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Biomedical subjects

G Kostka

Publications and source records attributed to G Kostka.

36 records · Page 2Linked to original sources
PubMed1993

Modulation of p145c-kit function in cells of patients with acute myeloblastic leukemia.

The function of the steel factor receptor, p145c-kit, in patient-derived acute myeloblastic leukemia (AML) cells was investigated. Steel factor stimulation of AML cells coexpressing p145c-kit and the progenitor cell antigen CD34 resulted in complete receptor down-regulation, a marked decrease of CD34 antigen expression, and the induction of the granulocyte lineage antigen CD15. These changes in surface marker expression paralleled morphological differentiation to granulated blasts and promyelocytes. Interestingly, the same phenotype was achieved by IL-3 stimulation of AML cells. p145c-kit extracellular domain-specific antibodies had either blocking or enhancing effects on ligand binding, receptor phosphorylation and down-regulation, and induction of cell proliferation. Correlations of these phenomena with distinct effects of antibody stimulation on cell substrate phosphorylation provide clues for the dissection of the p145c-kit signal and the analysis of its relevance for AML.

Animals↗
PubMed1993

Characterization of hemopoietic cell populations from human cord blood expressing c-kit.

Human cord blood or bone marrow cells expressing the CD34 surface antigen include a population of pluripotent progenitors. We identified and isolated a subpopulation of cells coexpressing CD34 and c-kit, a transmembrane receptor with tyrosine kinase activity. Novel monoclonal antibodies (16A6, 14A3, 3D6) directed against the extracellular domain of c-kit were used for immunofluorescence labeling and sorting of low-density mononuclear cells (MNCs) from umbilical cord blood and bone marrow. The frequency of c-kit-labeled MNCs from cord blood (mean 5.0% +/- 2.1%, n = 16) was similar to that from adult bone marrow (mean 3.7% +/- 1.3%, n = 4). On average, 1.4% of CD34-positive cells were recorded in cord blood and 2.1% in bone marrow MNCs. Roughly 60% of CD34-positive cells coexpressed c-kit. The ability of CD34+/c-kit+ cells to form multilineage colonies (CFU-GEMM) was assayed after sorting with an antibody that did not show any significant effect on c-kit ligand (RL) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation. For CD34+/c-kit+ cells, we found a 20- to 50-fold enrichment as against total MNCs, and a 2-fold enrichment if compared with the CD34+/c-kit-population. To study expression of c-kit in lymphocytic precursors, monoclonal anti-CD7 or anti-CD10 antibodies were used simultaneously. In contrast to CD34-expressing cells, however, no consistent double-labeled subpopulation of lymphocytic cells was detected. Furthermore, coexpression of CD38 (73% +/- 14%, n = 4) or CD33 (29% +/- 12%, n = 5) on a majority of c-kit-positive cells showed their lineage commitment to erythropoiesis and granulocytopoiesis.

ADP-ribosyl Cyclase↗
PubMed1993

[Toxicity of aldehydes used as disinfectants].

The paper presents a review of current scientific literature on the toxicology of aldehydes used as cold disinfectants. A particular attention was paid towards the mutagenic, teratogenic and cancerogenic potential as well as occupational hazard and risk for patients exposed to aldehydes.

Aldehydes↗
PubMed1992

Differential effects of carboxy-terminal sequence deletions on platelet-derived growth factor receptor signaling activities and interactions with cellular substrates.

Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptor's mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF-activated signaling pathways and demonstrate that phospholipase C gamma phosphorylation is not required for mitogenesis and transformation.

3T3 Cells↗
PubMed1989

Characterization and partial purification of a high-molecular-mass, calmodulin-binding, tyrosyl-phosphorylated protein from lymphocyte plasma membranes.

A plasma membrane phosphoprotein with a species-dependent molecular mass of 108 or 112 kDa (P108/112) was analyzed in lymphoid cells from rats and humans. After 24 h lectin stimulation its in vitro phosphorylation was raised to an important extent. Phosphotyrosine was analyzed by 2-D electrophoresis. Calmodulin was bound by P108/112 in a Ca2+-dependent manner. P108/112 remained insoluble after extraction with detergent, high salt, EDTA, or high pH. After chlorethanol extraction it was partially purified by gel filtration. P108/112 shows conspicuous similarities with the 110-kDa protein from chicken intestine microvilli.

Animals↗
PubMed1986

Ultraviolet light-induced crosslinking of two major phosphoproteins and poly(A)+RNA from free polyribosomes; changes in phosphorylation by inhibitors of transcription and translation.

Polyribosomes were isolated without the use of detergents, irradiated with ultraviolet light and labelled in the presence of (gamma-32P) adenosine 5'-triphosphate. Poly(A)+RNA-protein structures separated by chromatography on oligo (dT)-cellulose contained up to 1o crosslinked proteins as shown by SDS-polyacrylamide gel electrophoresis. These included a 71 kDa poly(A)-bound species and two major phosphoproteins of 66 and 13o kDa. Pretreatment of rats with inhibitors of transcription and translation caused different and significant alterations in the labelling of the two phosphoproteins, suggesting that phosphorylation of proteins closely associated with mRNA may be involved in the regulation of the stability of this RNA or its binding to structural elements in the cell.

Amanitins↗
PubMed1985

Phosphoproteins crosslinked to poly(A) + heterogeneous nuclear RNA after irradiation with ultraviolet light.

Isolated liver nuclei or whole lymph node lymphocytes stimulated with concanavalin A in culture were irradiated with ultraviolet light. The crosslinked structures of poly(A)+ heterogeneous nuclear RNA and protein were purified on oligo(dT)-cellulose after labelling irradiated nuclei in the presence of adenosine 5'-[gamma-32P]triphosphate and analysed by SDS-polyacrylamide gel electrophoresis. The liver and lymphocyte nuclear proteins included about 17-19 species of 35-150 kDa and were shown to produce quite similar electrophoretic band patterns. Two proteins of 110-120 and 40-42 kDa were phosphorylated. Using partial proteolytic digestion the large-size crosslinked phosphoprotein has been identified as the 110 kDa component described previously (Schweiger, A. and Kostka, G. (1984) Biochim. Biophys. Acta 782, 262-268). The 40-42 kDa band was presumably related to the group C species of main proteins associated with heterogeneous nuclear RNA. In crosslinked nuclear structures from rats treated with low doses of alpha-amanitin for 1 h the relative amount of the 110-120 kDa phosphoprotein was reduced while the labelling with [32P]ATP was almost abolished.

Animals↗
PubMed1985

Biphasic increase in the in vivo phosphorylation of nuclear 110-kDa protein during early lymphocyte transformation.

A major 110-kDa phosphoprotein in rat liver and hepatoma (P 110) was identified in nuclear 0.2 M HCl extracts from rat lymph node cells. Stimulation with concanavalin A altered both the amount of P110 and, more strikingly, its in vivo phosphorylation in a typical biphasic manner with an initial maximum after 10-14 h. RNA synthesis showed a similar biphasic increase. Inhibition of hnRNA synthesis (5,6 dichlorobenzimidazoleriboside), but not of DNA synthesis (hydroxyurea), depressed both the amount of P110 and its phosphorylation markedly.

Animals↗
PubMed1984

Concentration of particular high molecular mass phosphoproteins in rat liver nuclei and nuclear matrix decreases following inhibition of RNA synthesis by alpha-amanitin.

Purified liver nuclei were isolated from rats treated with non-lethal doses of alpha-amanitin, actinomycin D, galactosamine or cycloheximide. The nuclei were incubated in the presence of adenosine 5'-[gamma-32P]triphosphate, and digested with DNAase or DNAase plus high salt concentrations to prepare nuclear residual structures. Using SDS-polyacrylamide gel electrophoresis followed by autoradiography, samples from untreated rats were shown to contain major phosphoproteins in the range 76-260 kDa, with a prominent triplet of bands with 110, 117 and 128 kDa. Treatment of animals with alpha-amanitin or high doses of actinomycin D and galactosamine caused a significant decrease in the concentration of a few phosphorylated species, including the 110 kDa protein in whole nuclei, and their disappearance from the nuclear matrix or residual ribonucleoprotein structures after 1-3 h. The changes were reversible, complete recovery being observed after 5 h in the case of alpha-amanitin. No similar results were obtained with nuclei from rats treated with the translation inhibitor cycloheximide. The data are discussed in view of a possible effect of certain high molecular mass phosphoproteins on reactions of the heterogeneous nuclear RNA/mRNA pathway in the cell.

Amanitins↗
PubMed1984

Synthesis of 2',5'-oligoadenylate by rat liver nuclear matrix protein.

Nuclear matrix was prepared from unstimulated rat liver by treatment of nuclei with DNAse and 0.4 M NaCl and was further extracted with 2.0 M NaCl. Proteins were bound to poly(rI):(rC)-agarose, incubated with (alpha-32P) adenosine 5'-triphosphate and 2',5'-linked oligoadenylate was isolated from the supernatant. The substance inhibited amino acid incorporation in a reticulocyte translation system and was identified after enzymatic treatment followed by thin-layer chromatography on PEI-cellulose. The possible function of 2',5'-oligo(A) synthetase in the maturation of pre-mRNA associated with nuclear matrix is discussed.

Adenine Nucleotides↗
PubMed1983

High molecular mass phosphoproteins in the rat liver nuclear matrix identification of a prominent 110,000 Dalton species.

Purified rat liver nuclei were labelled in vitro in the presence of (32P) ATP and submitted to sequential extraction with DNAse, 0.4 or 2.0 M NaCl and Triton X-100. The residual or matrix structures contained 8-10 phosphoproteins between 76 and 260 kd including a triplet of major bands with 110, 117 and 128 kd. The 110 kd species was purified by chromatography on oligo(dT)-cellulose. It was shown to be identical with the 110 kd phosphoprotein of rat liver or Morris hepatoma free polyribosomes using the technique of limited digestion with S. aureus protease V 8.

Animals↗
PubMed1982

ADP-ribosylation of proteins associated with heterogeneous nuclear RNA in rat liver nuclei.

Purified rat liver nuclei were incubated in vitro in the presence of [adenylate-32P]nicotinamide adenine dinucleotide. The label was rapidly incorporated into trichloroacetic acid-insoluble material and also detected in particles carrying heterogeneous nuclear RNA. The particles were isolated by density gradient centrifugation, and their size determined to be 30-40 S from parallel experiments using nuclei labelled with [3H]uridine 5'-triphosphate under similar conditions. Treatment of the 30-40 S-particles with enzymes of different specificities showed that the label was tightly bound to proteins, not incorporated into nuclei acids and not utilized in phosphorylation of proteins. The label was detached by phosphodiesterase I from snake venom and identified as ADP-ribose and adenosine 5'-phosphate present at a ratio of 7.5 to 1 using thin layer chromatography on poly(ethyleneimine)-cellulose. Radioactively labelled (ADP-ribosylated) proteins were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. They included several major species of the ribonucleoprotein with molecular weights of 36000, 39000 and 42000, and a limited number of high molecular weight polypeptides.

Adenosine Diphosphate↗
PubMed1981

Properties of a 110000 molecular weight protein in rat liver hnRNP particles.

Particles carrying heterogeneous nuclear RNA (30--40 S-particles) were isolated from rat liver nuclei and the particle proteins separated by sodium dodecylsulfate gel electrophoresis. Some properties of a 110000 molecular weight component (P 110/103) were studied in detail: (i) P 110/103 was labeled to a 4-5 times higher specific activity than the major particle proteins in the presence of [14C]-amino acids in vivo. (ii) In nuclei incubated with [3H]- or [32P]-nicotinamide adenine dinucleotide P 110/103 was labeled presumably by ADP-ribosylation. (iii) A protein with the same molecular weight as P 110/103 and isolated from the nuclear extract by affinity chromatography was phosphorylated in vitro.

Amino Acids↗
PubMed1980

Composition of hnRNA-associated proteins in rat liver is specifically altered after cycloheximide treatment.

Rats were treated with non-lethal doses of alpha-amanitin or cycloheximide. Nuclei were prepared and the particles carrying heterogeneous nuclear RNA (30--40 S-particles) isolated by density gradient centrifugation. In the case of alpha-amanitin the yield of particles was reduced to about 45%. Cycloheximide affected the composition of proteins associated with the nuclear RNA. In particular, the concentration of a 110 000 molecular weight protein as determined by sodium dodecylsulfate gel electrophoresis was reduced to 20--30% after 2 h and then increased to 150--180% of the control before it approached the normal level after 30 h. Possible mechanisms underlying these changes are discussed.

Amanitins↗
PubMed1977

Preparation in undenatured form of the main protein bound to heterogeneous nuclear RNA in liver and hepatoma cells.

Particles carrying heterogeneous nuclear RNA (30 S-particles) were prepared from rat liver and Zajdela hepatoma ascites cell nuclei after ultrasonic disruption. The ribonucleoprotein structures were disintegrated in the presence of 100mM spermidine. Using chromatography on Sepharose-polyadenylate a protein component has been obtained which possessed high affinity for heterogeneous nuclear RNA, polyuridylate and polyadenylate, and double-stranded DNA. This protein was the main species of the ribonucleoprotein studied; it showed bands with molcular weights of 37000 and 40000 respectively in SDS gel electrophoresis. The RNA-binding proteins isolated from liver and hepatoma had identical molecular weights and the same affinity for Sepharose-polyadenylate used in the isolation.

Animals↗
PubMedDate not supplied

Early hepatic changes induced by nuarimol in rats.

It is commonly believed that in short-term tests hepatic cytochrome P-450b inducers stimulate liver enlargement and mitogenesis in the absence of overt hepatotoxic effects. In this investigation male Wistar rats received naurimol (an organochlorine pesticide) in one, three and five oral doses of 31.5, 62.5 and 125 mg kg-body wt. day-, whereupon the effects on liver were determined. The early effects were dose-dependent increases in p-nitroanisole metabolism, hepatocyte proliferation (DNA synthesis and mitotic activity) and liver weight. Five administrations of the lowest does (31.5 mg kg-1 body wt. day-1) did not change liver weight, despite increased p-nitroanisole metabolism and hepatocyte proliferation. In contrast to p-nitroanisole metabolism and hepatomegaly, proliferation was only transient and disappeared even when treatment continued. The increase in binuclear hepatocytes and signs of necrosis suggested that the hepatomitogenic effect of nuarimol reflected a regenerative response, which may simulate the proliferation caused by partial hepatectomy.

Administration, Oral↗