Intracellular polyamine patterns during encystment of Physarum flavicomum

In Physarum flavicomum Berk., growing amoebae convert to dormant cysts under conditions of nutrient imbalance. Exogenous adenine inhibits the process and the cells produce an elevated intracellular concentration of S-adenosylmethionine. Evidence indicates that the increased level of S-adenosylmethionine is responsible for the disruption of the normal developmental process. One of the biological functions of S-adenosylmethionine is in polyamine synthesis and it is known that a well-controlled intracellular concentration of polyamines is essential for normal cell growth and differentiation. In this study, high-performance liquid chromatography was used to determine the intracellular polyamine patterns in growing cells, adenine-treated and normal encysting cells, and dormant cysts. Putrescine and spermidine were the most abundant polyamines found in the cells; growing cells had the highest level, adenine-treated cells had a 1.5 to 2.0 times higher level than normal encysting cells, while cysts had the lowest (only 3 and 12% of that of growing cells). Cadaverine and N1-acetylspermidine were found in all the cells and their levels decreased during encystment. Acetylputrescine was found in growing cells only and acetylspermine was found in all cells except cysts. Acetylcadaverine, N8-acetylspermidine, 1,3-diaminopropane, and spermine were not detected in any of the cells.     

Polyamines biosynthesis and oxidation in free-living amoebae

Summary. In this paper we describe the polyamine biosynthesis and oxidation processes, giving an overview about recent results in free-living Amoebae.The protozoa polyamine levels are different in comparison with mammalian cells. Also, the polyamine levels in protozoa cells change if these species are pathological or not for the human beings. All the amoeba strains show high concentrations of 1,3-diaminopropane (DAP), spermidine and acetylspermidine while spermine is absent. In these amoeba a considerable polyamine oxidase activity has been found, which acts on N⁸-acetylspermidine, but not on free polyamines. This enzyme is responsible, together with polyamine acetylase, of DAP synthesis whose function is not well known.     

Formation of N1-acetylspermidine in rat liver after treatment with carbon tetrachloride

A single intraperitoneal injection of carbon tetrachloride produced a significant increase in the concentration of N¹-acetylspermidine in rat liver. The concentration of N¹-acetylspermidine was maximal at the same time after injection at which other workers reported maximal conversion of spermidine to putrescine and maximal acetylase activity in liv liver extracts. N¹-acetylspermidine was not detectable in livers of untreated animals and at 45 hours after injection with monoacetylation of polyamines precedes their degradation by polyamine oxidases. Spleen, lungs and erythrocytes of untreated animals contained detectable amounts of the monoacetyl polyamines. Treatment with carbon tetrachloride did not produce changes in the concentrations of the monoacetyl polyamines in these tissues.     

Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3- and SAM3-deficient cells. The K(m) values for putrescine and spermidine of DUR3 were 479 and 21.2 mum, respectively, and those of SAM3 were 433 and 20.7 mum, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr(250), Ser(251), and Thr(684) by polyamine transport protein kinase 2.     

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N⁶-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry. Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin. Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus. Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process. Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.     

Cytotoxicity of polyamines to Amoeba proteus: Role of polyamine oxidase

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H₂O₂, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N ¹-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N ¹-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3–20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid ( 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity ( 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.Abbreviations DAO diamine oxidase - DFMO DL--difluoromethylornithine - DP 1-3-diaminopropane - IC₅₀ 50% inhibition concentration - MAO monoamine oxidase - N ¹-ACSP; N ¹-acetylspermine - N¹-ACSPD N ¹-acetylspermidine - N ⁸-ACSPD N ⁸-acetylspermidine - ODC ornithine decarboxylase - PAO(s) polyamine oxidase(s) - PUT putrescine - SP spermine - SPD spermidine     

The effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers

Senescence of Petunia hybrida L. flowers is accompanied by a climacteric pattern in ethylene production and a rapid decline in the levels of putrescine and spermidine during the preclimacteric phase. The decrease in spermidine is caused by the decline in the availability of putrescine which is initially synthesized from L-arginine via agmatine and N-carbamoylputrescine. Inhibition of putrescine and polyamine synthesis resulted in a rapid drop in the levels of putrescine and spermidine without resulting in a concomitant increase in ethylene production. These results indicate that polyamine synthesis is not involved in the control of ethylene synthesis through its effect on the availability of S-adenosylmethionine, and is confirmed by the results obtained with pollinated flowers. Treatment with polyamines may stimulate or suppress ethylene production in the corolla, depending on the concentrations applied. In unpollinated flowers the onset of the climacteric rise in ethylene production was accelerated after treatment with polyamines. However, in pollinated flowers this process was delayed as a result of treatment with low concentrations of polyamines. The effects of exogenous polyamines on ethylene production in both pollinated and unpollinated flowers indicate that ethylene synthesis in these flowers is not regulated by a feedback control mechanism. Although polyamines do not play a key role in the control of ethylene production during the early stages of senescence through their effect on the availability of S-adenosylmethionine, it appears that they play an important role in some of the other processes involved in senescence.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MGBG methylglyoxal bis-(guanylhydrazone) - SAM S-adenosylmethionine     

Somatic embryo development in carrot is associated with an increase in levels of S-adenosylmethionine,S-adenosylhomocysteine and DNA methylation

Almost homogeneous populations representing different developmental stages of somatic embryos (globular, torpedo-shaped, plantlets) and vacuolated cells were obtained from a cell suspension culture of carrot. The concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and methylated DNA were determined in embryos at different developmental stages and were found to increase during somatic embryogenesis. The highest increase during embryogenesis was a 5-fold increase in the level of SAM. A considerable increase in the methylation index (SAM/SAH ratio) was also found. We propose that the levels of SAM and SAH may be involved in the control of somatic embryogenesis by affecting the level of DNA methylation, which in turn might cause differential changes in gene activation. An increase in the level of SAM may be a prerequisite for progression of embryogenesis and the development of complete embryos.     

Excretion of acetylated and free polyamines by polyamine depleted Chinese hamster ovary cells

1. Cultured Chinese hamster ovary cells (CHO) and their ornithine decarboxylase deficient mutant cells (C55.7) were found to excrete small amounts of N8-acetylspermidine and free polyamines, putrescine and spermidine into the culture medium. 2. The concentration of N8-acetylspermidine in the control cells was 2-3% of that of spermidine. In the medium, however, the amount of N8-acetylspermidine was about 2-fold that of spermidine and 2- to 3-fold higher than the intracellular amount. N1-acetylspermidine or acetylated spermine were never detected in the cells or in the media. 3. Confluent CHO cells treated with 2 mM difluoromethylornithine stopped the excretion when the intracellular spermidine concentration had decreased to 20% of control while there was no decrease in spermine concentration. At low cell density, neither polyamine depleted CHO cells nor the C55.7 cells excreted any polyamines into the culture media.     

Identification of a novel acetylated form of branched-chain polyamine from a hyperthermophilic archaeon Thermococcus kodakarensis

Long/branched-chain polyamines are unique polycations found in thermophiles. The hyperthermophilic archaeon Thermococcus kodakarensis contains spermidine and a branched-chain polyamine, N⁴-bis(aminopropyl)spermidine, as major polyamines. The metabolic pathways associated with branched-chain polyamines remain unknown. Here, we used gas chromatography and liquid chromatography-tandem mass spectrometry analyses to identify a new acetylated polyamine, N⁴-bis(aminopropyl)-N¹-acetylspermidine, from T. kodakarensis; this polyamine was not found in other micro-organisms. The amounts of branched-chain polyamine and its acetylated form increased with temperature, indicating that branched-chain polyamines are important for growth at higher temperatures. The amount of quaternary acetylated polyamine produced was associated with the amount of N⁴-bis(aminopropyl)spermidine in the cell. The ratio of acetylated to non-acetylated forms was higher in the stationary phase than in the logarithmic growth phase under high-temperature stress condition.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Pattern and concentration of free and acetylated polyamines in urine of cirrhotic patients.

The pattern and concentration of urinary, free, monoacetylated and total polyamines were determined in 31 cirrhotic patients, divided into three classes according to Child's classification, and in 28 healthy subjects. Cirrhotic patients had increased levels of free, monoacetylated and total polyamines. They also showed a significant increase in N1-acetylspermidine to N8-acetylspermidine molar ratio. Urinary polyamine excretion was not related to the severity of liver disease nor to the values of laboratory liver function tests. Furthermore, polyamine excretion was not significantly different in cirrhotics with or without diabetes or IGT, while plasma insulin and glucagon levels were increased in all cirrhotic patients. The results suggest that enhanced polyamine biosynthesis and catabolism, particularly N1-acetylation, occur in cirrhotic patients, probably due to hepatic regeneration and/or increased levels of insulin and glucagon.     

Investigation of polyamine metabolism by high-performance liquid chromatographic and gas chromatographic profiling methods

Measurements of polyamines, polyamine conjugates and their metabolites in tissues, cells and extracellular fluids are used in biochemistry, (micro)biology, oncology and parasitology. Decarboxylation of ornithine yields putrescine. Aminopropylation of putrescine yields spermidine, and aminopropylation of spermidine yields spermine. Spermidine and spermine are retroconverted to putrescine and spermidine, respectively, by initial N-acetylation and subsequent polyamine oxidation. The intermediate N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine are the major urinary N-acetylpolyamines. Polyamines and N-acetylpolyamines are terminally degraded to non-α-amino acid metabolites by oxidative deamination and aldehyde dehydrogenation. Chromatography with on-line detection is the most commonly applied profiling method for polyamines, N-acetylpolyamines and their non-α-amino acid metabolites. Cation-exchange and reversed-phase high-performance liquid chromatography require pre- or post-column derivatisation, followed by UV-Vis spectrophotometric or fluorimetric detection. Isolation and derivatisation precedes gas chromatography with flame-ionisation, nitrogen-phosphorus, electron-capture or mass spectrometric detection. High-performance liquid chromatography and gas chromatography of polyamines are not competitive techniques, but rather supplementary.     

Regulation of polyamine transport by polyamines and polyamine analogs

Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in V_max to levels ～4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor-induced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N¹, N¹²-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1-hr exposure to 10 μM BESPM, V_max values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400–500 pmol/10⁶ cells—about 15–20% of the total polyamine pool (～2.8 nmol/10⁶ cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 μM produced an increase in SPD and SPM V_max values, whereas concentrations 3 μM and higher produced a marked suppression of these values. Cells treated with 3 μM BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15–20%) changes in intracellular polyamine pools.     

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]     

Recent advances in the understanding of polyamine functions during plant development

Suggested roles for polyamine function, and the evidence for these functions, is reviewed. These include membrane stabilization, free radical scavenging, effects on DNA, RNA and protein synthesis, effects on the activities of RNase, protease and other enzymes, the interaction with ethylene biosynthesis, and effects on second messengers. It is concluded that in addition to interacting with plant hormones, polyamines are able to modulate plant development through a fundamental mechanism(s) common to all living organisms.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - ADC arginine decarboxylase - Chl chlorophyll - DAP diaminopropane - DFMA DL--difluoromethylarginine - DFMO DL--difluoromethylornithine - PAs polyamines - Put putrescine - SAM S-adenosylmethionine - Spd spermidine - Spm spermine     

S-Adenosylmethionine metabolism and its relation to polyamine synthesis in rat liver. Effect of nutritional state, adrenal function, some drugs and partial hepatectomy

S-Adenosylmethionine metabolism and its relation to the synthesis and accumulation of polyamines was studied in rat liver under various nutritional conditions, in adrenalectomized or partially hepatectomized animals and after treatment with cortisol, thioacetamide or methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}. Starvation for 2 days only slightly affected S-adenosylmethionine metabolism. The ratio of spermidine/spermine decreased markedly, but the concentration of total polyamines did not change significantly. The activity of S-adenosylmethionine decarboxylase initially decreased and then increased during prolonged starvation. This increase was dependent on intact adrenals. Re-feeding of starved animals caused a rapid but transient stimulation of polyamine synthesis and also increased the concentrations of S-adenosylmethionine and S-adenosylhomocysteine. Similarly, cortisol treatment enhanced the synthesis of polyamines, S-adenosylmethionine and S-adenosylhomocysteine. Feeding with a methionine-deficient diet for 7-14 days profoundly increased the concentration of spermidine, whereas the concentrations of total polyamines and of S-adenosylmethionine showed no significant changes. The results show that nutritional state and adrenal function play a significant role in the regulation of hepatic metabolism of S-adenosylmethionine and polyamines. They further indicate that under a variety of physiological and experimental conditions the concentrations of S-adenosylmethionine and of total polyamines remain fairly constant and that changes in polyamine metabolism are not primarily connected with changes in the accumulation of S-adenosylmethionine or S-adenosylhomocysteine.     

The Effects of Inhibitors of Polyamine and Ethylene Biosynthesis on Senescence, Ethylene Production and Polyamine Levels in Cut Carnation Flowers

The role of polyamine metabolism in the regulation of senescenceand ethylene evolution was examined in cut carnations. Endogenousconcentrations of spermine and spermidine did not change asflowers aged, but putrescine, their immediate biosynthetic precursor,increased dramatically and paralleled a sharp rise in ethylene.When D-arginine, difluoromethylarginine and methylglyoxal bis(guanylhydrazone)were used to inhibit specific steps in polyamine synthesis,ethylene production and the onset of senescence were promoted.In contrast, inhibition of ethylene by aminooxyacetic acid increasedthe level of spermine, presumably by increasing the availabilityof aminopropyl groups derived from S-adenosylmethionine (SAM)and required for the synthesis of polyamines from putrescine.These results suggest that the ethylene and polyamine biosyntheticpathways compete for SAM during senescence of carnation flowers. (Received September 1, 1983; Accepted January 7, 1984)     

Ethylene evolution during ripening of detached tomato fruit: Its relation with polyamine metabolism

Ethylene and polyamine metabolism, both sharing a common precursor, S-adenosylmethionine (SAM), were investigated during detached tomato (Lycopersicon esculentum Mill. nothovar F1 Lorena) fruit ripening. Putrescine (PUT) was found to be the major polyamine in the fruits, always over 100 nmols/g FW, while spermidine (SPD) was between 7% and 3% of the level of PUT. Spermine (SPM) was not detected at any stage of ripening. The level of PUT and SPD, did not change significantly during ripening in spite of the almost continuous synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor, and only at the last stage of ripening was a drastic decrease in SPD content observed. The results obtained show that the onset of ACC synthesis and its accumulation within the tissue is not a consequence of a decrease in SPD synthesis.     

Variations in S-adenosylmethionine, S-adenosylhomocysteine and adenosine concentrations in rat liver.

The hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and adenosine (Ado) in the rat were examined diurnally and as a function of fasting. Ado concentrations increased continuously throughout the fasting period; concentrations after 2 days of fasting were 7.5-fold higher than control values. Diurnally, the concentration of Ado was highest during the light hours. SAM and the ratio of SAM/SAH were reduced greater than 50% due to fasting and exhibited a significant daily rhythm which appeared to be related to dietary methionine availability. Hepatic SAM concentrations decreased continuously during the light hours and increased during the dark period to levels 7.3-fold greater than the lowest light values. The concentration of SAH was altered in a similar fashion yet to a much lesser degree such that the ratio of SAM/SAH paralleled the changes in the concentration of SAM. The SAM/SAH ratio exhibited a 4.5-fold difference between the peak and nadir values.