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E J Schlaeger

Publications and source records attributed to E J Schlaeger.

At least 19 recordsLinked to original sources
PubMed2000

Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections.

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S:)-4-Carboxyphenylglycine, (S:)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [(3)H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM: [(3)H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 microM: kainate, revealing the distribution of mGlu1+5 receptors. The latter were almost completely inhibited by the group I agonist (S:)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively.

Animals↗
PubMed1997

Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin.

A human prothrombin cDNA has been engineered to obtain a cDNA coding for a secreted form of human prethrombin-2. The secreted prethrombin-2 has been produced in a mammalian expression system using DXB11 cells, a mutant strain of CHO cells in which the dihydrofolate reductase gene has been deleted, and an expression vector carrying the dihydrofolate reductase cDNA. Methotrexate-induced gene amplification favored an efficient production of the recombinant protein which accumulated in the culture medium of the DXB11 cells. Growth in suspension of the stable transformants in an airlift fermenter resulted in the production of 25 mg/L recombinant prethrombin-2. The recombinant protein was purified using single-step affinity chromatography on a recombinant-hirudin column and activated by agarose gel-immobilized ecarin. All purified recombinant prethrombin-2 was activated and the generated recombinant thrombin showed catalytic properties identical to those of plasma-derived alpha-thrombin. This expression system can be used to prepare mutants of prethrombin-2 for structure-function studies investigating thrombin interactions with substrate proteins, inhibitors, and cell membranes.

Animals↗
PubMed1996

The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties.

The tryptic meat digest Primatone RL is a low-cost medium supplement of a complex nature which serves as a source of amino acids, oligopeptides, iron salts, some lipids and other trace low molecular weight substances. Its addition to mammalian and insect cell culture media significantly improves the cell growth properties of many cell lines. In this work the growth promoting effects of Primatone RL are described in more detail using different mouse hybridomas, a mouse myeloma cell line, and human promyelocytic leukemia HL-60 cells. The positive effects on cell growth induced by Primatone were observed in the presence of serum but were even more pronounced in serum-free culture. In addition the adaptation time from high serum to low (1%) or serum-free growth in the presence of Primatone is also significantly reduced. Primatone RL, when added to HL and DHI medium, improves cell growth under low serum or serum-free conditions by increasing the maximum cell numbers and in particular the viability of the culture. The observed decrease in cell death (apoptosis) induction leads to a significant improvement in antibody (recombinant protein) production by increasing the volumetric yields during long-term batch culture. The so-called anti-apoptotic effects of Primatone RL for mouse hybridomas, which is concentration dependent, is not fully understood.

Animals↗
PubMed1995

Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ib alpha.

A fragment (residues His1-Val289) of the alpha chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation. The recombinant GP Ib alpha fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 micrograms of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib alpha fragment.

Animals↗
PubMed1994

Isolation of a heregulin-like growth factor secreted by estrogen receptor-negative MDA-MB-231 human breast cancer cells that stimulates estrogen receptor-positive cells.

A 52 kDa peptide growth factor secreted by the estrogen receptor (ER)-negative human breast cancer cell line MDA-MB-231 was purified to homogeneity. It induced transient membrane ruffling, lamellipodia formation, cell motility and proliferation exclusively of ER-positive human breast cancer cells. Partial sequencing revealed a high homology to the protein family of heregulins. However, the obtained amino acid sequences of the new factor were not completely identical to any of the members of the heregulin family. This finding together with the observation that the successful purification protocol was significantly different from that used to isolate other members of the heregulin family indicate the isolation of a novel heregulin-like proliferation factor for ER-positive human breast cancer cells.

Amino Acid Sequence↗
PubMed1994

The integrin alpha IIb-beta 3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit.

Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469). Sf9 insect cells were infected with the corresponding baculovirus, and the produced soluble recombinant proteins were purified using an RGD-like affinity column. The bound receptor fragments were specifically eluted with RGDS and existed as a heterodimeric complex (rec alpha IIbH-t beta 3) with an apparent M(r) of 160,000. In an immunocapture assay, the monoclonal antibody pl-55, which only recognizes the functionally active form of alpha IIb-beta 3, bound to the purified complex. Rec alpha IIbH-t beta 3 specifically bound 125I-fibrinogen with an affinity comparable with that of purified platelet alpha IIb-beta 3. Electron micrographs of rotary-shadowed rec alpha IIbH-t beta 3 showed that the complex had the characteristic globular head, but the two rodlike tails were 4-6 nm shorter than those found in intact alpha IIb-beta 3. Thus, the cysteine-rich repeats of beta 3 are not required for assembly, stability, and functional activity of this integrin.

Amino Acid Sequence↗
PubMed1993

Expression, purification and crystallization of fully active, glycosylated human interleukin-5.

Recombinant human interleukin-5 (hIL-5) has been expressed at high levels and produced in large quantities in baculovirus infected Sf9 insect cells. The glycosylated protein was purified using immuno-affinity chromatography and gel filtration. Purified hIL-5 has been crystallized using standard vapour diffusion techniques with PEG as a coprecipitant. The crystals belong to the C2 space group and diffract to 2 A.

Animals↗
PubMed1992

Propagation of a mouse myeloma cell line J558L producing human CD4 immunoglobulin G1.

Transfected mouse myeloma cells are of increasing interest for the production of a wide variety of solubilised recombinant fusion proteins. A stably transfected J558L mouse myeloma subclone (J558L-CD4) secreting human CD4-immunoglobulin type G1 receptor (CD4-H gamma 1) was employed as a model system for cell suspension culture and expression of chimaeric molecules. Cells were grown up to 3-5 x 10(6) cells/ml in serum-free and protein-reduced DHI medium consisting of a mixture of DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL and Pluronic F68. Primatone RL was the essential growth-promoting factor in protein-free medium. The soluble CD4-H gamma 1 receptor, the production of which was not growth-associated, accumulated in the medium to concentrations of 40 micrograms/ml with a specific formation rate of 0.18 micrograms/10(6) cells/h in conventional cultures. The cell density was further increased by growing the cells in dialysis tubing or by using a perfusion system with cell retention. Because of the continuous exchange of nutrients and metabolic end-products average concentrations of 35 x 10(6) cells/ml were achieved. CD4-H gamma 1 accumulated in the dialysis tubing up to 1.3 mg/ml. After an initial rapid growth period, a ten-fold reduction in specific nutrient consumption rates and metabolic end-product formation was observed. Chimaeric proteins purified by protein G chromatography from conventional and perfusion cultures were indistinguishable when compared by SDS-PAGE, limited proteolysis and isoelectric focusing analysis (isoelectric point: 8.5-8.6).

Amino Acids↗
PubMed1992

Growth study of lactate and ammonia double-resistant clones of HL-60 cells.

The possibility that lactate and ammonia accumulation may have less detrimental effect on cell growth than usually admitted is investigated. We report here the isolation of several HL-60 subclones able to proliferate in the presence of 60 mM sodium lactate and 4 mM ammonium chloride, concentrations usually considered to be toxic for cell proliferation. Growth kinetics and final cell densities of these clones in suspension cultures were similar to the HL-60 cell population in control medium as well as in medium containing ammonia and lactate in which control cells were unable to grow. The metabolic pattern of the double-resistant clones revealed that lactate and ammonia formation was inhibited in the presence of lactate and ammonia in the medium, while alanine production and arginine consumption were enhanced irrespective of the medium.

Ammonium Chloride↗
PubMed1991

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.

Animals↗
PubMed1991

Purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells.

The extracellular domain of the mouse interferon gamma receptor comprising amino acids 17-243 of the protein was produced in Spodoptera frugiperda cells infected with a recombinant baculovirus. The receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. The purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%. From each purification run, two pools of soluble receptor with the same interferon gamma binding capacity were isolated. Under reducing electrophoretic conditions the protein of pool I migrates as two bands of molecular masses 32 and 34 kDa and of pool II as two bands of 30 and 32 kDa. The soluble receptor of both pools carries a heterogeneous glycosylation. After deglycosylation it appears as one protein band of 27 kDa. N-linked carbohydrates contribute about 6 kDa and O-linked carbohydrates 1 kDa to its molecular mass. The nonreduced protein specifically binds interferon gamma on ligand blots and in a solid-phase binding system and competes for the binding of radiolabeled interferon gamma to the cell surface receptor. The soluble mouse interferon gamma receptor exists as a monomer in physiological buffer and binds interferon gamma in its dimeric form. It is stable at room temperature and against tryptic digestion, but is very sensitive to proteinase K digestion. The soluble mouse interferon gamma receptor produced in the insect/baculovirus expression system may prove useful to study the function of interferon gamma receptor as an antagonist of endogenous interferon gamma in the treatment of immunological and inflammatory disorders.

Amino Acids↗
PubMed1991

Cloning of the DNA-binding subunit of human nuclear factor kappa B: the level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor alpha.

The DNA binding subunit of nuclear factor kappa B (NF-kappa B), a B-cell protein that interacts with the immunoglobulin kappa light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor alpha (TNF alpha), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-kappa B protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-kappa B binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNF alpha or phorbol ester. Thus, both factors not only activate NF-kappa B protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-kappa B.

Amino Acid Sequence↗
PubMed1991

Recombinant soluble tumor necrosis factor receptor proteins protect mice from lipopolysaccharide-induced lethality.

The in vivo efficacy of human recombinant soluble tumor necrosis factor (TNF) receptor protein to prevent and to treat lipopolysaccharide (LPS)-induced lethal toxicity in D-galactosamine-treated mice was investigated. Chimeric proteins of the receptor extracellular domains fused to the hinge region of human IgG3 were expressed in myeloma cells (rsTNFR-h gamma 3). The fusion proteins had a disulfide-bonded dimeric structure. Upon intravenous injection, their serum concentration decreased relatively slowly after an initial phase of rapid elimination. D-galactosamine-sensitized mice were fully protected from the toxic effects of LPS, if the animal were pretreated with rsTNFR-h gamma 3 at 20 micrograms/animal. Partial protection was seen at significantly lower doses and when rsTNFR-h gamma 3 was given up to 3 h after LPS.

Animals↗
PubMed1990

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells.

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.

Amino Acid Sequence↗
PubMed1990

Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies.

The pleiotropic cyto/lymphokine tumor necrosis factor (TNF) exerts its functions by binding to specific cell-surface receptors. We have prepared two sets of monoclonal antibodies (mAbs) against TNF-binding proteins from the HL-60 (htr-mAb series) and U-937 (utr-mAb series) cell lines. The htr antibodies inhibit the binding of 125I-labeled TNF-alpha to HL-60 cells only partially, whereas they block the TNF-alpha binding to several adenocarcinoma cell lines (HEp-2, HeLa, and MCF7) almost completely. In contrast, the utr antibodies have no effect on TNF-alpha binding to the adenocarcinoma cell lines but partially inhibit TNF-alpha binding to HL-60 and U-937 cells. However, htr-9 and utr-1 antibodies in combination fully inhibit the TNF-alpha binding to HL-60 and U-937 cells. The binding of TNF-beta to HEp-2 and U-937 cells is also inhibited by htr and utr antibodies. Neither htr nor utr mAb has an effect on the TNF-sensitive murine cell lines L929 and WEHI 164. Flow cytometry studies show that mAbs htr-9 and utr-1 detect two distinct TNF-binding sites on human cell lines. Immunologic blot and immunoprecipitation analyses indicate that mAbs htr-9 and utr-1 recognize proteins of approximately 55 kDa and 75 kDa, respectively. These data provide evidence for the existence of two distinct TNF receptor molecules that contribute to varying extent to the TNF binding by different human cells.

Antibodies, Monoclonal↗
PubMed1990

Optimization of culture conditions for high cell density proliferation of HL-60 human promyelocytic leukemia cells.

The purpose of the present investigation was to optimize the culture conditions in suspension of the HL-60 cell line for high-density production. The optimized HL medium was a mixture of RPMI-1640, DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL, Pluronic F68, ethanolamine and selenite. Under these conditions, whether serum was added or not, cells grew to up to 8 x 10(6) cells ml-1, which was at least three times higher than the maximum cell density usually described. Glucose and four amino acids: cystine, glutamine, methionine and serine, were highly consumed and disappeared quickly from the medium. Nutrient supply and metabolic end-product accumulation were the most probable growth-limiting factors. Different propagation systems were used to increase the cell density further. Cells were grown in dialysis tubing where relatively high levels of nutrients and low levels of waste products were maintained, leading to cell densities of 60 x 10(6) to 70 x 10(6) cells ml-1. A perfusion-culturing method with cell retention was found to be most effective for high-density production of HL-60 cells at a 21 as well as a 60 1 fermentor scale. Average concentrations of 40 x 10(6) to 50 x 10(6) cells ml-1 were achieved. Expression and distribution of both tumor necrosis factor receptors (55 and 75 x 10(3) Mr) on the surface of HL-60 cells were analysed as a control of the physiological integrity of the cells during the perfusion course; 1.3 micrograms of tumor necrosis factor receptors/10(10) cells was regularly expressed on the surface of HL-60 cells.

Ammonia↗
PubMed1987

Proteolytic activity in the culture supernatants of mouse hybridoma cells.

When serum-reduced or serum-free culture supernatants were incubated for 16 hours at 37 degrees C, more than 70-80% of original proteins were digested as measured by gel electrophoretic analysis. The proteolytic activity, which was only observed at pH values lower than 4.5, was reduced in conditioned medium containing higher concentrations of fetal calf serum. During incubation large amounts of the monoclonal antibody (IgG1) were cleaved giving F(ab')2 fragments. The results reported here strongly suggest that the conditioned medium of mouse hybridoma cultures contain probably two cellular (acid) proteases with apparent MW of 45-50 K and 90-95 K, respectively. The similarities with the lysosomal cathepsin D are discussed.

Animals↗