A rapid chemiluminescence-based method for the determination of total polyamines in biological samples
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Ornithine decarboxylase antizyme
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Diamine oxidase (DAO, EC 1.4.3.6) activity was examined in relation to polyamine content in Helianthus tuberosus L. during the first synchronous cell cycle induced in vitro by 2,4,-dichloro-phenoxyacetic acid in tuber slices and during the in vivo formation of the tuber. The optimal pH, buffer and dithiothreitol concentrations for the enzyme extraction and assay were determined. When added in the assay mixture, catalase enhanced DAO activity, while polyvinylpyrrolidone had no effect; both aminoguanidine and hydrazine inhibited enzyme activity. The time course of the reaction, based on the recovery of Δ1-pyrroline from labeled putrescine in lipophilic solvents, showed that it was linear up to 30 minutes; the Km of the enzyme for putrescine was of the order of 10−4 molar. During the first cell cycle, DAO activity exhibited a peak at 15 hours of activation while putrescine content gave a peak at 12 hours. During tuber formation (from August till October) DAO activity was relatively high during the first phase of growth (cell division), decreased until flowering (end of September-early October), and then newly increased during the cell enlargement phase preceding the entry into dormancy (November). Maximum putrescine content was observed at the end of October. The increase in DAO activity paralleled the accumulation of putrescine. This indicates a direct correlation between the biosynthesis and oxidation of putrescine which, as already demonstrated in animal systems, occur simultaneously in physiological stages of intense metabolism such as cell division or organ formation.
Helianthus
Polyamine oxidase
Polyamine
Dithiothreitol
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Putrescine, spermidine and spermine are polyamines found in all eukaryotic cells. At cellular pH's they are cations, being attached to polyanions such as DNA, RNA and phospholipids. Some PAS are conjugated with phenolic acids. Massive polyamine biosynthesis precedes DNA biosynthesis, and cells deprived of polyamines are locked into the G1 stage of the cell cycle. In plants, polyamines can affect rates of growth, the nature of differentiation, and patterns of senescence. Polyamine titers are influenced by light, hormones and environmental stresses, which generally increase arginine decarboxylase (ADC) activity. Stress stimuli lead to massive putrescine accumulation. Some phytopathogenic fungi have only one pathway to putrescine formation, via ornithine decarboxylase (ODC) while higher plants have both ODC and ADC. Since specific inhibition of the ODC pathway can be accomplished chemically, some plant diseases can be prevented without adverse effects on the host plant.
Polyamine
Ornithine decarboxylase antizyme
Arginine decarboxylase
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Ornithine decarboxylase antizyme
Polyamine
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Catabolism
Amine oxidase (copper-containing)
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Ornithine decarboxylase (ODC) activity and polyamine levels were measured during early development of the frog, Microhyla ornata. ODC activity was found to be high and it showed three major peaks during the first 60 hr of development. Putrescine and spermidine levels increased gradually during the above period with little change in spermine. Treatment of developing embryos with exogenous putrescine and spermidine prevented the normal increase in ODC activity. Spermine did not have any significant effect. Addition of ornithine also prevented the increase in ODC activity. Experiment using exogenous ornithine and alpha-methylornithine revealed that formation of putrescine and/or spermidine from ornithine is necessary for the suppression of ODC to occur. Suppression of ODC takes place even if conversion of putrescine to spermidine is blocked, indicating that putrescine, independent of its conversion to spermidine, also plays a role in ODC regulation.
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Polyamine
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ABSTRACT The aim of this study was to investigate the effect of cell spreading on the induction of ornithine decarboxylase and the rate of putrescine uptake in anchorage-dependent and anchorage-independent cells. Plating non-transformed IEC-6 epithelial cells at high versus low cell density restricted cell spreading from 900 μm2 to approximately 140 μm2, blunted the transient induction of ornithine decarboxylase activity from 202 to 32 pmol 14CO2/mg protein per hour and reduced the rate of [14C]putrescine uptake from 46 to 23 pmol/105 cells per hour. The mean spreading area of the cell population was controlled by coating tissue culture dishes with the nonadhesive polymer, polyHEMA. Ornithine decarboxylase activity and putrescine uptake correlated with cell spreading with minimal spreading (263 μm2) corresponding to an 83% decrease in ornithine decarboxylase activity and 51% decrease in the rate of putrescine uptake. Adding the RGD peptide, Gly-Arg-Gly-Glu-Ser-Pro to the medium of sparsely plated cells resulted in rapid reductions in cell spreading concomitant with dose-dependent decreases in ornithine decarboxylase activity and putrescine uptake. Finally, minimizing cell spreading by depriving cells of sub-stratum contact completely abolished serum-induced increases in ornithine decarboxylase and reduced the rate of putrescine uptake by 47%. In contrast to IEC-6 cells, ornithine decarboxylase of neoplastic HTC-116 cells was constitutively expressed with basal and stimulated activity (193 and 982 pmol 14CO2/mg protein per hour, respec-tively) completely independent of cell adhesion. Putrescine uptake, however, was abolished in the absence of cell adhesion. These data suggest that the induction of ornithine decarboxylase activity and the rate of putrescine uptake correlate with spreading of anchorage-dependent IEC-6 cells and that ornithine decarboxylase activity, but not putrescine uptake, appears to be independent of spreading of neoplastic HTC-116 cells.
Ornithine decarboxylase antizyme
Polyamine
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Amine oxidase (copper-containing)
Polyamine
Polyamine oxidase
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Objective To study the regulation of natural polyamines(putrescine,spermidine and spermine) on ornithine decarboxylase gene transcription in regenerating rat hepatocytes and to assess the roles of polyamines in liver regeneration. Methods Rat regenerating liver was induced by surgical partial hepatectomy(PH).The expressive levels of ODC mRNA in regenerating liver were measured by in situ hybridization analysis.Exogenous polyamines(dissolved in 0.9% NaCl) were administered subcutaneously to adult male Sprague-Dawley rats. Results ODC mRNA expression was always lower in high dose(20 mg/kg body weight) putrescine-treated rats compared with that in control rats during the observed regeneration,especially at the 2(nd),4(th) and 10(th) hours(P0.05) after PH;but it was higher in low dose(0.02 mg/kg body weight) putrescine-treated group compared with that in control group only at the 4(th) and 12(th) hours(P0.05) post-PH.The mRNA levels in 0.15 mg/kg spermidine-treated group were below the control group in the whole experiment except at the 10(th) hour post-PH;however,0.03 mg/kg spermidine treatment,first statistically decreasing the mRNA level in the 2 hours after PH,promoted the increase in ODC mRNA at the 4(th) and 6(th) hours after PH respectively.The effect of spermine on ODC mRNA expression was similar to that of spermidine.Conclusion Polyamines(especially spermine and spermine) of different concentrations are involved in feedback regulating ODC gene transcription in rat regenerating hepatocytes.
Polyamine
Liver Regeneration
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