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F Wagner

Publications and source records attributed to F Wagner.

At least 19 recordsLinked to original sources
PubMed1992

Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity.

The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.

Cell Line↗
PubMed1992

Phorbol ester-induced amino-terminal phosphorylation of human JUN but not JUNB regulates transcriptional activation.

Phorbol ester tumor promoters activate gene transcription by regulating both the synthesis and posttranslational modification of the activator protein 1 (AP-1) transcription factor, c-Jun and JunB are components of the mammalian AP-1 complex. Here we demonstrate that in U-937 human leukemic cells, phorbol esters stimulate the phosphorylation of the amino terminus of human c-Jun (JUN) but not human JunB (JUNB). Mutational analysis indicates that serine-63 and -73, which reside within the putative regulatory domain of JUN, are required for both constitutive and phorbol 12-myristate 13-acetate-inducible N-terminal JUN phosphorylation. To determine the functional role of this N-terminal phosphorylation, we prepared several chimeric proteins containing the N-terminal 84 amino acids (positions 5-89) of human JUN or murine JUNB fused to the yeast GAL4 DNA-binding domain. This region was found to be sufficient for the phorbol ester-inducible transcriptional activity of JUN, but not JUNB. This induction was abolished by the mutation of serine-63 and -73 to leucine residues. Thus, we propose that phorbol esters enhance the trans-activation potential of JUN, but not JUNB, by the phosphorylation of the N-terminal regulatory domain of JUN.

Amino Acid Sequence↗
PubMed1992

Upstream regions of the c-jun promoter regulate phorbol ester-induced transcription in U937 leukemic cells.

To understand the mechanism by which phorbol esters (PMA) stimulate c-jun transcription in human leukemic cell line U937, we have mutated specific enhancer sequences within the c-jun promoter. We find in the region of DNA from -132 to +170 containing Sp1, C-TF and AP-1 sequences that mutation of the AP-1 sequence alone is not sufficient to abrogate transcription, and mutation of the Sp1 sequence increases transcription 4-fold. Although mutation of the CTF site had no effect, CTF and AP-1 mutations together totally abrogate PMA-induced transcription. In comparison mutations of either of these sites alone or together in a construct containing -1639/+740 of the c-jun promoter had no effect on transcription. Because this data suggested the possibility of other upstream control regions, we sequenced the promoter from -142 to -1639. This sequence demonstrates a greater than 70% homology between human, and mouse c-jun promoters for the region from -142 to -441, and a second AP-1-like site in the -183 to -192 region. Mutation of this site did not influence transcription by PMA. By making constructs containing varying portions of the promoter, we have identified the region between -142 and -711 to be responsible for mediating PMA-induced c-jun transcription.

Base Sequence↗
PubMed1992

Francis X. Dercum.

Explore the source record for details and available documents.

History, 20th Century↗
PubMed1992

Ethanol inhibits interferon-gamma secretion by human peripheral lymphocytes.

Clinical and epidemiological evidence exists that subjects who chronically abuse alcohol are disposed to infections and certain types of cancer. In vitro inhibition of mitogen-induced lymphocyte proliferation has been shown suggesting a direct immunosuppressive effect of ethanol. Using human peripheral blood mononuclear cells we could demonstrate in vitro for the first time that even low ethanol concentrations of 6 and 12.5 mM significantly inhibit spontaneous and mitogen-induced secretion of interferon-gamma. This effect was more pronounced with lower mitogen stimulation and it increased in a dose dependent manner when higher ethanol concentrations were used. Inhibition of cell proliferation as measured by 3H-thymidine incorporation did not parallel the inhibition of interferon-gamma secretion. As this lymphokine exerts a great number of immunostimulating effects, diminished secretion might well contribute to the immune defect observed in alcoholics.

Alcohol Drinking↗
PubMed1991

Regulation of c-jun expression and AP-1 enhancer activity by granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates the growth and differentiation of human hematopoietic progenitor cells by activating transcription of specific genes. The mechanism by which binding of GM-CSF to its receptor stimulates gene expression remains unknown. To examine this process in more detail, we have transfected human monocytic leukemia cells U937 with a plasmid containing an AP-1 enhancer element and a chloramphenicol acetyltransferase recorder gene and treated them with GM-CSF. We find that GM-CSF stimulates a 2-3-fold increase in chloramphenicol acetyltransferase activity over a concentration range 1-1,000 units/ml. Northern and Western blot analysis demonstrates that the mechanism by which GM-CSF stimulates AP-1 enhancer activity involves increases in c-jun and c-fos mRNA levels, and increases in Jun protein. In a similar fashion the treatment of normal human monocytes with GM-CSF also induced increases in total cellular c-jun. Because protein kinase C plays a crucial role in activating c-jun transcription we examined the role of this enzyme in mediating the effects of GM-CSF. Treatment of U937 cells with inhibitors of protein kinase C including staurosporine 10 nM and H-7 50 microM, or down-regulation of protein kinase C by phorbol ester pretreatment blocks the induction of c-jun by GM-CSF. However, HA which does not block protein kinase C had no effect on GM-CSF stimulation of c-jun RNA levels. In addition, GM-CSF treatment causes the rapid translocation of protein kinase C to the particulate fraction which was maximal by 5 min and returned to base line by 80 min. These data suggest that the binding of GM-CSF to its receptor stimulates increases in c-jun mRNA and protein and activates AP-1 enhancer activity. These effects may be at least in part mediated by activation of protein kinase C.

Alkaloids↗
PubMed1991

Haemodynamic effects of glyceryl trinitrate following repeated application of a transdermal delivery system with a phasic release profile.

The haemodynamic effects and plasma concentrations of glyceryl trinitrate (GTN) and its dinitrate metabolites were investigated in 8 healthy male volunteers during 5 days of application of a new transdermal delivery system (TDS) with time-dependent release characteristics, which were considered to prevent or to diminish development of nitrate tolerance. On the first and fifth day of administration the following haemodynamic parameters were determined: digital pulse ratio of height of systolic peak to height of dicrotic wave (i.e. a/b-ratio), heart rate and systolic blood pressure under orthostatic conditions. Peak plasma concentrations of GTN were 139 and 155 pg.ml-1 on the first and fifth day of treatment, and the corresponding trough concentrations (i.e. 24 h after administration) were 52.5 and 36.6 pg.ml-1, respectively. Compared to placebo, the area under the effect curve of the a/b-ratio of the digital pulse was increased on the first (25.6%) and fifth day (13%). A significant increase of heart rate and a decrease of systolic blood pressure were seen only on the first day of treatment. The haemodynamic effects of sublingual GTN 0.8 mg were reduced by 69% (a/b-ratio) and 52% (standing heart rate) on the fifth day compared to the pretreatment values. Thus, the phasic release of GTN from the new TDS can be demonstrated by the time course of the plasma concentrations of GTN and its metabolites. Nevertheless, following repeated administration the hemodynamic effects are blunted.

Administration, Cutaneous↗
PubMed1991

Differences in the antiischaemic effects of molsidomine and isosorbide dinitrate (ISDN) during acute and short-term administration in stable angina pectoris.

The acute and short-term effects of treatment with 10 consecutive doses of isosorbide dinitrate 40 mg t.i.d. and molsidomine 8 mg t.i.d. in slow release formulations were investigated in 10 patients with angiographically documented coronary artery disease and stable angina pectoris according to a randomized, double-blind, double-dummy, cross-over study design using conventional symptom-limited exercise testing. Acute exercise testing 3 h following the first dose of ISDN and molsidomine showed a significant reduction of maximal ST segment depression and of the area above the ST segments. Time to occurrence of 0.1 mV ST segment depression, exercise duration, time to onset of angina and exercise tolerance increased significantly. On the fourth treatment day with ISDN and molsidomine an attenuation of these antiischaemic effects was seen. The mean effects on ST segment depression, area above ST segments, time to occurrence of 0.1 mV ST segment depression, exercise duration, time to onset of angina and exercise tolerance were reduced by 40%, 44%, 47%, 58%, 54% and 65%, respectively, in patients administered ISDN and by 33%, 48%, 58%, 59%, 45% and 60% in those given molsidomine. Thus, following sustained short-term therapy the antiischaemic effects of both drugs seem to be attenuated. In this report no marked differences were found between ISDN and molsidomine.

Angina Pectoris↗
PubMed1990

Multiple doses of diacylglycerol and calcium ionophore are necessary to activate AP-1 enhancer activity and induce markers of macrophage differentiation.

In contrast to phorbol esters, multiple doses of diacylgycerols are needed to differentiate U937 human monoblastic leukemic cells to a macrophage-like phenotype. Although both of these agents similarly activate protein kinase C in vitro, it is not known why these agents appear to have differing biologic effects. One possibility is that they regulate gene transcription in slightly different ways. Regulation of gene transcription by phorbol esters is complex and involves the stimulation of the transactivating proteins Jun and Fos which form dimers and bind to the AP-1 enhancer elements (5'-TGAGTCA-3'). To understand whether diacylglycerols regulate gene transcription similarly to phorbol esters and to examine whether activation of AP-1 enhancer activity is correlated with differentiation, we have treated U937 human monoblastic leukemic cells with these agents and examined activation of transcription from AP-1 enhancer elements. We find that, although a single dose of diacylglycerol, like phorbol esters, is sufficient to elevate mRNA levels of both the c-jun and c-fos protooncogenes, in contrast to phorbol esters there is no increase in either Jun protein or activation of AP-1 enhancer activity. However, multiple doses of this agent given over 24 h stimulate repeated elevations in c-jun and c-fos mRNA, increases in Jun protein, and enhancer activation. Treatment of U937 cells with ionomycin, a calcium ionophore, also stimulates an increase in c-jun mRNA, but neither activates AP-1 enhancer activity nor stimulates differentiation of these cells. However ionomycin functions to enhance the effects of diacylglycerols both on transcriptional activation and U937 differentiation. These results suggest a complex regulation of AP-1 enhancer activity in U937 cells by diacylglycerols involving both transcriptional and post-transcriptional regulatory mechanisms. Maximal activation of AP-1 enhancer elements, and not changes in jun and fos mRNA, is correlated with increases in markers of U937 differentiation. These changes may be important in the early events leading to differentiation of hematopoietic cells.

Blotting, Northern↗
PubMed1990

Effect of thyrotropin-releasing hormone on immune functions of peripheral blood mononuclear cells.

The tripeptide thyrotropin-releasing hormone (TRH) works as a hypothalamic hormone, but is found also outside the brain in intrinsic nerve fibers of the gastrointestinal tract. There is evidence that TRH modulates the activity of immunocompetent cells, although there are only very few data on TRH-mediated immune effector functions. Since we could recently show that TRH inhibits monocyte activities we were also interested in other possible TRH modulated immune functions. Peripheral blood mononuclear cells (PBMC) from ten healthy subjects were cultured for 7 days and pulsed with 0.125 and 0.250 microgram/ml Pokeweed mitogen (PWM). 10(-12) to 10(-6) M TRH was added simultaneously with PWM. Lymphocyte proliferation [(3H]thymidine incorporation), interferon-gamma (IFN-gamma) activity (RIA) and immunoglobulin activities (IgG, IgM, IgA; ELISA) were determined in the supernatants. We could demonstrate a TRH-dependent decrease in PWM-pulsed IgG activity with significant (alpha = 0.05) values at 10(-8) and 10(-10) M (-29 +/- 6%/-16 +/- 3% for PWM 0.125 microgram/ml and -17 +/- 9%/-11 +/- 9% for PWM 0.250 microgram/ml). This inhibitory effect could be abolished by an anti-TRH antiserum. There was no TRH effect on IgM and IgA activities, IFN-gamma activity and lymphocyte proliferation compared with the PWM stimulated values alone. The described TRH effect on the polyclonal IgG response by PBMC gives further evidence for a functional link between the immune system and the endocrine system, although its underlying mechanism is not yet clear.

Enzyme-Linked Immunosorbent Assay↗
PubMed1990

Lack of effect of cefixime on the metabolism of vitamin K1.

It seems that cephalosporins bearing a N-methyl-thio-tetrazole or a methyl-thiadiazole moiety in their molecule can cause hypoprothombinemia in patients via inhibition of the metabolism of vitamin K1 if they are in addition in a vitamin K1-deficient state. The authors therefore studied the effects of two different oral doses (200 and 400 mg) of the cephalosporin cefixime on the metabolism of vitamin K1 in healthy volunteers, because the accumulation of vitamin K1-2,3-epoxide in plasma is a sensitive marker of coumarin-like activity of drugs. The results indicate that the development of hypoprothrombinemia due to an impairment of the metabolism of vitamin K1 by cefixime seems unlikely because only trace amounts of vitamin K1-2,3-epoxide could be determined in the plasma of the subjects investigated.

Administration, Oral↗
PubMed1990

Effect of the addition of microbial surfactants on hydrocarbon degradation in a soil population in a stirred reactor.

The hydrocarbon degradation rate could be doubled by the addition of sophorose lipids as biosurfactants in a model system containing 10% soil and a 1.35% hydrocarbon mixture of tetradecane, pentadecane, hexadecene, 1,2,4-trimethylcyclohexane, pristane (2,6,10,14-tetramethylpentadecane) phenyldecane and naphthalene suspended in mineral salts medium. The adaptation phases for two degradation phases were shortened, and the extent of degradation and final biomass were increased. The added biosurfactants were degraded after they had facilitated degradation of all hydrocarbon components.

Biodegradation, Environmental↗
PubMed1990

Relationship between pharmacokinetics and hemodynamic tolerance to isosorbide-5-mononitrate.

Healthy male volunteers received three different dose regimens of a controlled-release form of isosorbide-5-mononitrate (IS-5-MN; 60 mg per tablet). Dose regimen I consisted of a single daily dose of 60 mg given for 5 days. Dose regimen II was started with a dose of 60 mg, followed by 30 mg 12 h later and thereafter every 8 h. The last dose, on the 5th day was again 60 mg. In dose regimen III 60 mg followed by 30 mg 6 h later were administered every day for 5 days. The peripheral arterial and venous effects of IS-5-MN during the first and last dosing interval were followed by changes in the finger pulse curve, standing systolic blood pressure, heart rate, and venous distensibility. Plasma concentrations of IS-5-MN were measured frequently following the first and the last dose. Following dose regimen I all hemodynamic effects produced by the first dose were maintained during the study. The maximal plasma concentrations were about 400 ng/ml and the trough value, lower than 100 ng/ml. Following dose regimen II the hemodynamic effects of IS-5-MN and sublingual glyceroltrinitrate were completely abolished on the 5th day. Trough plasma concentrations were approximately 300 ng/ml during the entire study period. Following dose regimen III pronounced hemodynamic effects were seen on the 1st day. However, a significant attenuation of the hemodynamic effects was measured on the 5th day, when trough plasma concentrations were between 100 and 230 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Effect of substance P on immunoglobulin and interferon-gamma secretion by cultured human duodenal mucosa.

Recently, we have demonstrated a substance P (SP)-dependent modulation of in vitro IgM and interferon-gamma (IFN-gamma) secretion by human peripheral blood mononuclear cells, as well as lymphokine activities in supernatants of cultured duodenal mucosa. Therefore we investigated other local immunoregulatory effects of SP. Duodenal biopsies of 7 healthy subjects were cultured with Pokeweed mitogen (PWM, 1 microgram/ml) for 4 days at 37 degrees C in 1 ml medium each. SP was added in concentrations ranging from 10(-12)M to 10(-6)M on day 1. Fresh media with fresh PWM were added every day. IgG, IgM, IgA (ELISA) and IFN-gamma (RIA) were determined in the culture supernatants. Values were referred to 5 mg biopsy weight and expressed as % change in basal PWM pulsed secretion, or as units/ml. 10(-6) M and 10(-12) M SP increased secretion of all immunoglobulin isotypes. Compared to controls, 10(-6) M and 10(-12) M SP led to an increase in IgM secretion of up to 73 +/- 23% and 41 +/- 32% and to an increase in IgA secretion up to 96 +/- 35% and 25 +/- 33%, respectively (alpha = 0.02 for both isotypes at 10(-6) M). 10(-12) M to 10(-6) M SP led to a significant dose-dependent increase in IFN-gamma secretion from 7.08 +/- 1.65 up to 21.8 +/- 12.6 units/ml/5 mg. The maximum effect could be seen on culture days 3 and 4. We were able to demonstrate for the first time that SP stimulates PWM pulsed immunoglobulin and IFN-gamma secretion by human duodenal immunocompetent cells. These results support the hypothesis of local neuropeptidergic-immune interactions.

Adult↗