scholarly journals Human myeloma cells acquire resistance to difluoromethylornithine by amplification of ornithine decarboxylase gene

1987 ◽  
Vol 242 (1) ◽  
pp. 199-203 ◽  
Author(s):  
P Leinonen ◽  
L Alhonen-Hongisto ◽  
R Laine ◽  
O A Jänne ◽  
J Jänne
Keyword(s):  
Effective Dose ◽  
Ornithine Decarboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Synthesis Rate ◽  
Irreversible Inhibitor ◽  
Myeloma Cells ◽  
Cultured Human Cells ◽  
Enzyme Molecules ◽  
Dose Dependent

Stepwise increments of the concentration of 2-difluoromethylornithine (DFMO), a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (ODC), resulted in a selection of cultured human IgG-myeloma cells (Sultan cell line) capable of growing in the presence of up to 3 mM-DFMO. This capacity was associated with 10-fold increase in ODC activity in the dialysed extracts of drug-resistant myeloma cells, markedly enhanced synthesis rate for ODC enzyme molecules, as revealed by a 20 min [35S]methionine labelling of cellular proteins, followed by specific immunoprecipitation and SDS/polyacrylamide-gel electrophoresis, dose-dependently increased expression of ODC mRNA in resistant cells (effective dose causing 50% inhibition), dose-dependent amplification of ODC gene sequences in a 9-kilobase-pairs EcoRI genomic DNA fragment, and (v) a 10-fold increase in the ED50 (effective dose causing 50% inhibition) for the anti-proliferative action of DFMO in these myeloma cells. These results represent one of the few gene amplifications described in cultured human cells.

scholarly journals Polyamine-stimulated alteration of the ornithine decarboxylase molecule in Physarum polycephalum

1983 ◽  
Vol 214 (2) ◽  
pp. 345-351 ◽  
Author(s):  
J L A Mitchell ◽  
J M Wilson
Keyword(s):  
Ornithine Decarboxylase ◽  
Gel Electrophoresis ◽  
Physarum Polycephalum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Radioactivity ◽  
Covalent Attachment ◽  
Two Dimensional ◽  
Irreversible Inhibitor ◽  
Enzyme Modification ◽  
High Specific Radioactivity

The molecular mechanism for polyamine-stimulated feedback modification of ornithine decarboxylase isolated from Physarum polycephalum was investigated by using two-dimensional polyacrylamide-gel electrophoresis. Partially purified A-form enzyme was converted into the B-form enzyme by isolated fractions of the Physarum A-B-converting protein, and the substrates and products were subsequently labelled by covalent addition of alpha-difluoro[14C]methylornithine, an enzyme-activated irreversible inhibitor. The active (A-form) and inactive (B-form) states of this enzyme were found to have the same Mr value, 52 000, yet they differed noticeably in their pI values, 5.45 and 5.65 respectively. In further experiments, the use of high-specific-radioactivity [3H]spermidine to stimulate this enzyme modification was shown not to result in the covalent attachment of this polyamine to ornithine decarboxylase. These results demonstrate that the polyamine-induced modification of ornithine decarboxylase in Physarum is not due to any of the mechanisms previously suggested for ornithine decarboxylase inactivation in this and other eukaryotes, namely phosphorylation, covalent polyamine addition or the non-covalent association of a specific low-Mr protein.

scholarly journals Multiple regulation of ornithine decarboxylase in enzyme-overproducing cells

1993 ◽  
Vol 289 (2) ◽  
pp. 581-586 ◽  
Author(s):  
T Kameji ◽  
S Hayashi ◽  
K Hoshino ◽  
Y Kakinuma ◽  
K Igarashi
Keyword(s):  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Synthesis Rate ◽  
Translational Efficiency ◽  
Irreversible Inhibitor ◽  
Variant Cell ◽  
Mrna Content ◽  
The Stability ◽  
Variant Cell Line ◽  
Fm3a Cells

We have isolated from mouse FM3A cells a variant cell line, termed EXOD-1, that overproduces ornithine decarboxylase (ODC). The cells were resistant to alpha-difluoromethylornithine, an irreversible inhibitor of the enzyme, and produced the enzyme protein to the extent of approx. 3-6% of total cytosolic protein. The rate of ODC synthesis in this cell line accounted for 25-50% of the rate of total protein synthesis. The amounts of the ODC gene and its mRNA in the variant cells were both about 60 times as much as those in wild-type FM3A cells. Upon removal of the inhibitor, the growth of the ODC-overproducing cells was stimulated approx. 2-fold. Under these conditions, the rate of ODC synthesis increased about 4-fold on day 1 and then decreased to near the original level by day 3. The amount of ODC mRNA increased about 1.7-fold on day 1 and 2.5-fold on day 3. No correlation was observed between changes in ODC synthesis rate and in ODC mRNA content, suggesting a translational repression of ODC mRNA due to accumulation of polyamines. In fact, the cellular contents of putrescine and spermidine markedly increased and that of spermine inversely decreased during the same period. Pulse-chase experiments showed that the accumulation of putrescine and spermidine also elicited a rapid degradation of ODC. Excess amounts of newly synthesized putrescine and cadaverine were excreted into the medium, whereas spermidine, spermine and acetylated polyamines were undetectable there. We conclude that ODC regulation upon removal of the inhibitor is dependent on at least three steps, namely the level of mRNA, the translational efficiency of mRNA and the stability of the enzyme, the last two of which are involved in cellular polyamines.

Increased Expression of u-PA and u-PAR on Monocytes by LDL and Lp(a) Lipoproteins – Consequences for Plasmin Generation and Monocyte Adhesion

1999 ◽  
Vol 81 (04) ◽  
pp. 594-560 ◽  
Author(s):  
Florence Ganné ◽  
Marc Vasse ◽  
Jean-Louis Beaudeu ◽  
Jacqueline Peynet ◽  
Arnaud François ◽  
...  
Keyword(s):  
Fold Increase ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Atheromatous Plaque ◽  
Monocyte Adhesion ◽  
Blood Monocytes ◽  
Proteolytic Cascade ◽  
Ecm Degradation ◽  
Dose Dependent Increase ◽  
Dose Dependent

SummaryMonocyte-derived foam cells figure prominently in rupture-prone regions of atherosclerotic plaque. As urokinase/urokinase-receptor (u-PA/u-PAR) is the trigger of a proteolytic cascade responsible for ECM degradation, we have examined the effect of atherogenic lipoproteins on monocyte surface expression of u-PAR and u-PA. Peripheral blood monocytes, isolated from 10 healthy volunteers, were incubated with 10 to 200 µg/ml of native or oxidised (ox-) atherogenous lipoproteins for 18 h and cell surface expression of u-PA and u-PAR was analysed by flow cytometry. Both LDL and Lp(a) induced a dose-dependent increase in u-PA (1.6-fold increase with 200 μg/ml of ox-LDL) and u-PAR [1.7-fold increase with 200 μg/ml of ox-Lp(a)]. There is a great variability of the response among the donors, some of them remaining non-responders (absence of increase of u-PA or u-PAR) even at 200 μg/ml of lipoproteins. In positive responders, enhanced u-PA/u-PAR is associated with a significant increase of plasmin generation (1.9-fold increase with 200 μg/ml of ox-LDL), as determined by an amidolytic assay. Furthermore, monocyte adhesion to vitronectin and fibrinogen was significantly enhanced by the lipoproteins [respectively 2-fold and 1.7-fold increase with 200 μg/ml of ox-Lp(a)], due to the increase of u-PAR and ICAM-1, which are receptors for vitronectin and fibrinogen. These data suggest that atherogenous lipoproteins could contribute to the development of atheromatous plaque by increasing monocyte adhesion and trigger plaque weakening by inducing ECM degradation.

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

1976 ◽  
Vol 35 (02) ◽  
pp. 350-357 ◽  
Author(s):  
Hana Bessler ◽  
Galila Agam ◽  
Meir Djaldetti
Keyword(s):  
Protein Synthesis ◽  
Fold Increase ◽  
Human Platelets ◽  
Polystyrene Latex ◽  
Latex Particles ◽  
The Third ◽  
Platelet Protein ◽  
Dose Dependent ◽  
Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

Genotoxicity and Cell Proliferative Activity of 3-Chloro-4-(Dichloromethyl)-5-Hydroxy-2(5H)-Furanone [MX] in Rat Glandular Stomach

1992 ◽  
Vol 25 (11) ◽  
pp. 341-345 ◽  
Author(s):  
C. Furihata ◽  
M. Yamashita ◽  
N. Kinae ◽  
T. Matsushima
Keyword(s):  
Cell Proliferation ◽  
Body Weight ◽  
Dna Synthesis ◽  
Ornithine Decarboxylase ◽  
Tap Water ◽  
Fold Increase ◽  
Single Strand ◽  
Glandular Stomach ◽  
Decarboxylase Activity ◽  
Pyloric Mucosa

MX is a strong direct acting mutagen on Salmonella typhimurium TA100 and is present in chlorinated tap water which contains organic compounds. MX was administered orally to 7-week-old male F344 rats, and its geno-toxicity in the pyloric mucosa of stomach was examined by analysis of DNA single strand scissions by the alkaline elution method. The effect of MX on cell proliferation was examined by assays of the inductions of replicative DNA synthesis and ornithine decarboxylase. MX at closes of 20-48 mg/kg body weight induced DNA single strand scissions dose-dependently (p<0.02) in the pyloric mucosa of the stomach 2 h after its administration. Moreover at doses of 10-60 mg/kg body weight, it induced up to 21-fold increase in replicative DNA synthesis (p<0.01) 16 h after its administration. At doses of 10-60 mg/kg body weight, it induced up to 100-fold increase in ornithine decarboxylase activity with a maximum 16 h after its administration. These results suggest that MX is genotoxic and induces cell proliferation in the glandular stomach of rats.

Biochemical studies of intrauterine components of the tammar wallaby Macropus eugenii during pregnancy

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 325-338
Author(s):  
Elizabeth J. Thornber ◽  
Marilyn B. Renfree ◽  
Gregory I. Wallace
Keyword(s):  
Protein Concentration ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Tammar Wallaby ◽  
Macropus Eugenii ◽  
Tenfold Increase ◽  
Specific Proteins ◽  
Wet Weight

The in vitro uptake and incorporation of [3H]ui idine by blastocysts of the tammar wallaby showed a 16- and 30-fold increase from day 0 to day 10 after removal of pouch young, respectively. Two of the six non-expanded blastocysts recovered on day 5 showed a tenfold increase in incorporation. During the first ten days after removal of pouch young the diameter of the blastocyst increased threefold. Endometrial exudate from gravid uteri had a higher protein concentration than exudate from nongravid uteri (39·5 ± 0·9 and 32·0 ± 2·0 mg/ml (mean ± s.e.m.), respectively). Endometrial exudates from uteri where the blastocyst was actively growing were found to contain six uterine-specific proteins. These were separated by gradient polyacrylamide gel electrophoresis. Two of the proteins were pre-albumins and the others were larger molecules (M.W. 153000–670000). Two proteins were only present at particular stages of pregnancy: the other four were present at all stages from diapause to birth, in exudate from gravid and nongravid uteri. The specific binding of progesterone and androstenedione to proteins in endometrial exudates or uterine flushings from pregnant wallabies was less than one per cent of the value obtained from day-5 pregnant rabbits. The ability of mouse blastocysts to take up and incorporate [3H]uridine into acidinsoluble material increased threefold in the presence of day-10 endometrial exudates from wallabies. However, this was less than ten percent of the values obtained in the presence of bovine serum albumin. The concentration of calcium in endometrial exudates increased from 23·6 to 45·2 μg/ml during pregnancy; in endometrium it remained at 88·7 μg/g (wet weight) throughout pregnancy, and in plasma it was 53·3 μg/ml. The concentration of zinc in endometrial exudates was 4·5 μg/ml; in endometrium it decreased from 21·8 to 13·3 μg/g (wet weight) during pregnancy and in plasma it was 0·6 μg/ml.

scholarly journals Effect of inflammation-mediated endothelial metabolic shift on endothelial barrier function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
M Aslam ◽  
H Idrees ◽  
C W Hamm ◽  
Y Ladilov
Keyword(s):  
Endothelial Cells ◽  
Glucose Uptake ◽  
Barrier Function ◽  
Atp Synthesis ◽  
Fold Increase ◽  
Endothelial Barrier ◽  
Synthesis Rate ◽  
Endothelial Barrier Function ◽  
Metabolic Switch ◽  
Barrier Integrity

Abstract Background The integrity of the endothelial cell barrier of the microvasculature is compromised by inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial metabolism as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial metabolism and its role in the maintenance of endothelial barrier integrity. Methods The study was carried out on cultured human umbilical vein endothelial cells and rat coronary microvascular endothelial cells. Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFα for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). Total cellular ATP concentration was measured using luminescence-based commercial kit (ATPLite, PerkinElmer). Mitochondrial mass was analysed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Results Treatment of human endothelial cells with TNFα resulted in significant suppression of mitochondrial and upregulation of glycolytic ATP synthesis rate, suggesting a metabolic switch. This was accompanied by a reduction in mitochondrial content (mtDNA/nDNA), reduction in total cellular ATP levels, an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFα caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFα-induced endothelial barrier failure. On the other hand pharmacological activation of AMPK, a potent inducer of mitochondrial biogenesis, could attenuate TNFα-induced but not 2DG-induced endothelial hyperpermeability. Conclusion The study demonstrates that TNFα induces metabolic switch towards glycolysis in endothelial cells. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFα-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Justus-Liebig University GiessenDZHK (German Centre for Cardiovascular Research), partner site Rhein-Main, Bad Nauheim, Germany

scholarly journals Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 810-817
Author(s):  
KJ Balazovich ◽  
JE Smolen ◽  
LA Boxer
Keyword(s):  
Protein Kinase ◽  
Phorbol Esters ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Dose Dependent

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the “translocation” of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

scholarly journals Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 913-919 ◽  
Author(s):  
R J Sargeant ◽  
M R Pâquet
Keyword(s):  
Glucose Transporter ◽  
Insulin Treatment ◽  
Fold Increase ◽  
Glucose Transporters ◽  
Synthesis Rate ◽  
Insulin Stimulation ◽  
Transporter Proteins ◽  
Specific Manner ◽  
Glucose Transporter Proteins ◽  
Synthesis And Degradation

The effect of continuous insulin stimulation on the rates of turnover and on the total cellular contents of the glucose-transporter proteins GLUT1 and GLUT4 in 3T3-L1 adipocytes was investigated. Pulse-and-chase studies with [35S]methionine followed by immunoprecipitation of GLUT1 and GLUT4 with isoform-specific antibodies revealed the half-lives of these proteins to be 19 h and 50 h respectively. Inclusion of 100 nM insulin in the chase medium resulted in a decrease in the half-lives of both proteins to about 15.5 h. This effect of insulin was specific for the glucose-transporter proteins, as the average half-life of all proteins was found to be 55 h both with and without insulin stimulation. The effect of insulin on the rate of synthesis of the glucose transporters was determined by the rate of incorporation of [35S]methionine. After 24 h of insulin treatment, the rate of synthesis of GLUT1 and GLUT4 were elevated over control levels by 3.5-fold and 2-fold respectively. After 72 h of treatment under the same conditions, the rate of synthesis of GLUT1 remained elevated by 2.5-fold, whereas the GLUT4 synthesis rate was not different from control levels. Western-blot analysis of total cellular membranes revealed a 4.5-fold increase in total cellular GLUT1 content and a 50% decrease in total cellular GLUT4 after 72 h of insulin treatment. These observations suggest that the rates of synthesis and degradation of GLUT1 and GLUT4 in 3T3-L1 adipocytes are regulated independently and that these cells respond to prolonged insulin treatment by altering the metabolism of GLUT1 and GLUT4 proteins in a specific manner.

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