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A J Sytkowski

Publications and source records attributed to A J Sytkowski.

At least 55 records · Page 3Linked to original sources
PubMed1990

Characterization of biologically active, platelet-derived growth factor-like molecules produced by murine erythroid cells in vitro and in vivo.

Platelet-derived growth factor (PDGF) is an important serum regulator of erythropoiesis in vitro. We have now obtained evidence suggesting that PDGF-like molecules may also modulate erythropoiesis in vivo. Western blot analysis of cytoplasmic extracts from Rauscher murine erythroleukemia cells and phenylhydrazine-treated mouse splenic erythroid cells revealed the presence of several PDGF-like proteins. The presence of PDGF-like proteins in the cytoplasm of these two erythroid cell types was confirmed by immunohistochemical staining. Using a serum-free biologic assay, PDGF-like biological activity was found in cell lysates and conditioned medium of both Rauscher cells and phenylhydrazine-treated mouse erythroid cells. Subcellular localization experiments revealed the biological activity to be concentrated in the cytosolic fraction. Using a series of antibodies to hematopoietic growth factors we demonstrated that PDGF-like biological activity was specifically immunoprecipitated by both monoclonal and polyclonal anti-human PDGF antibodies but not by antibodies to burst-promoting activity, granulocyte-macrophage colony-stimulating factor, IL-3, or erythropoietin. Taken together, the data are consistent with the hypothesis that PDGF-like molecules play a role in the regulation of mammalian erythropoiesis in vivo.

Animals↗
PubMed1987

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide.

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.

1-Deoxynojirimycin↗
PubMed1987

Site-specific antibodies to human erythropoietin: immunoaffinity purification of urinary and recombinant hormone.

The 19-amino acid domain Ala111----Pro129 of human erythropoietin was identified as an accessible surface antigen based on the binding of radio-iodinated and of unmodified hormone to antibodies prepared against a synthetic peptide of homologous sequence. The specificity and affinity of this binding was sufficient to provide for the use of anti-peptide antibodies in the preparation of an immunosorbent for the purification of urinary, and of recombinant human erythropoietin. Immobilization of anti-peptide antibodies using agarose activated either with CNBr or with N-hydroxysuccinimido groups largely inactivated binding sites for erythropoietin. In contrast, antibodies crosslinked to N-acetyl-DL-homocysteine agarose through the hetero-bifunctional reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate retained their antigen-binding capacity virtually completely and provided a superior immunosorbent for hormone. Urinary erythropoietin with a specific bioactivity of 100 U/A280 was prepared initially by chromatography on phenyl-Sepharose. Subsequent immunoaffinity chromatography resulted in a 350-fold purification with 46.2% recovery yielding erythropoietin with a specific bioactivity of 35,200 U/A280 (44,300 U/mg). Radioiodination of this purified protein and subsequent SDS-polyacrylamide gel electrophoresis indicated that this preparation contained a single major component (Mr 30,000) which co-migrated in gels with unmodified biologically active hormone. Recombinant erythropoietin, which was prepared by the cloning of the human erythropoietin gene and its expression in COS cells using the SV40-derived vector pSV2, was purified by the same scheme. Chromatography on phenyl-Sepharose of medium derived from transfected cells (400 U/ml, 170 U/A280) provided for a 3.6-fold purification of recombinant hormone with an apparent recovery of 122%. This erythropoietin bound to the anti-peptide antibody gel and was purified to a specific bioactivity of 10,370 U/A280 with 55% recovery. The procedure described here for attaching antibodies to a solid support maximizes their antigen-binding capacity and is generally applicable. The development of an anti-peptide immunosorbant for human erythropoietin provides a valuable means for isolating hormone for use in studies of its receptor and its presently unresolved mechanism of action.

Amino Acid Sequence↗
PubMed1987

Erythropoietin rapidly alters phosphorylation of pp43, an erythroid membrane protein.

The phosphorylation of a prominent 43-kDa phosphoprotein (pp43) in the membranes of normal murine erythroid cells was reduced markedly by exposure of the membranes to highly purified erythropoietin. A virtually identical reduction of pp43 phosphorylation was seen when erythropoietin-responsive Rauscher murine erythroleukemia cell membranes were exposed to the hormone. This effect was both time-dependent, occurring within 30 min after erythropoietin exposure, and concentration-dependent. Phosphoamino acid analysis revealed that pp43 is phosphorylated on serine residues. The results provide the first evidence that rapid alterations in membrane protein phosphorylation may serve as a trans-membrane signal for erythropoietin.

Adenosine Triphosphate↗
PubMed1987

Immunochemical studies of human erythropoietin using site-specific anti-peptide antibodies. Identification of a functional domain.

Anti-peptide antibodies that bind to the amino terminus of human erythropoietin (residues 1-26) do not inhibit the hormone's biological activity, indicating that this region of the protein does not play a role in receptor recognition (Sytkowski, A. J., and Fisher, J. W. (1985) J. Biol Chem. 260, 14727-14731). We have now identified six other regions of the primary sequence that are relatively hydrophilic and, therefore, have a higher probability of being accessible to such antibody probes. Antibodies raised against synthetic peptides homologous to five of these regions, corresponding to residues 40-59, 80-99, 99-118, 111-129, and 131-150 recognize erythropoietin, confirming the prediction based upon relative hydrophilicity. Antibodies to a carboxyl terminal peptide 147-166 failed to bind the hormone, presumably due to steric hindrance imposed by a disulfide bond between Cys161 and one of the other cysteinyl residues. The antibodies were affinity purified on the relevant immobilized peptide and their capacity to inhibit (neutralize) erythropoietin's activity was assessed. Only anti-peptide 99-118 and anti-peptide 111-129 antibodies inhibited erythropoietin. This effect was reversed by excess peptide, demonstrating that the neutralizing action of the antibody was due to its antigen-specific binding. The results strongly suggest that the portion of erythropoietin's amino acid sequence represented by these peptides plays a functional role in the hormone's action, most probably by forming part of the receptor-binding domain.

Amino Acid Sequence↗
PubMed1987

Identification and characterization of a differentiation-specific antigen on normal and malignant murine erythroid cells.

Rauscher murine erythroleukemia cells grow continuously in vitro and undergo terminal differentiation in response to the physiological inducer erythropoietin. In the course of this developmental process they express many erythroid-specific markers. In order to investigate the expression of cell surface determinants during Rauscher cell differentiation we generated monoclonal antibodies to uninduced cells. Using an anti-Rauscher cell monoclonal antibody, we have identified a cell surface determinant, designated ERY-1, that is present on normal murine erythroid cells. This determinant is apparently absent from the early progenitor BFU-E, but is present on the more mature progenitors CFU-E and CFC-E. It disappears during erythroid maturation and is absent from the mature erythrocyte. This pattern of ERY-1 expression is exhibited with remarkable fidelity during the erythropoietin-induced differentiation of Rauscher cells. Such differentiation-specific expression of the ERY-1 determinant suggests that it may play a functional role in erythropoiesis.

Animals↗
PubMed1986

Hybridoma production by simplified avidin-mediated electrofusion.

A simplified method for producing cell hybrids by avidin-mediated electrofusion has been developed. First, biotin was attached both to immunogen and to the surface of murine myeloma cells using N-hydroxysuccinimidobiotin. Biotinylated immunogen was incubated with splenocytes derived from immunized mice and allowed to bind to surface immunoglobulins of B cells. Using streptavidin, biotinylated myeloma cells then were bridged to those B cells bearing biotinylated immunogen. Selective fusion of bridged cells was accomplished by their limited exposure to high-voltage potentials. A high frequency of hybridomas was obtained all of which secreted high titers of antibodies to a selected model immunogen, keyhole limpet hemocyanin.

Animals↗
PubMed1986

Detection of low-density cell-surface molecules using biotinylated fluorescent microspheres.

Biotinylated fluorescent microspheres have been developed as a reagent for studying antigens and receptors expressed at the cell surface. Labeling of antigen or receptor was accomplished by crosslinking biotinylated microspheres through streptavidin to corresponding biotinylated antibodies or ligands. Detection of labeled cells by flow microfluorimetry provided an extremely sensitive means for the analysis and potential manipulation of heterogeneous cell populations. The data indicate that cells bearing fewer than 200 surface antigen-antibody complexes per cell are readily detectable by this approach. Crosslinked to a selected biotinylated peptide immunogen, biotinylated fluorescent microspheres also allowed the labeling and detection of hybridoma cells bearing antigen-specific surface immunoglobulin.

Animals↗
PubMed1985

Isolation and characterization of an anti-peptide monoclonal antibody to human erythropoietin.

A site-specific monoclonal antibody to human erythropoietin has been developed. It is secreted by a hybridoma cell line derived from the fusion of murine myeloma cells with the splenocytes of a mouse that had been immunized with a 26-residue synthetic peptide antigen homologous to the amino-terminal sequence of the hormone. The antibody binds specifically to peptide, 125I-erythropoietin, and biologically active erythropoietin. The equilibrium dissociation constants of the antibody-erythropoietin and the antibody-peptide interactions are identical, Kd = 6.7 X 10(-9) M, suggesting strong conformational similarity or identity of the epitope as expressed on the peptide and the hormone. Immune complexes formed between the antibody and either human or rat erythropoietin exhibit full biologic activity. However, the antibody does not recognize the baboon, sheep, or canine hormones, indicating antigenic differences or structural variation among these erythropoietins. These results indicate that the amino-terminal region of erythropoietin is not involved in receptor binding. Furthermore, they form a basis for the study of the structure and function of the hormone using anti-peptide antibodies.

Animals↗
PubMed1985

Identification of a surface epitope of human erythropoietin with anti-peptide antibodies.

Antibodies reactive with human erythropoietin were isolated from the serum of rabbits immunized with a twenty-six amino acid synthetic polypeptide corresponding to a proposed NH2-terminal sequence of the hormone. As shown by inhibition with peptide fragments, those antibodies that bound to erythropoietin recognized the (8-15) domain, strongly suggesting tht this region is exposed on the hormone's surface. This was confirmed by affinity purification of these antibodies on immobilized fragment (8-15). These results provide insight into the tertiary structure of human erythropoietin and suggest uses for the sequence-specific antibodies in labeling the hormone.

Amino Acid Sequence↗
PubMed1984

A novel radioimmunoassay for human erythropoietin using a synthetic NH2-terminal polypeptide and anti-peptide antibodies.

Antibodies prepared by immunizing rabbits with a synthetic polypeptide representing the first 26 amino acids of the proposed NH2-terminal sequence of human erythropoietin cross-react with peptide, highly purified 125I-labeled erythropoietin and biologically active erythropoietin. We have now developed a rapid and convenient radioimmunoassay for the hormone using 125I-labeled peptide and anti-peptide antibodies. This peptide/anti-peptide approach completely circumvents the relative lack of availability of highly purified 125I-labeled erythropoietin and of erythropoietin for immunization purposes and makes a radioimmunoassay for the hormone universally available to investigators. This concept may be applied as a general method for designing radioimmunoassays of molecules available in severely restricted quantities.

Amino Acid Sequence↗
PubMed1984

The beta-adrenergic receptor adenylate cyclase complex of Rauscher murine erythroleukemia cells and its response to erythropoietin-induced differentiation.

Rauscher murine erythroleukemia cells, grown continuously in vitro, undergo erythroid differentiation in response to the hormone erythropoietin. Therefore, they serve as an important model system with which to examine critical biochemical aspects of this developmental process. Intact, uninduced Rauscher cells possess a functional beta-adrenergic receptor-adenylate cyclase complex. The adrenergic agonists, isoproterenol, epinephrine, and norepinephrine, exhibited activation constants (Kact) of 0.1, 0.5, and 20 mumol/L, respectively. Thus, the beta-receptor-cyclase complex of Rauscher cells is apparently one of the most sensitive of all erythroid cells reported thus far. The epinephrine-stimulated cyclic adenosine monophosphate (cAMP) response was inhibited by propranolol, alprenolol, and hydroxybenzylpindolol, with inhibition constants (KI) of 3.8, 2.2, and 0.1 nmol/L, respectively. Using [125I]-iodohydroxybenzylpindolol as ligand, uninduced Rauscher cells were shown to possess 1,100 receptors/cell, with an equilibrium dissociation constant (KD) of 400 pmol/L. Erythropoietin, but not dimethylsulfoxide, induction caused a specific increase in receptor density to 3,300/cell on differentiating Rauscher cells. This is the first demonstration of membrane receptor regulation by erythropoietin that may be important in the complex interplay of hormonal effects during erythropoiesis.

Adenylyl Cyclases↗
PubMed1984

Erythropoietin: a prime regulator of red cell differentiation.

Erythropoietin, a glycoprotein hormone that regulates erythroid differentiation, has been recognized for several decades. Recently, the study of this hormone has increased markedly. It has been purified and partially sequenced. There are several monoclonal and site-specific polyclonal antibodies to it that have revealed important structural information. The gene appears to have been cloned. Current efforts to understand its mode of action will yield answers to questions of importance to both basic scientists and clinicians.

Animals↗
PubMed1984

Secretion of erythropoietin-like activity by clones of human renal carcinoma cell line GKA.

Human renal carcinoma cell line GKA was derived from a patient with the paraneoplastic syndrome of erythrocytosis and secretes erythropoietin-like activity into its growth medium (Sytkowski, A. J., Richie, J. P., and Bicknell, K. A. Cancer Res., 43: 1415-1419, 1983). In order to derive homogeneous sublines with higher secretory rates, we cloned line GKA. Over 100 clones were generated, and 21 secreted erythropoietin-like activity, up to 6-fold higher than the uncloned line. This activity stimulated the growth and differentiation of CFU-E derived colonies in plasma clot culture. However, the secreted erythropoietin-like activity did not cross-react in a sensitive radioimmunoassay utilizing highly purified 125I-labeled human urinary erythropoietin and heterologous anti-human urinary erythropoietin antiserum. These results suggest that line GKA secretes an erythropoietic stimulating factor distinct from the hormone erythropoietin.

Cell Line↗
PubMed1983

Site-specific antibodies to human erythropoietin directed toward the NH2-terminal region.

Site-specific antibodies to human erythropoietin have been raised in rabbits immunized with a synthetic polypeptide composed of the putative 26 NH2-terminal amino acids of the hormone. The immunogenic peptide was coupled to bovine serum albumin. Antibodies specific for peptide were detected by enzyme-linked immunosorbent assay. They immunoprecipitated both highly purified 125I-labeled erythropoietin and biologically active erythropoietin. The immunoprecipitation of 125I-labeled erythropoietin was inhibited by unlabeled erythropoietin and by peptide, demonstrating their crossreactivity. The antibodies did not neutralize erythropoietin's biological activity. These results indicate that a portion of the NH2-terminal region of erythropoietin is exposed on the surface of the protein at some distance from the receptor-binding domain. These antibodies will be important in further studies of the hormone and its mechanism of action.

Amino Acid Sequence↗