Changes in polyamine synthesis and concentrations during chick embryo development

We have measured the activities of the two rate controlling enzymes in polyamine synthesis, L-ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAMDC), and the concentrations of the polyamines, putrescine, spermidine and spermine, in the developing chick embryo from laying to hatching. The embryo exhibited major peaks in the ODC and SAMDC activities as well as in the concentrations of all three polyamines at 15 h (gastrulation), 23-30 h (early organogenesis), days 4-5 (mid-organogenesis), and days 12-17 (organ growth and maturation). In the 4 and a half-day-old embryo, ODC activity and polyamine concentrations were about twice as high in the head region as compared to the trunk region. In the 14-day-old embryo, the highest ODC and SAMDC activities were found in lung, intestine and kidney, and there was a positive correlation between the enzyme activities and the growth rates of most organs/tissues.     

Polyamines and somatic embryogenesis in two Vitis vinifera cultivars

Polyamine content and activities of enzymes of polyamine biosynthesis were assayed during somatic embryogenesis in Vitis vinifera callus cultures of Chardonnay and Brachetto 'a grappolo lungo' (Brachetto g.l.) cultivars. The analyses were carried out on embryogenic callus samples, embryos at different stages and developing plants. Polyamine content, both in the free and PCA-soluble conjugated form, was higher in Brachetto g.l. than in Chardonnay, and putrescine was present at higher concentrations than the other polyamines. In all samples of both cultivars, ornithine decarboxylase activity (ODC, EC 4.1.1.17) was higher than arginine decarboxylase (ADC, EC 4.1.1.19), with a maximum in developing plant roots. S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) activity displayed a similar trend. The activities of all three enzymes were detected both in the supernatant and pellet fractions, indicating for the first time the presence of SAMDC activity in the particulate fraction. Particularly in the Chardonnay cultivar, an increase in the mRNAs expression patterns of ODC and SAMDC during morphogenesis from small embryos to plantlets was detected by northern blot, suggesting a direct correlation with enzymatic activities.     

Changes in cellular polyamine contents and activities of their biosynthetic enzymes at each phase of the cell cycle in by-2 cells

We analyzed changes in polyamine contents and the activities of biosynthetic enzymes during each phase of the cell cycle for a synchronized population of BY-2 cells. Based on our analysis of H³-thymidine incorporation flow cytometry, and the mitotic index, the M and G₂ phases seemed to occur at 8 h and from 2.5 to 8 h, respectively, after the release of aphidicolin. The respective activities of arginine decarboxylase (ADC), Ornithine decarboxylase (ODC), and S-adenosyl methionine decarboxylase (SAMDC) at the beginning (7.4, 11.2, and 5.5 nmol mg^-1 protein h^-1) were increased to 22.6, 22.1, and 15.1 nmol mg^-1 protein h^-1. However, those increases do not coincide with the general change in polyamines reported from animal cells. In addition, the bi-phasic activation of polyamine biosynthetic enzymes, such as those found in the general animal model, was observed with ADC and ODC but not with SAMDC. These results suggest that the general animal model for explaining polyamine changes and SAMDC activation in the cell cycle cannot be applied to BY-2 cells. Further, our flow-cytometric analysis of cell populations may be a useful tool for evaluating the effects of polyamines on cell cycle progression in BY-2 cells.     

Selenium and polyamine metabolism: different effect of selenite on liver and bursa of Fabricius ornithine decarboxylase activity

1. When injected i.p., sodium selenite promoted a marked increase of rat liver ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities; when administered with the diet for 6 weeks, a less marked increase in liver ODC was observed, whereas SAMDC was not significantly changed. 2. Protein synthesis was involved in the observed modifications. The rate of ODC inactivation was also changed. 3. ODC increase was accompanied by an enhanced putrescine concentration in liver. 4. A marked increase of ODC, accompanied by an enhancement of putrescine, was promoted by selenite (i.p.) also in chicken liver, together with an enhancement of glutathione concentration. Spermidine acetyltransferase (SAT) was also increased. 5. In the bursa of Fabricius, SAT activity was also increased, whereas ODC was decreased. However the expected modifications in polyamine concentration were not observed. 6. Decrease of ODC activity in the bursa was not due to an antizyme. 7. In vitro, selenite concentrations known to inhibit cell proliferation (greater than 1 microgram/ml) inhibited both ODC and SAT activities; at lower concentration, SAT activity was enhanced.     

Induction of ornithine decarboxylase and S-adenosylmethionine decarboxylase by sulfobromophthalein in rats

The administration of sulfobromophthalein (BSP, 0.5 mmol/kg, ip.) increased ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities to 30-fold and 5-fold, respectively, of the controls at 12 hr in the liver of rats. Parallel to the increase in ODC, there was an increase in hepatic putrescine content. However, spermine content tended to decrease. BSP increased ODC and SAMDC activities and putrescine content, but decreased spermine content, in a dose-dependent manner. Pretreatment of rats with actinomycin D and cycloheximide almost completely blocked the BSP-mediated increase of ODC and SAMDC activities. Pretreatment with glutathione (GSH) failed to inhibit BSP-mediated increase of ODC and SAMDC activities. In addition, the administration of BSP-GSH conjugate (0.5 mmol/kg, iv.) did not produce the increase of ODC and SAMDC activities. Pretreatment with phenobarbital and 3-methylcholanthrene did not inhibit BSP-mediated increase of ODC and SAMDC. The results indicate that BSP could cause changes in hepatic polyamine content due to the induction of ODC and SAMDC.     

UV-B辐射增强对水稻多胺代谢及内源激素含量的影响

研究表明,在处理前期（7－14d),增强的UV－B辐射能使供试水稻汕成63（Sy63）的精氨酸脱羧酶（ADC）、鸟氨酸脱羧酶（ODC）和s－腺苷蛋氨酸脱羧酶（SAMDC）活性分别平均增加165．74％、104．60％和89．60％,南川（NC）的这3种酶活性分别增加59．91％、41．30％和23．68％,IR65600－85只表现出ADC和ODC活性分别平均提高115．93％、14．45％,SAMDC活性却下降33．01％,在处理后期（21－28d）,Sy63的ADC－ODC活性分别对照平均增加89．72％、3．71％,NC则分别增加73．95％、27．38％,IR656000－85表现为ADC活性增加94．41％,ODC活性却下降13．57％,就SAMDC而言,处理后期（21－28d）,三者分别下降40．06％、19．20％和38．21％,多胺氧化酶（PAO）活性变化趋势恰好相反,从而引起多胺（PA）含量特别是腐胺（Put）含量明显上升,此外,UV－B辐射增强能使IAA和GA11／3含量在整个处理期间（7－28d）供试水稻品种（组合）Sy63分别平均下降58．92％和45．48％,NC分别减少43．31％和56．20％,IR65600－85则分别降低38．69％和47．33％,所有供试水稻品种（组合）的ZRs含量表现为处理前期（7－14d）有所降低,处理后期（21－28d）则明显提高,就ABA含量而言,整个处理时间（7－28d）,3个品种（组合）均比对照显著或后期（21－28d）则明显提高,就ABA含量而言,整个处理期间（7－28 d）,3个品种（组合）均比对照显著或极显著增加,三者分别提高了14．4%、99．6％和56．7％,从而显著降低IAA／ABA、GA1／3／ABA和ZRs/ABA的比值,影响水稻生长发育。     

Direct inhibitory effect of gonadotropin releasing hormone in the uterus of rat

Estradiol induced increase in ornithine decarboxylase (ODC) and glucosamine-6-phosphate synthase activities of rat uterus were inhibited by simultaneous treatment with gonadotropin releasing hormone (GnRH) or its agonists. The direct inhibitory effect of GnRH analogs was found to be dose dependent. It was observed that a higher dose of GnRH analog was needed to cause inhibition of glucosamine-6-phosphate synthase when compared to ODC activity. The inhibitory effect of GnRH was not observed if its injection was delayed following estradiol treatment. These results show that the extra-pituitary inhibitory effects of GnRH involves enzymes associated with cell proliferation.     

Polyamine biosynthetic decarboxylase activities following estradiol-17β or estriol stimulation of the immature rat uterus

Following a single intraperitoneal injection of 0.5 μg estradiol-17β (E2) into immature female rats uterine ornithine decarboxylase (ODC) activity increased to a peak at 4 hours postinjection. It decreased to intermediate levels by 6 hours and remained elevated until returning to control levels by 18 hours. When either 0.5 μg estriol (E3) or 0.05 μg E2 was injected, activity increased to a 4 hour ODC peak then decreased to control levels by 10 hours. The decrease to intermediate levels of ODC activity after dosing with 0.5 μg E2 occurred at the same time activity decreased to control levels following treatment with either 0.05 μg E2 or 0.5 μg E3.S-Adenosyl methionine decarboxylase (SAMDC) activity had increased by 4 hours following an injection of 0.5 μg E2 and remained elevated until 16 hours then decreased to control levels. An injection of 0.05 μg E2 or 0.5 μg E3 stimulated only a 4 hour peak after which time SAMDC decreased to control levels by 14 hours. After an Injection of 5.0 μg E2 SAMDC activity had increased by 4 hours and remained elevated for the remainder of the experiment (16 hours).Decreases in ODC activity following 4 and 10 hours may reflect a decrease in nuclear estrogen receptor levels. The ODC activity seen here following 0.5 μg E2 injection is similar in timing to that seen in other proliferating systems and may be due to a common mechanism.     

The effect of submergence,ethylene and gibberellin on polyamines and their biosynthetic enzymes in deepwater-rice internodes

Submergence and treatment with ethylene or gibberellic acid (GA₃) stimulates rapid growth in internodes of deepwater rice (Oryza sativa L. cv. Habiganj Aman II). This growth is based on greatly enhanced rate of cell-division activity in the intercalary meristem (IM) and on increased cell elongation. We chose polyamine biosynthesis as a biochemical marker for cell-division activity in the IM of rice stems. Upon submergence of the plant, the activity of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) in the IM increased six- to tenfold within 8 h; thereafter, SAMDC activity declined. Arginine decarboxylase (ADC; EC 4.1.1.19) showed a similar but less pronounced increase in activity. The activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in the IM was not affected by submergence. The levels of putrescine and spermidine also rose in the IM of submerged, whole plants while the concentration of spermine remained low. The increase in SAMDC activity was localized in the IM while the activity of ADC rose both in the node and the IM above it. The node also contained low levels of ODC activity which increased slightly following submergence. Increased activities of polyamine-synthesizing enzymes in the nodal region of submerged plants probably resulted from the promotion of adventitious root formation in the node. Treatment of excised rice-stem sections with ethylene or GA₃ enhanced the activities of SAMDC and ADC in the IM and inhibited the decline in the levels of putrescine and spermidine. We conclude that SAMDC and perhaps also ADC may serve as biochemical markers for the enhancement of cell-division activity in the IM of deepwater rice.Abbreviations ADC arginine decarboxylase - GA gibberellin - IM intercalary meristem - ODC ornithine decarboxylase - SAM S-adenosylmethionine - SAMDC SAM decarboxylase     

Ornithine and S-adenosylmethionine decarboxylase activities and polyamine contents in developing muscle tissues and primary cultures of normal and polymyopathic hamsters

Polyamine (putrescine, spermidine, and spermine) contents and ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylase activities have been assessed in an age-dependent manner, in normal and polymyopathic (dystrophic) hamster skeletal muscle, heart, and tongue extract and in primary tongue myoblast and skin fibroblast cultures. At 2 weeks of age, polyamine contents were significantly elevated in all of the dystrophic hamster tissues studied when compared with their age-matched controls. The degree of this elevation decreased with the age of the animals, generally, to a level where no significant difference in polyamine contents could be noted between normal and dystrophic hamster tissues. ODC and SAMDC activities in whole tissue extracts were consistently highest in 2-week-old muscle extracts and also declined with age. However, no significant changes in ODC or SAMDC activities were evident in any of the dystrophic muscle tissues studied when compared with their age-matched controls. Polyamine contents in dystrophic hamster myoblast and fibroblast primary cultures were also during proliferation (1 and 2 days after the initial seeding) compared with cultures prepared from normal hamsters. ODC and SAMDC activities in primary myoblast and fibroblast cultures clearly reflected the rate of cell proliferation, with highest activities found in subconfluent cell cultures. However, in general, no significant dystrophic-related abnormality in ODC or SAMDC activity was evident in proliferating myoblast or fibroblast cultures. These results suggest that the elevated polyamine contents of dystrophic hamster tissues and primary cultures may be due to a deficiency in polyamine catabolism or transport.     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities of S49 lymphoma cells by agents increasing cyclic AMP

We have examined the regulation of two key enzymes that control polyamine biosynthesis-L-ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) - by agents increasing cAMP in S49 lymphoma cells. Incubation of wild type S49 cells with beta-adrenergic agonists (terbutaline or isoproterenol) inhibited ODC and SAMDC activities rapidly (less than 2 hr). more quickly than these agents arrested the cells in the G1 phase of the cell cycle. The beta-adrenergic antagonist propranolol blocked inhibition of ODC activity produced by isoproterenol, but only if added simultaneously or less than 4 hr after the agonist. Incubation of wild type S49 cells with cholera toxin or PGE1 also inhibited ODC activity. Decreases in ODC activity produced by beta-adrenergic agonists, cholera toxin, PGE1 or dibutyryl cAMP were all enhanced by the phosphodiesterase inhibitor Ro 20-1724. Results of studies of ODC and SAMDC activity in S49 variants having lesions in the pathway of cAMP generation and action were as follows: kin- cells (which lack cAMP-dependent protein kinase activity) showed no inhibition of ODC by any agent; AC- cells (which have absent nucleotide coupling units in their adenylate cyclase system) only demonstrated inhibition in response to dibutyryl cAMP; UNC cells (which have deficient coupling of hormone receptors and adenylate cyclase) only demonstrated inhibition in response to dibutyryl cAMP and cholera toxin, and beta-depleted cells (which have a decreased number of beta-adrenergic receptors) responded as did wild type cells except for absent response to isoproterenol. We conclude that inhibition of ODC and SAMDC activity in S49 cells is an early response to agents that increase cAMP and that this action occurs via the "classical" pathways of activation of adenylate cyclase and protein kinase. These results in S49 cells contrast with evidence in other systems in which cAMP has been suggested to enhance polyamine biosynthesis, perhaps through alternative mechanisms.     

Temporal Profiles of Proteins Responsive to Transient Ischemia

The responses of long and short half-lived proteins to ischemia were measured in rat brain during 6 days of recovery from 30 min of transient forebrain ischemia produced by four-vessel occlusion. At the end of the ischemic interval, the neocortical activities of four vulnerable enzymes [ornithine (ODC) and S-adenosylmethionine (SAMDC) decarboxylases, and RNA polymerases I and II] were unchanged, but within 30 min of reperfusion, their activities dropped by 25-50%. The loss of substance P in the striatum and substantia nigra was slower, reaching about 50% by 12 h. On the other hand, the activities of 5 long half-lived enzymes did not change in the neocortex at 5 and 15 h of reperfusion and regional protein concentrations were essentially unaffected over 6 days survival. The rate and extent of normalization of the amounts or activities of the vulnerable proteins varied. RNA polymerase II and ODC activities were restored within 4 h, and ODC showed a biphasic increase in activity, with peaks at 10 h and 2-3 days. RNA polymerase I and SAMDC activities were restored by 18 h and 5 days, respectively, whereas substance P concentrations did not completely recover, even at 6-15 days. The greater the regional reduction of blood flow during ischemia, the larger the net change (gain or loss) of SAMDC or ODC activity and the longer the time required to normalize the activities of these enzymes. The average rate of proteolysis, assessed by measuring the rate of clearance of 14C from protein prelabeled with [14C]bicarbonate, was abnormal during the first 2 days of reperfusion. Postischemic changes in both protein synthesis and degradation could affect the amounts of some of the proteins responsive to transient ischemia.     

Polyamine Biosynthesis and Effect of Dicyclohexylamine during the Cell Cycle of Helianthus tuberosus Tuber

Polyamine content and the activities of their main biosynthetic enzymes, ornithine decarboxylase (ODC, EC 4.1.1.17), arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50), and arginase (EC 3.5.3.1.), were examined in crude extracts of Helianthus tuberosus tuber slices during the first synchronous cell cycle, induced by synthetic auxin, with or without the addition of 1 or 5 millimolar dicyclohexylamine (DCHA), an inhibitor of spermidine synthase. In the DCHA-treated slices a peak of accumulation of the drug was observed at 12 hours. Bound DCHA was also found. Free polyamine content generally increased, reaching a maximum at 12 to 18 hours in the S phase of the cycle; while spermidine content was decreased slightly with DCHA after 12 hours, putrescine almost doubled at 18 hours. Bound polyamines were also present. ODC and ADC showed a maximum activity at 15 and 18 to 21 hours, respectively, i.e. in the S phase; both activities increased slightly in the presence of 5 millimolar DCHA at or near the time of maximum activity. Arginase was initially very high and then rapidly decreased although a small peak of activity occurred at 15 hours. SAMDC, which had two peaks of activity, was initially inhibited by DCHA, and then stimulated, especially at 12 hours and in coincidence with the main peak, at 21 hours. Thus ODC, ADC, and SAMDC activities as well as polyamine titer increased before and during the S phase of the cell cycle and all declined during cell division. The slight inhibitory effect of DCHA was possibly due to its degradation in the tissue and to the fact that putrescine could substitute for the function(s) of spermidine.     

Polyamine titer and biosynthetic enzymes during tuber formation ofHelianthus tuberosus

During the formation ofHelianthus tuberosus tubers the activities of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC), examined in medullary parenchyma cells, increase with the increase in weight of the tuber. The ornithine decarboxylase (ODC) activity is about 100-fold less with respect to ADC activity, and it was detected only during the deceleration phase of the growth curve. Spermidine and spermine content are strictly related to the SAMDC activity and tuber growth. The increase of ADC and SAMDC activity is directly related to cell extension and increase in weight. The limited area of cell division in parenchyma tissue found during the first stage of tuber formation could justify the low ODC activity. The data suggest that ADC affects mainly growth processes, while ODC seems to be preferentially related to cell division.     

Essential role for polyamine biosynthesis in thyroxine stimulated pancreatic development in neonatal rats.

Administration of thyroxine to rat pups leads to precocious development of the pancreas. The role of ornithine decarboxylase (ODC) and polyamines in thyroxine-induced pancreatic maturation was examined. Rat pups (aged 5 days) were given daily subcutaneous injection of thyroxine (0.1 micrograms/g body wt.) until the day before death. Serial ODC activities were measured in pancreatic homogenates after 1, 2, 3, 4, 5, 6, 7 and 10 days of thyroxine treatment. There was a biphasic induction of ODC activities by thyroxine: an early peak appeared on day 2 of treatment followed by a decrease on day 4; a second peak was evident on day 5 and then a decrease to control values by day 7. Significant increases in tissue concentrations of putrescine and spermidine were observed concomitant with two peaks of ODC activity. Pancreatic amylase concentration, DNA and protein also showed a significant increase after thyroxine treatment. Difluoromethyl ornithine (DFMO), a specific ODC inhibitor, given orally (8% in drinking water) to nursing dams at postnatal day 5 for 5 days caused an 83% inhibition of pancreatic ODC activity in thyroxine-treated pups when compared to thyroxine-treated pups not exposed to DFMO. Concomitantly, the thyroxine-induced increases in pancreatic weight, protein and amylase activity were suppressed. Our results suggest that increases in ODC activities and polyamine levels are critical intermediary steps in the precocious induction of pancreatic development by thyroxine.     

Polyamine biosynthesis in the developing rabbit palate

Levels of putrescine, spermidine, and spermine and their biosynthetic enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) were measured in the developing rabbit palate between day 14 and day 18 of gestation. DNA, RNA, and protein synthesis were also measured during this time period to determine if a correlation exists between polyamine biogenesis and macromolecular synthesis. ODC activity was found to be twice as high on day 14 as on the succeeding days of gestation, while SAMDC activity did not change significantly. Levels of putrescine and spermine were higher on day 14 by 22% and 30%, respectively, than levels on day 18. Spermidine concentration did not change. DNA synthesis remained relatively constant between days 14 and 18 of gestation, suggesting that there is no peak in cell proliferation during this period. RNA synthesis was elevated significantly on day 14 and protein synthesis was significantly higher on both days 14 and 16. This data indicates that there is no correlation between polyamine synthesis and cell proliferation during this period of palatal development, but polyamines could play a regulatory role in RNA and/or protein synthesis.     

Comparative studies of the effects of stilbene compounds on hepatic ornithine decarboxylase and S-adenosylmethionine decarboxylase induction in rats

Trans-Stilbene oxide (TSO, 2 mmol/kg, ip.) induced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) to 60-fold and 5-fold of the controls, respectively, in the liver of rats. Parallel to ODC induction, there was a marked increase in putrescine content to 50-fold of the control levels. Cis-Stilbene oxide (CSO), a stereoisomer of TSO, also produced the induction of ODC and SAMDC and the increase in putrescine content. There was no difference in the ability to induce ODC and SAMDC between TSO and CSO with respect to the extents of induction and the time needed to reach maximal levels. Trans-Stilbene (TS), a mother compound of TSO, did not show such an effect on ODC, while cis-stilbene (CS) induced both ODC and SAMDC. Treatment with glutathione inhibited TSO- and CSO-mediated induction of ODC and SAMDC. These findings add new information concerning the abilities of TSO, CSO and CS on hepatic polyamine metabolism.