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S O Doskeland

Publications and source records attributed to S O Doskeland.

15 recordsLinked to original sources
PubMed2003

Search for new cyclic AMP-binding proteins.

Today, there is evidence that the cAMP-dependent kinases (PKA) are not the only intracellular receptors involved in intracellular cAMP signalling in eukaryotes. Other cAMP-binding proteins have been recently identified, including some cyclic nucleotide-gated channels and Epac (exchange protein directly activated by cAMP) proteins. All these proteins bind cAMP through conserved cyclic nucleotide monophosphate-binding domains. However, all putative cAMP-binding proteins having such domains, as revealed by computer analysis, do not necessarily bind cAMP, indicating that their presence is not a sufficient criteria to predict cAMP-binding property for a protein.

Amino Acid Sequence↗
PubMed2001

Apoptotic effect of cyanobacterial extract on rat hepatocytes and human lymphocytes.

Toxic cyanobacterial blooms are an increasing problem in Poland. The production of cyanobacterial toxins and their presence in drinking and recreational waters represent a growing danger to human and animal health. This is connected with the increase of cyanobacterial biomass caused by excessive eutrophication of the water ecosystem. There is evidence that cyanobacterial hepatotoxins can act as a potent promoter of primary liver cancer. The apoptotic effect of microcystins in Polish cyanobacterial bloom samples on rat hepatocytes and human lymphocytes was observed using light and fluorescence microscopy, flow cytometry, and electrophoretic analysis. The incubation time needed to observe the first morphological apoptotic changes in hepatocytes was approximately 30 min; however, the characteristic biochemical changes in DNA were not observed even after 120 min. In lymphocyte cultures the morphological changes characteristic for apoptosis were observed after 24 h of incubation and a 48-h incubation was found to be optimal for analysis of internucleosomal DNA fragmentation, which is one of the main biochemical hallmarks of programmed cell death. These cells are an easily isolated and inexpensive material for medical diagnostics. Therefore the apoptotic changes, together with the clastogenic effect seen in lymphocyte cultures, are proposed as a future analytical method for these toxins.

Animals↗
PubMed2000

Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP-induced apoptosis to granulocytic differentiation.

The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.

Animals↗
PubMed2000

Apoptotic effect of cyanobacterial blooms collected from Polish water reservoirs.

Recently in many countries, including Poland, the problem of toxicity of cyanobacterial blooms has been of great importance. In many cases it is connected with the increase of microcystins (MCYSTs) concentration in fresh water. This problem is caused by excessive eutrophication of drinking and recreational water bodies. In humans, the most frequent symptoms of the MCYST effect are: cutaneous rash, fever, vomiting, diarrhoea, gastroenteritis and acute damage of the liver. The aim of this work was to estimate apoptotic effects of five different cyanobacterial hepatotoxic extracts containing MC-LR and other variants of MCYSTs (MC-RR, MC-YR, and MC-WR). These effects were analysed in rat hepatocytes--primary target of cyanobacterial hepatotoxins. Morphological changes in hepatocytes were examined by means of fluorescence and differential interference contrast microscopy with the DNA-specific dye, Hoechst 33342. The hepatocytes were treated with each cyanobacterial extracts containing MC-LR in the range between 100 nM-2000 nM for 30 min, 60 min and 120 min. The first characteristic apoptotic changes: shrinking and budding of cells were seen after 30 min, MC-LR = 100 nM. During the next 30 min the percentage of apoptotic cells increased by over 50%, MC-LR at concentrations ranging from 100 to 250 nM (the value dependent on a bloom sample). Highly condensed chromatin and apoptotic bodies were observed in 85-90% of hepatocytes after 120 min of treatment with MC-LR in concentration of 1000 nM. The apoptotic changes in rat hepatocytes confirm the high cytotoxic potential of cyanobacterial bloom samples collected during different months and years from reservoirs of drinking and recreational water in central Poland.

Apoptosis↗
PubMed1998

Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2.

Microinjection of cytochrome c induced apoptosis in all the cell types we tested (IPC-81, Swiss 3T3, Clone 8 fibroblasts, NRK, H295, Y1, HEK 293). The apoptotic phenotype induced by injected cytochrome c was characterized by externalization of phosphatidyl serine, cell detachment from substratum and from neighbor cells, and had the classic ultrastructural features of membrane budding, chromatin condensation and cell shrinkage. Depending on the cell type and concentration of cytochrome c, the induction of apoptosis was remarkably rapid. The development of apoptosis was prevented by the caspase inhibitor Z-VAD.fmk. Four of the cell types (Clone 8, Swiss 3T3, NRK, Y1) were transfected with bcl-2 and these all showed a markedly decreased sensitivity towards injected cytochrome c. Our data suggest that extramitochondrial cytochrome c is a general apoptogen in cells with a functioning caspase system. They also indicate that, in preventing apoptosis, Bcl-2 acts not only at the level of regulation of cytochrome c release from mitochondria, but can also interfere with caspase activation induced by cytochrome c microinjected directly into the cytoplasm.

3T3 Cells↗
PubMed1997

Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.

Animals↗
PubMed1994

Two distinctly regulated events, priming and triggering, during retinoid-induced maturation and resistance of NB4 promyelocytic leukemia cell line.

In t(15;17) acute promyelocytic leukemia, all-trans retinoic acid (RA) induces leukemic cell maturation in vitro and remission in acute promyelocytic leukemia patients, but in vivo treatments invariably lead to relapse with resistance to RA. NB4, a maturation-inducible cell line, and NB4-RAr sublines (R1 and R2) displaying no maturation in the presence of RA have been isolated from a patient in relapse. We show that resistance to maturation is not a mere unresponsiveness to RA: rather, R1 "resistant" cells do respond to RA (1 microM) by sustained growth, become competent to undergo terminal maturation, and up-regulate CD11c/CD18 integrins. Interestingly, maturation of "resistant" cells, rendered competent by RA, can be achieved by cAMP-elevating agents (prostaglandin E, isoproterenol, cholera toxin, or phosphodiesterase inhibitor) or stable agonistic cAMP analogs such as (SP)-8-chloroadenosine cyclic 3',5'-phosphorothioate. This shows that activation of cAMP-dependent protein kinase (cA kinase) can override the RA resistance and suggests interdependent RA and cAMP signaling pathways in acute promyelocytic leukemia maturation. No such cooperation was observed in the R2 resistant cells, though their cA-kinase was functional. (RP)-8-Chloroadenosine cyclic 3',5'-phosphorothioate, which by displacing endogenous cAMP inhibits the basal cA-kinase activity, decreased the response of sensitive cells to RA. This raises the possibility that cA-kinase plays a key role in the maturation also of RA-sensitive cells. Our results define two discrete steps in the maturation process: an RA-dependent priming step that maintains proliferation while cells become competent to undergo maturation in response to retinoids and a cAMP-dependent step that triggers RA-primed cells to undergo terminal maturation. Uncoupling RA and cAMP action might cause the so-called "resistance."

Antigens, CD↗
PubMed1980

Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions.

We sought procedures which would allow a rapid concentration in high yield of bacterial antigens from tissue fluids of patients and which could be applied also to protein-rich fluids like serum. Ethanol precipitation at a subzero temperature with albumin added as an antigen coprecipitant made it possible to achieve a more than 20-fold concentration of antigen in 15 min and a 200-fold concentration in 45 min. Heat-stable antigens could be concentrated from protein-rich fluids (like serum) after the sample had been deproteinized by boiling. Such heating (100 degrees C, 3 min) also liberated bacterial polysaccharides from antibody complexes and elminated the nonspecific interference of serum in enzyme-linked immunosorbent assay.

Antigen-Antibody Complex↗
PubMed1977

Binding proteins for adenosine 3':5'-cyclic monophosphate in bovine adrenal cortex.

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.

Adenosine↗
PubMed1977

Factors affecting the binding of [3H]adenosine 3':5'-cyclic monophosphate to protein kinase from bovine adrenal cortex.

Inorganic salts, several proteins and traces of protein precipitants were tested to find out by what mechanisms they modulate the binding of cyclic [3H]AMP to protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37). The separation of free and bound cyclic AMP by (NH4)2SO4 precipitation was unaffected by the above agents and was more reliable than the Millipore filtration technique. Several binding sites for cyclic AMP were revealed in adrenal-cortex extract. When this extract was used as binding reagent in an assay for cyclic AMP, the standard curve was distorted in the presence of KCl because the salt affected the different binding sites to a varying extent. At high ionic strenth the protein kinase isoenzyme I dissociated and showed an extraordinarily high affinity for cyclic AMP. Trichloroacetate and perchlorate at very low concentrations were able to dissociate the protein kinase and modulate its binding characteristics as well. A progressive decrease in the cyclic AMP-binding capacity occurred on prolonged incubations. The binding protein was protected against inactivation by 2-mercaptoethanol, EDTA and several proteins. It was more resistant to denaturation when complexed to cyclic AMP. The enhancement of cyclic AMP binding by bovine serum albumin was investigated in some detail and appeared to be a pure stabilizing effect. It is proposed that the competitive-binding assays for cyclic AMP based on protein kinase be conducted at high ionic strength and in the presence of stabilizers (protein, EDTA, 2-mercaptoethanol). The interference from agents that may dissociate the protein kinase or influence its stability will thus be decreased.

Adrenal Cortex↗
PubMed1977

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.

Adenosine↗
PubMed1976

Adenosine 3':5'-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver.

Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.

Animals↗
PubMed1975

Protein kinases activated by cAMP in the genital tract of spayed mice treated with oestradiol-17beta.

The cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), has been studied in the vaginal epithelium, vaginal stroma, endometrium, and whole uterus of spayed mice treated with oestradiol-17 beta, and in the vaginal epithelium and uterus of spayed mice. Two protein kinase isoenzymes (PK I and PK II) were found in whole uterus, endometrium, and vaginal stroma. Vaginal epithelium contained only one isoenzyme (PK II). Oestradiol treatment increased PK I relative to PK II in the uterus. The isoenzyme pattern in the vaginal epithelium was unaltered after such treatment. The total protein kinase activity was 70% higher in uterine extracts (cytosol) than in extracts from vaginal epithelium. Oestradiol treatment did not influence the total protein kinase activity in either tissue.

Animals↗