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A J Sytkowski

Publications and source records attributed to A J Sytkowski.

At least 73 records · Page 4Linked to original sources
PubMed1983

Erythropoietin-induced differentiation of Rauscher erythroleukemia cells.

We have shown morphologic and biochemical evidence for erythroid differentiation in the erythropoietin-responsive cell line Rauscher murine erythroleukemia. These cells synthesize adult and presumably embryonic hemoglobin in response to erythropoietin and dimethyl sulfoxide. Clear differences are observed in some clones between different inducers. The molecular basis for these differences, which may relate to differential modes of inducer action, must be clarified. These cells possess a functional beta-adrenergic receptor/adenylyl cyclase complex remarkably similar to that found on erythrocytes of several species. The receptor density is up-regulated specifically by erythropoietin and, as such, is the first description of membrane receptor regulation by this hormone. Thus, we now have the opportunity to investigate the development of the receptor-cyclase complex on the differentiating erythroid cell and to elucidate its role in the complex interplay of erythropoietin and other hormonal effects during erythropoiesis.

Animals↗
PubMed1983

New human renal carcinoma cell line established from a patient with erythrocytosis.

A continuous human renal carcinoma cell line (GKA) has been established from a patient with the paraneoplastic syndrome of erythrocytosis. The cells are epithelioid and anchorage dependent and have a doubling time in vitro of 48 to 72 hr. They exhibit a modal karyotype of 45,XX with abnormalities in chromosomes 3 and 9 and an absent chromosome 17 as determined by quinacrine mustard staining. Line GKA secretes erythropoietin activity into its growth medium, consistent with the biology of the tumor in vivo. This unique cell line will permit an investigation of the cellular physiology of this carcinoma and should result in clonal sublines with high erythropoietin-secretory activities.

Cell Line↗
PubMed1981

Characterization of multiple erythroid progenitors available in large quantity from rabbit marrow.

We have examined the characteristics of erythroid progenitors from rabbit marrow and assessed the potential of this species as a source from which substantial quantities of erythroid progenitors could be obtained. Plasma clot cultures of rabbit marrow demonstrate colonies consistent with erythroid colony forming units (CFUE) by day 2 and colonies resulting from two types of burst forming units (BFUE) seen on days 3 and 8-9, respectively. Characteristic colony morphologies were noted from each cell type as well as differential sensitivity to erythropoietin (Ep) with discrete maximum responses for each progenitor. Distinct buoyant density characteristics were noted when the precursors were subjected to isopyknic separation on linear density gradients of polyvinylpyrrolidone-coated silica. The total nucleated marrow cells obtained from each animal ranged from 1.7-4.7 X 10(9). The average yields for each progenitor wee 1.71(+/-0.43) X 10(7) for CFUE, 1.99(+/-0.36) X 10(6) for 3 day BFUE and 1.24(+/-0.11) X 10(6) for 8-9 day BFUE.

Animals↗
PubMed1980

Erythroid differentiation of clonal Rauscher erythroleukemia cells in response to erythropoietin or dimethyl sulfoxide.

Clonal lines of Rauscher erythroleukemia cells exhibited selective responses to two inducers of differentiation, erythropoietin and dimethyl sulfoxide. There were substantial quantitiative differences between clones that reponded to both inducers. Several clones differentiated only in response to erythropoietin. Erythropoietin stimulated cell proliferation and differentiation whereas dimethyl sulfoxide inhibited proliferation, suggesting dissimilar modes of action.

Animals↗
PubMed1980

The biochemistry of erythropoietin: an approach to its mode of action.

The elucidation of the mechanism of action of erythropoietin depends upon a detailed assessment of its effects at the molecular level. We have now begun to examine the effects of human erythropoietin and other inducers on clonal lines of Rauscher murine erythroleukemia cells. Over 100 clonal lines have been examined by assessing the hemglobinization of colonies grown in plasma clot culture in response to erythropoietin and dimethylsulfoxide. Many of the clones respond to both inducers. However, some clones respond only to erythropoietin. The cells also differentiate in suspension culture, exhibiting striking morphological changes characteristic of erythroid development. This system should serve as an excellent model for the study of control mechanisms in erythropoiesis.

Animals↗
PubMed1979

Cadmium-109 as a probe of the metal binding sites in horse liver alcohol dehydrogenase.

The noncatalytic and catalytic zinc atoms of horse liver alcohol dehydrogenase, [(LADH)Zn2Zn2] or LADH, have been replaced differentially with 109Cd by equilibrium dialysis, resulting in two new enzymatically active species, [(LADH)109Cd2Zn2] and [(LADH)109Cd2109Cd2]. The UV difference spectra of the cadmium enzymes vs. native [(LADH)Zn2Zn2] reveal maxima at 240 nm with molar absorptivities, delta epsilon 240, of 1.6 X 10(4) M-1 cm-1 per noncatalytic 109Cd atom and 0.9 X 10(4) M-1 cm-1 per catalytic 109Cd atom, consistent with coordination of the metals by four and two thiolate ligands, respectively, strikingly similar to the 250-nm charge-transfer absorbance in metallothionein. Carboxymethylation of the Cys-46 ligand to the catalytic metal in LADH presumably lowers the overall stability constant of the coordination complex and results in loss of catalytic 109Cd or catalytic cobalt but not catalytic zinc from the enzyme.

Alcohol Oxidoreductases↗
PubMed1978

Cobalt exchange in horse liver alcohol dehydrogenase.

The preparation of metal hybrid species of horse liver alcohol dehydrogenase is made possible by the development of carefully delineated systems of metal in equilibrium metal exchange employing equilibrium dialysis. The conditions which are optimal for the site-specific replacement of the catalytic and/or noncatalytic zinc atoms of the native enzyme by cobalt are not identical with those which are utilized for substitution with 65Zn. Thus, while certain 65Zn hybrids can be prepared by exploiting the differential effects of buffer anions, the cobalt hybrids are generated by critical adjustments in the pH of the dialysate. Factors which may determine the mechanism of metal replacement reactions include acid-assisted, ligand-assisted, and metal-assisted dechelation, steric restriction, and ligand denticity as well as physicochemical properties of the enzyme itself. The spectral characteristics of the catalytic and noncatalytic cobalt atoms reflect both the geometry of the coordination complexes and the nature of the ligands and serve as sensitive probes of these loci in the enzyme.

Alcohol Oxidoreductases↗
PubMed1976

Human liver alcohol dehydrogenase: purification, composition, and catalytic features.

Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.

Alcohol Oxidoreductases↗
PubMed1976

Chemical reactivities of catalytic and noncatalytic zinc or cobalt atoms of horse liver alcohol dehydrogenase: differentiation by their thermodynamic and kinetic properties.

Horse liver alcohol dehydrogenase (EC 1.1.1.1) contains one catalytic and one noncatalytic pair of zinc atoms that can be replaced selectively with cobalt and/or 65zince. We have now prepared "hybird" metalloenzymes by specifically replacing one or both pairs of zinc atoms with 65zinc and/or cobalt. Their differential chemical reactivities serve to characterize the metal atoms at either site. The spectral and kinetic properties of the resultant 65zinc, cobalt, and hybrid enzymes, as well as those of their complexes with 1,10-phenanthroline, identify the metal atoms that are at the catalytic sites and differentiate them from those at the noncatalytic sites. All data are in complete agreement with the results of the x-ray crystal structure analysis. Remarkably, under the conditions used, chemical reactivity, as gauged by thermodynamic methods under equilibrium conditions, identifies the catalytic metal atoms as those which are reactive to 1,10-phenanthroline, while this reagnet does not affect the noncatalytic pair. Under dynamic conditions the kinetics of the metal-metal exchange reveals the converse to be true: the chemical reactivity of the noncatalytic atoms is much higher and, hence, they exchange more rapidly. The results are examined in terms of thermodynamic and kinetic properties of metal complex ions which serve as the basis of possible mechanisms underlying these observations.

Alcohol Oxidoreductases↗
PubMed1974

Cholinergic augmentation of lymphocyte-mediated cytotoxicity. A study of the cholinergic receptor of cytotoxic T lymphocytes.

Cholinergic agonists have previously been shown to augment the ability of sensitized lymphocytes to injure cells bearing the sensitizing alleantigens. The cholinergic receptor of the attacking lymphocyte population has been studied with pharmacological manipulation of an in vitro system that quantitates the injury mediated by sensitized attacking cells upon target cells. The data reveal that muscarinic ligands are several orders of magnitude more potent than nicotinic agents in altering cytotoxicity.

Animals↗