitor (alpha 1PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of alpha 1PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian alpha 1PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, alpha 1PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the alpha 1PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of alpha 1PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.

Amino Acid Sequence↗