Cadaverine, an Essential Diamine for the Normal Root Development of Germinating Soybean (Glycine max) Seeds

When the polyamine content of soybean (Glycine max) seeds was examined during the early stages of germination, the major polyamine in the cotyledons was found to be spermidine, followed by spermine; while very low concentrations of cadaverine were found. In the embryonic axes, however, cadaverine was the main polyamine and its content markedly increased 24 hours after the start of germination. When the germination of the seeds was performed in the presence of 1 millimolar α-difluoromethylornithine (DFMO), a marked decrease in the cadaverine content was found, while the other polyamines were not affected. This decrease of the cadaverine content was already noticeable after the first hours of germination. In the presence of DFMO, a pronounced elongation in the roots of the seedlings and a marked decrease in the appearance of secondary roots as compared with controls, was observed. This abnormal rooting of the seedlings caused by DFMO was almost completely reverted by the addition of 1 millimolar cadaverine. The latter also increased the appearance of secondary roots in the seedlings. The decrease in the cadaverine content produced by DFMO could be traced to a strong inhibition of lysine decarboxylase. A temporal correlation between the increase in cadaverine content and the increase in lysine decarboxylase activity was found. Both reached a maximum at the second day of germination. The activity of diamine oxidase, the cadaverine degrading enzyme, started to increase at the third day and reached a maximum between the fourth and fifth day of germination. DFMO increased the activity of diamine oxidase by about 25%. Hence, the large decrease in cadaverine content produced by DFMO has to be attributed to the in vivo suppression of lysine decarboxylase activity. Ornithine decarboxylase activity was also suppressed by DFMO, but putrescine and spermidine contents were not affected, except in the meristematic tissues. The obtained results suggest an important role for cadaverine in the normal rooting process of soybean seedlings.     

Distribution of polyamines and their related catabolic enzyme in etiolated and light-grown leguminosae seedlings

Diamine-oxidase (DAO; EC 1.4.3.6) activity and di-and polyamine levels were estimated along the epicotyl and root of light-grown and etiolated lentil (Lens culinaris Medicus) and pea (Pisum sativum L.) seedlings. The activity of DAO was higher in etiolated epicotyls than in lightgrown ones. In both species there was a positive correlation between DAO activity and the diamine (putrescine and cadaverine) levels along the whole epicotyl and root. Polyamine (spermine and spermidine) distribution seemed to be associated with the meristematic and elongating zone of the epicotyl and root. The physiological function of DAO is discussed in relation to its possible role in providing hydrogen peroxide to peroxidase-dependent reactions occurring in the cell wall.Abbreviations CAD cadaverine - DA diamine - DAO diamine oxidase - PA polyamine - PUT putrescine - SPD spermidine - SPM spermine     

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.     

Effects of dietary polyamines and clofibrate on metabolism of polyamines in the rat

The activities of catalase, polyamine oxidase, diamine oxidase, ornithine decarboxylase, and peroxisomal β-oxidation were assayed in homogenates from liver and small intestinal mucosa of rats which had been fed either a diet very low in polyamines or a diet containing five times the levels of dietary polyamines (putrescine, spermine, and spermidine) found in a standard rat diet. In rats fed the high polyamine diet, hepatic activities of catalase and polyamine oxidase were significantly decreased. Levels of the other activities were unchanged, except that intestinal ornithine decarboxylase was decreased. In rats treated simultaneously with clofibrate, the high polyamine diet restored activities of catalase, ornithine decarboxylase, and polyamine oxidase back to levels found in rats fed the low polyamine diet. The expected increase in activity of peroxisomal β-oxidation was observed, although this was somewhat diminished in rats fed the high polyamine diet. Intestinal diamine oxidase activity was stimulated by clofibrate, particularly in rats fed the high polyamine diet. For the duration of the experiment (20 days), levels of putrescine, spermine, and spermidine in blood remained remarkably constant irrespective of treatment, suggesting that polyamine homeostasis is essentially independent of dietary supply of polyamines. It is suggested that intestinal absorption/metabolism of polyamines is of significance in this respect. Treatment with clofibrate appeared to alter polyamine homeostasis.     

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.     

Competition of homologous substrates, putrescine and cadaverine, in the reaction catalyzed by pea diamine oxidase.

The determination of diamine oxidase activity with the ninhydrin reagent was used for monitoring of simultaneous oxidation of two homologous substrates, putrescine and cadaverine, which give different colour products (519 and 417 nm). We measured the reaction rates of oxidation of both substrates in different proportion and compared them with the total reaction rate determined by the guaiacol method. The substrates show competition with inhibition constants of putrescine against cadaverine of 0.14 mmol.l-1 and cadaverine against putrescine of 6.4 mumol.l-1.     

Effect of fat free diet during the last week of pregnancy upon the linoleic and arachidonic acid content of fetal organ lipids and placenta lipids of the rat

Pregnant Wistar rats were fed a fatfree diet from day 16--22 of pregnancy. On day 22, the fatty acid components of cholesterol esters, triglycerides, free fatty acids and phospholipids of maternal (brain, muscle, serum, white adipose tissue, liver) and fetal (brain, carcass, serum, liver) tissues, including the placenta, were examined gaschromatographically for the participation of linoleic and arachidonic acid. In all fetal and maternal organs the linoleic acid levels in the fatty acid patterns were strongly reduced. The alterations nearly always involved all the lipid fractions of a tissue and were mostly equal within a tissue. The strongest decreases of linoleic acid occurred in the placenta, and the weakest, in the lipids of maternal muscle and maternal adipose tissue. The linoleic acid alterations were principally similar in fetal and the corresponding maternal tissues, while being less pronounced in case of maternal muscle. The participation of arachidonic acid in the fatty acid pattern is completely retained in the lipids of fetal organs, and is even enhanced in those of the placenta.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Polyamine synthesis from proline in the developing porcine placenta

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about polyamine synthesis in the porcine placenta during conceptus development. The present study was conducted to test the hypothesis that arginine and proline are the major sources of ornithine for placental polyamine production in pigs. Placentae, amniotic fluid, and allantoic fluid were obtained from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, and 110 of the 114-day gestation (n = 6 per day). Placentae as well as amniotic and allantoic fluids were analyzed for arginase, proline oxidase, ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), proline transport, concentrations of amino acids and polyamines, and polyamine synthesis using established radiochemical and chromatographic methods. Neither arginase activity nor conversion of arginine into polyamines was detected in the porcine placenta. In contrast, both proline and ornithine were converted into putrescine, spermidine, and spermine in placental tissue throughout pregnancy. The activities of proline oxidase, OAT, and ODC as well as proline transport, polyamine synthesis from proline, and polyamine concentrations increased markedly between Days 20 and 40 of gestation, declined between Days 40 and 90 of gestation, and remained at the reduced level through Day 110 of gestation. Proline oxidase and OAT, but not arginase, were present in allantoic and amniotic fluids for the production of ornithine (the immediate substrate for polyamine synthesis). The activities of these two enzymes as well as the concentrations of ornithine and total polyamines in fetal fluids were highest at Day 40 but lowest at Days 20, 90, and 110 of gestation. These results indicate that proline is the major amino acid for polyamine synthesis in the porcine placenta and that the activity of this synthetic pathway is maximal during early pregnancy, when placental growth is most rapid. Our novel findings provide a new base of information for future studies to define the role of proline in fetoplacental growth and development.     

Gamma-aminobutyric acid (GABA) formation from putrescine in guinea-pig liver during ontogenesis

1. The changes in hepatic diamine oxidase (DAO) activity of the foetal and maternal origin and their relations to GABA formation during pregnancy in guinea-pigs are described. 2. Foetal DAO activity continuously increased while the maternal enzyme from the 45th day of gestation onwards decreased. 3. Conversion of putrescine to GABA via oxidative deamination has been detected in the earliest studied day i.e. the 34th.     

Response of tissue diamine oxidase activity to polyamine administration.

The administration to rats of putrescine (750 mumol/kg body wt.) caused in liver, kidney and heart an increase in putrescine at 1 h and in diamine oxidase (EC 1.4.3.6) activity within 3-6 h. An increase in spermidine was observed at 9 h in liver and at 6 h in kidney, whereas in heart there was no change. The increase in diamine oxidase activity by exogenous putrescine was prevented by the administration of actinomycin D and cycloheximide, suggesting that syntheses of mRNA and protein are involved. Equimolar doses of 1,3-diaminopropane, 1,5-diaminopentane and monoacetylputrescine stimulated, similarly to putrescine, hepatic, renal and cardiac diamine oxidase activity. After the injection of a non-toxic dose of spermidine (750 mumol/kg body wt.), the increase in diamine oxidase activity occurred at 9 h in all the tissues studied, when a substantial putrescine formation from spermidine occurred. sym-Norspermidine, which is unable to form putrescine, did not cause an increase in enzyme activity. The possibility that the tissue contents of putrescine might regulate diamine oxidase activity is discussed.     

Polyamine oxidase and tissue transglutaminase activation in rat small intestine by polyamines.

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.     

Polyamine catabolism in dormant embryos of the spindle tree (Euonymus europaeus L.) and in dormancy break obtained after treatment with gibberellic acid

Previously we showed that dormancy break of spindle tree embryos after gibberellic acid (GA₃) treatment was followed by an increase in arginine decarboxylase (ADC) activity (Béranger-Novat N. et al., Plant Sc. 102: 139–145, 1994). These results indicated that arginine decarboxylase pathway mediate hormone-induced growth responses in spindle tree embryos. In the present investigation we show that in GA₃-treated embryos diamine oxidase (DAO) increases immediately after putrescine content and the increase in DAO activity paralleles the accumulation of putrescine at the beginning of the culture (before the visible appearance of the radicle). In this system polyamine oxidase (PAO) increases immediately after DAO activity and follows closely the increase in spermidine content. These results demonstrate a direct correlation between the biosynthesis and oxidation of putrescine and spermidine. At every stage of development DAO and putrescine levels are lower than spermidine and PAO levels. Dormant embryos can be distinguished from GA₃-treated embryos by a complete lack of putrescine accumulation. In dormant embryos compared to GA₃-treated embryos DAO changed more or less in parallel and on the whole seemed to follow the same content and distribution, but the kinetics of the activation of DAOs were different in dormant embryos with a delay of 1.5 day for the first and 1 day for the second peak. During the first days of culture at least up to 4 days the distribution of spermidine and PAO in GA₃-treated embryos followed the same pattern observed in dormant embryos, but the levels of spermidine and PAO were greatly reduced in dormant embryos. On the other hand the kinetics of the activation of PAOs were different in dormant embryos with a delay of 1 day. The results suggest that dormant embryos are deficient in their ability to synthesize polyamines efficiently and support the view that spermidine catabolism (via PAO pool) is limiting in untreated embryos during the first days of culture.     

Ornithine decarboxylase, transglutaminase, diamine oxidase and total diamines and polyamines in maternal liver and kidney throughout rat pregnancy.

Ornithine decarboxylase (ODC; EC 4.1.1.17), transglutaminase (EC 2.3.2.13), diamine oxidase (DAO; EC 1.4.3.6) and total di- and poly-amines were studied in rat liver and kidney cortex throughout pregnancy. In liver, ODC activity exhibited two major peaks (4.5-5 times the control activities) on days 15 and 17. Also putrescine and spermidine increased biphasically (3-4-fold), but no variation in spermine content was observed. Transglutaminase activity showed slight variations only near the end of gestation. In kidney, ODC activity did not fluctuate significantly during pregnancy, whereas both transglutaminase activity and putrescine content showed three major increases, in very early, middle and late pregnancy. No significant variations in spermidine and spermine were observed. In both organs, DAO activity, very low or undetectable until day 10, dramatically increased (10- and 20-fold in kidney and liver respectively) in the second half of pregnancy, reaching maxima on days 16-17 and 19. The results obtained for transglutaminase, ODC and total di- and poly-amines are interpreted on the basis of hyperplastic and hypertrophic events in the liver and kidney respectively. The behaviour of DAO suggests that the enzyme plays an important role in the control of intracellular diamine concentration.     

Tissue distribution of cocaine in the pregnant rat

Cocaine hydrochloride was administered by single intraperitoneal (IP) doses to pregnant rats at day 18 or 19 of gestation. Plasma and tissue cocaine and norcocaine concentrations were measured by high-pressure liquid chromatography. Pharmacokinetic analysis of concentration versus time data showed rapid distribution of cocaine and its metabolite to maternal and fetal tissues. The area under the cocaine concentration versus time curve (AUC) in fetus compared to maternal plasma was 3.33. The half-life of cocaine in the maternal plasma and fetus was 46 and 55 minutes, respectively, similar to values reported for cocaine elimination half-life in human plasma. The order of cocaine concentrations was placenta greater than fetal liver greater than maternal heart greater than whole fetus greater than fetal brain greater than maternal brain = maternal plasma. Norcocaine concentrations were usually less than 20% of cocaine concentrations in plasma and tissues. These results support extensive fetal exposure to cocaine following administration to pregnant rodents. Pharmacodynamic studies of cocaine in pregnancy should consider the effects of the drug on the developing fetus.     

Ethanol perturbs polyamine metabolism in the phytopathogenic fungus Pyrenophora avenae

Biomass production by the plant pathogenic fungus Pyrenophora avenae was reduced following growth in 1, 3 and 6% ethanol. Although cadaverine concentration was not affected by growth in ethanol, putrescine and spermine concentrations were increased following growth in 3% ethanol and concentrations of spermidine and spermine were substantially increased following exposure to 6% ethanol. These changes were accompanied by significant increases in the activities of the polyamine biosynthetic enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase and in the flux of label from ornithine into the polyamines. Formation of the cadaverine derivatives aminopropylcadaverine and N,N-bis(3-aminopropyl)cadaverine was greatly increased in P. avenae exposed to 6% ethanol, probably via the action of lysine decarboxylase, S-adenosylmethionine decarboxylase and the aminopropyltransferases. There was also a doubling of polyamine oxidase activity following fungal growth in 6% ethanol.     

Role of diamine oxidase during the treatment of tumour-bearing mice with combinations of polyamine anti-metabolites.

Treatment of mice bearing L1210 leukaemia with 2-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase (EC 4.1.1.17), produced a profound depletion of putrescine and spermidine in the tumour cells. Sequential combination of methylglyoxal bis(guanylhydrazone), an inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), with difluoromethylornithine largely reversed the polyamine depletion and led to a marked accumulation of cadaverine in the tumour cells. Experiments carried out with the combination of difluoromethylornithine and aminoguanidine, a potent inhibitor of diamine oxidase (EC 1.4.3.6), indicated that the methylglyoxal bis(guanylhydrazone)-induced reversal of polyamine depletion was mediated by the known inhibition of diamine oxidase by the diguanidine. In spite of the normalization of the tumour cell polyamine pattern upon administration of methylglyoxal bis(guanylhydrazone) to difluoromethylornithine-treated animals, the combination of these two drugs produced a growth-inhibitory effect not achievable with either of the compounds alone.     

Selenomethionine induces polyamine biosynthesis in regenerating rat liver tissue

Summary. Our study was undertaken to elucidate the effects of selenomethionine (SeMet) on polyamine metabolism in regenerating rat liver tissue, as useful model of rapidly growing normal tissue. We have examined the levels of spermine, spermidine and putrescine in liver tissue. At the same time we have evaluated the activities of polyamine oxidase (PAO) and diamine oxidase (DAO), the catabolic enzymes of polyamine metabolism. The obtained results suggest that polyamine levels in regenerating liver tissue, at 7^th day after two-thirds partial hepatectomy, were higher in comparison with control group. The administration of selenomethionine to hepatectomized animals during seven days, in a single daily dose of 2.5 μg/100 g body weight, increases the amount of spermine and spermidine; the level of putrescine does not change under the influence of SeMet in regenerating rat liver tissue. PAO activity is lower in regenerating hepatic tissue than in control group. Supplementation of hepatectomized animals with SeMet significantly decreases the activity of this enzyme. DAO activity was significantly higher in hepatectomized and in operated animals treated with SeMet compared to the sham-operated and control ones. The differential sensitivity observed in our model of highly proliferating normal tissue to SeMet, compared with the reported anticancer activity of this molecule is discussed.