High Concentrations Of Polyamines Research Articles

Ornithine Decarboxylase Activity and Concentrations of Polyamines in Embryos of Diabetic Rats

Previous studies have demonstrated high concentrations of polyamines in neoplastic tissue and embryos and these compounds are therefore believed to play a role in cellular growth and embryonic development. Maternal diabetes causes embryonic dysmorphogenesis and alterations in embryonic polyamine concentrations may contribute to this process. In the present study we have measured the contents of DNA, putrescine, spermidine and spermine in embryos on days 10 and 11 of gestation in normal and diabetic rats. We also estimated the activity of ornithine decarboxylase (ODC) in embryos on days 9-11. We found that maternal diabetes causes delayed growth as reflected by decreased content of DNA on day 11 in the embryos of the diabetic group. Both the polyamine content and ODC activity were altered in the embryos of diabetic rats. Thus, the polyamines were increased on day 10 and decreased on day 11, and the ODC activity was decreased in a down-regulated manner in day-10 embryos of the diabetic rats. These findings suggest that polyamine metabolism is involved in the dysmorphogenesis of diabetic pregnancy.

Positron emission tomography study of human prostatic adenocarcinoma using carbon-11 putrescine

To evaluate [1- 11C]putrescine ([ 11C]PUT) as a potential tracer for imaging and characterization of human prostatic adenocarcinoma, positron emission tomography (PET) was performed in eight patients and three normal controls. In addition, four of the patients and the three normal controls also had a prostate scan with 2-deoxy-2-[ 18F]fluoro- d-glucose ( 18FDG). Three of the patients had undergone resection of the prostate tumor and all of the patients except for one had bone metastasis. Carbon-11 rapidly accumulated in prostate, bone and rectum after injection of [ 11C]PUT. Maximal uptake was achieved 5 min after injection with minimal washout during the 50 min study period. The uptake of carbon-11 in the prostate of normal controls was significantly higher than that in the patients. However, three of the four patients scanned for metastatic bone lesions showed higher uptake in bone metastasis than in normal bone. Quantitation of 18FDG uptake in the prostate was hindered by the high accumulation of activity in the urinary bladder. [ 11C]PUT does not appear to be a useful tracer for assessing proliferation of human prostate adenocarcinoma. Its utility in the imaging of other cancers with high polyamine concentration remains to be investigated.

Induction of ornithine decarboxylase by N-methyl-D-aspartate receptor activation is unrelated to potentiation of glutamate excitotoxicity by polyamines in cerebellar granule neurons.

Polyamines positively modulate the activity of the N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)--the key regulatory enzyme in polyamine synthesis--and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.

Polyamine changes during early development of Drosophila melanogaster

Concentration changes of the polyamines putrescine, spermidine and spermine have been measured throughout Drosophila embryogenesis. Spermidine is the predominant polyamine with concentrations 10–15-fold higher than spermine and putrescine respectively. High polyamine concentrations during periods of intravitelline nuclear division, cell formation and cell proliferation suggest the involvement of polyamines in continued embryonic growth. Additional evidence for this comes from the use of cyclohexylamine which is an inhibitor of spermidine synthase.

THE EFFECT OF POLYAMINES ON THE SENESCENCE OF CARNATION PETALS.

The interaction between polyamines and ethylene is still not clear for floral tissues. The aim of the present paper is to examine the senescence on the isolated petals of carnation (Dianthus caryophyllus cv. Desio) but not the whole flower in an attempt to clarify the exact role of polyamines. Petals were treated with putrescine (Put; 0.0, 1.0, 10mM), spermidine (Spd; 0.0, 1.0, 10mM), spermine (Spn; 0.0, 1.0, 10mM), Put+Spd (1.0mM), Put+Spn (1.0mM). The fresh weight of petals in all 10mM treatment was decreased less than those in the other treatments at all times but there were no significant differences. The differences in ethylene production were significant. In petals maintained in 10mM of polyamines, ethylene production was completely inhibited until 13 days and senescence was considerably retarded. However, ethylene productions in 1.0mM polyamines treatments were delayed 2-3 days with reduced amounts. These results suggest that high concentrations of polyamines retard senescence and completely inhibit ethylene production. ACC content, activities of ACC synthase and SAM decarboxylase were analyzed. Finally, the role of SAM in ethylene and polyamines biosynthesis will be discussed.

Polyamines in various rice (Oryza sativa) genotypes with respect to sodium chloride salinity

Physiological responses of various rice genotypes were studied in relation to salt (NaCl) stress. Cultivars CSR‐1 and Dular germinated well in different NaCl regimes compared to cvs Rupsail, Assam Getu and M‐1–48. At 100 mM NaCl, the lowest germination was observed in cv. M‐1–48. Cvs CSR‐1 and Dular were relatively effective in maintaining high concentrations of polyamines as well as arginine decarboxylase (EC 4.1.1.19) activity in coleoptiles and roots in a non‐stressed condition. The activities of two biodegradative enzymes, diamine oxidase (EC 1.4.3.6) and polyamine oxidase (EC 1.4.3.4), were lowest in cv. CSR‐1. The polyamine content was not significantly altered when seedlings of cv. CSR‐1 were exposed to 100 mM NaCl. However, in cv. M‐1–48 enhancement of arginine decarboxylase activity with concomitant accumulation of polyamines was observed. Leakage of metabolites and changes in the levels of Na+ and Cl− were prominent in cv. M‐1–48 under saline conditions. The results suggest a correlation between polyamine and salt stress‐induced responses in rice genotypes.

Polyamine Uptake by Human Colon Carcinoma Cell Line CaCo-2

The intracellular concentrations of the polyamines are highly regulated and high polyamine concentrations are associated with rapidly proliferating cells. Hormones, nutrients and growth factors that stimulate the proliferation of the intestinal epithelium, increase the intracellular polyamine concentration mainly by activating ODC expression. Other cell types stimulated to proliferate satisfy their requirement for polyamines by increasing polyamine uptake. In the present study, we investigated polyamine uptake by a human colon carcinoma cell line, CaCo-2. Uptake of putrescine, spermidine and spermine by CaCo-2 cells was saturable and temperature dependent and all polyamines appear to share a common carrier. The carrier of differentiated cells had an apparently higher affinity and lower activity than the carrier of replicating cells. Culture of CaCo-2 cells on porous filters showed that polyamine accumulation occurred mainly through the basolateral membrane in replicating cells, while an increase in the rate of apical uptake was observed after differentiation. A significant increase in polyamine uptake and in ODC expression resulted from fresh medium replacement, a well-known stimulus to proliferation; no change in uptake occurred after ODC inhibition by DFMO. We conclude that CaCo-2 cells are able to increase their polyamine concentration by both enhanced synthesis and increased polyamine uptake.

In vitro interactions between polyamines and pectic substances

Putrescine, spermidine and spermine induce a decrease in the pH value of 1 mM polygalacturonic acid or pectin solutions; spermidine and spermine also cause the precipitation of the polymers. The association constants between polyamines and polygalacturonic acid were in the order of 10(5) for putrescine and spermidine, and 10(6) for spermine. The number of galacturonic units per binding sites are proportional to the number of positive charges on the polyamine molecule. Low affinity binding sites appear at high polyamine concentrations. Calcium ions seem to compete weakly with spermine by lowering the association constant 4- to 6-fold. Two natural pectins tested, showed that methylation of the carboxylic groups influences only the number of galacturonic units per site but not the association constant.

Polyamine Uptake in Carrot Cell Cultures

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca(2+). The effects of Ca(2+) were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca(2+) and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca(2+) concentration range between 10 micromolar and 1 millimolar. La(3+) nullified the stimulatory effect of 10 micromolar Ca(2+) on putrescine uptake and that of 1 millimolar Ca(2+) on spermidine uptake. La(3+) at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule.

Differences in polyamine metabolism between carcinomatous and uninvolved human breast tissues.

The polyamine biosynthetic enzymes ODC and SAMDC show higher activity in carcinomatous human breast tissue than in uninvolved tissue of the same breast; the interconversion enzyme PAO shows significantly lower activity in carcinomatous than uninvolved tissue. The polyamine metabolism in carcinomatous human breast tissue thus appears to differ from that in uninvolved tissue. Intracellular polyamine concentrations, particularly spermine, are high in carcinomatous tissue. This increase and that of the biosynthetic enzymes suggest that a higher polyamine concentration is needed for carcinomatous cell growth. If this is the case, the lower PAO activity in carcinomatous tissue may be explained as a mechanism that conserves the high intracellular polyamine concentration.

Polyamine Uptake, Kinetics, and Competition among Polyamines and between Polyamines and Inorganic Cations

Polyamine uptake, the kinetics of this uptake, and the competition among polyamines and between polyamines and inorganic cations were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments using (14)C-labeled polyamines were carried out on single petals, at room temperaure (20 degrees C) and in the light. The results show that putrescine, spermidine, and spermine uptake was dependent on the external pH and occurred up to high external polyamine concentrations with K(m) values of 8.6, 1.2, and 2.1 millimolar, respectively, with spermidine being the most absorbed at low concentration (17 micromolar). Putrescine and spermidine did not seem to compete for the same site of absorption. Furthermore, putrescine and spermidine uptake was not inhibited by Ca(2+), Mg(2+), and K(+) at the same concentrations (17 micromolar), whereas 1.7 millimolar Ca(2+) inhibited and K(+) enhanced spermidine uptake. The intracellular localization of the absorbed putrescine was determined using two different methods. Very little label was found in the apoplast, while most of it was localized in the 98,500g supernatant. According to our data the vacuole, which represents a substantial part of Saintpaulia parenchyma cells, could be a site of putrescine accumulation. 2,4-Dinitrophenol and diethylstilbestrol did not inhibit uptake; however, at 0 degrees C there was a 35% inhibition of spermidine uptake, compared with the controls kept at 20 degrees C as well as a 68% inhibition with 20 millimolar NaSCN.

Effect of Photoperiod on Polyamine Metabolism in Apical Buds of G2 Peas in Relation to the Induction of Apical Senescence

Polyamine content and arginine decarboxylase activity of apical buds were measured to determine whether polyamines are required to prevent apical senescence in pea. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. High polyamine concentrations were found in the vigorous apices of plants grown under a short day photoperiod during which senescence is delayed. As the apex senesced in long days, the amounts of polyamines per organ declined in parallel with decreases in the size of the apical bud. However, a decrease in polyamine concentration, due mainly to a change in spermidine, occurred at the time of marked reduction in bud size and growth rate, but not before the onset of the early symptoms of senescence. No correlation was found with arginine decarboxylase. The results suggest polyamines may be required to support bud growth, but the photoperiodic mechanism which governs apical senescence of G2 peas does not exert control through polyamine metabolism.

Role of Polyamines in Gibberellin-Induced Internode Growth in Peas

To determine the requirement for polyamines in gibberellin (GA) induced internode growth polyamine content was measured in internodes of peas of various internode phenotypes (slender, tall, dwarf, nana) with and without applied gibberellin (GA(3)) and polyamine synthesis inhibitors. Polyamines were assayed as dansyl derivatives which were separated by reverse phase high performance liquid chromatography and detected by fluorescence spectrophotometry. The amounts of polyamines in the different genetic lines of peas, which differed in internode lengths and extractable GA content, correlated with the extent of internode elongation. High polyamine concentrations were associated with young internodes and decreased with internode expansion. Extremely short internodes of nana plants without GA exhibited equal or higher amine concentrations relative to internodes of other lines of peas and GA-stimulated nana seedlings. The polyamine synthesis inhibitors, alpha-difluoromethylornithine and alpha-difluoromethylarginine, independently or in combination, inhibited polyamine accumulation and internode elongation of tall peas and GA-stimulated nana plants. Agmatine and putrescine restored growth and endogenous polyamine content to variable degrees. However, exogenous polyamines were not effective in promoting growth unless intracellular amines were partially depleted.These results suggest that polyamines do not have a role in cell elongation, but may be required to support cell proliferation. Polyamines do not mediate the entire action of GA in internode growth of peas since GA induction of growth involves both cell division and cell elongation, whereas polyamines appear to affect cell division only.

Effect of spermine on interaction of DNA polymerase α from the loach ( Misgurnus fossilis) eggs with DNA

Polyamines (putrescine, spermidine and spermine) cause a marked increase in the activity of the loach Misgurnus fossilis DNA polymerase α on activated (gapped) DNA. The stimulatory effect increases in the order: putrescine, spermidine, spermine. Kinetic analysis shows that spermine does not change the affinity of the polymerase for dTTP, but it decreases the enzyme affinity for DNA. The apparent K m of the polymerase for activated DNA progressively increases from 14 to 1200 μM (nucleotide), if the concentration of spermine rises up to 2 mM, while V max reaches a maximum at 0.5 mM spermine and then drops at higher polyamine concentrations. Native calf thymus DNA and especially single-stranded DNA from phage M13 appear to be inhibitors of α-polymerase activity on gapped DNA. Dixon plots suggest simple competitive inhibition of the polymerase activity by single- or double-stranded DNA and absence of cooperativity in the interaction of the polymerase with DNA. Hill-plot analysis is compatible with the interpretation that there is only one DNA binding site on each DNA polymerase α molecule. Spermine, even at low concentrations, decreases sharply the affinity of the enzyme for double-stranded DNA, while the enzyme affinity for single-stranded DNA changes insignificantly. Another result of spermine action is the destabilization of the polymerase-DNA complex. The ratio of the ‘static affinity’ of the enzyme to its ‘kinetic affinity’ decreases 2.2-fold in the presence of 0.5 mM spermine. As a result, the sensitivity of DNA synthesis to 3′-deoxy-3′-aminothymidine 5′-triphosphate and to 1-β- d-arabinofuranosylcytidine 5′-triphosphate decreases in the presence of the polyamine. Both spermine effects, the decrease in the ‘nonproductive binding’ of the polymerase to double-stranded regions in DNA and the destabilization of the polymerase-DNA complex, presumably account for the increase in the activity of the loach α-polymerase on activated DNA.

Cellular content and biosynthesis of polyamines during rooster spermatogenesis.

The natural polyamines spermine and spermidine, and the diamine putrescine, were extracted from rooster testis cells separated by sedimentation at unit gravity, and from vas-deferens spermatozoa. The ratios spermine/DNA and spermidine/DNA were kept relatively constant throughout spermatogenesis, whereas the ratio putrescine/DNA rose in elongated spermatids. The cellular content of spermine, spermidine and putrescine decreased markedly in mature spermatozoa. Two rate-limiting enzymes in the biosynthetic pathway of polyamines, ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, showed their highest activities at the end of spermiogenesis and were not detectable in vas-deferens spermatozoa. A marked reduction in cell volume during spermiogenesis without a parallel decrease in the cellular content of polyamines suggests the possibility that the marked changes in chromatin composition and structure occurring in rooster late spermatids could take place in an ambience of high polyamine concentration.

Polyamines, hydrolases, (PAP, LAP, SDH, plasmin) TSH, T3, T4 and C-peptide in benign hyperplasia of the prostate.

High concentrations of polyamines have been found in the normal human prostate. The profile of these amines appeared significantly changed in benign hypertrophy of the prostate. An increase of spermine and a fall of putrescine were always found in patients with a hypertrophied prostate weighing more than 30 g. Alterations of plasmin in these tissues seemed to reflect changes in the matrix; abnormalities of thyroid and pancreatic function documented by changes in the serum levels of TSH and c-peptide which are thought to be further evidence of a mesenchymal-epithelial interaction in the pathogenesis of benign prostate hypertrophy.

Effect of polyamines on the reduction and adsorption of DNA on the mercury electrode

The influence of the addition of a polyamine to solutions of doublestranded ( ds) or denatured DNA's and RNA's was studied by means of polarographic techniques. It was found that, depending on the composition of the background electrolyte, the addition of spermine or spermidine may result either in an increase or in a decrease of the derivative (differential) pulse—polarographic peak of the DNA. The experimental data suggest that low concentrations of polyamines may play a role similar to that of ammonium ions in the electrolyte if the ammonium ion concentration is relatively low. At sufficiently high ammonium ion and polyamine concentrations, the height of the derivative pulse—polarographic peak of ds DNA was decreased and its summit potential ( U s ) was shifted to more negative values as a result of the addition of the polyamine. Similarly, the descending part of the dependence of the voltammetric peak height on the initial potential was shifted to more negative potentials. These results show that, in spite of the fact that the polyamine stimulates DNA adsorption at the negatively charged electrode surface (due to efficient screening of the DNA negative charges), it can cause a decrease of the derivative pulse polarographic peak of ds DNA. It is assumed that the known stabilizing effect of polyamines on the DNA double—helical structure is responsible for this decrease. The addition of spermine to ds RNA solution had little effect on the height of the ds RNA derivative pulse—polarographic peak. It was demonstrated that the difference between U s of the peak II R of ds RNA and U s of III R of denatured RNA was so large that these two peaks (produced by both RNA's at the same concentrations) did not overlap. Experimental data presented in this paper are in good agreement with our previous results do not support the model of ds DNA polarographic reduction proposed by BERG. 8 According to his model DNA reduction is independent of DNA conformation.

The regulation of mouse liver ornithine decarboxylase by metabolites

The enzyme ornithine decarboxylase ( l-Ornithine carboxy-lyase, EC 4.1.1.17), has been partially purified from the livers of mice subjected to partial hepatectomy (6–8 h previously). Mouse liver ornithine decarboxylase requires pyridoxal phosphate, and dithiothreitol for maximal activity. The enzyme has a pH optimum of 7.3, it is inhibited in the presence of 0.3 M phosphate, glycine, Tricine and Tris. It shows no dependence on metal ions and is inhibited by high salt concentrations, particularly ammonium salts. The kinetics of the enzyme have been studied with putrescine (and analogs), spermidine and spermine, in the presence of both high and low levels of pyridoxal phosphate. High concentrations of pyridoxal phosphate inhibit the enzyme. The enzyme is also inhibited by low concentrations of putrescine (1 mM). As the concentration of putrescine increased to 10 mM, non-competitive inhibition was observed, this could be reversed by addition of higher levels of pyridoxal phosphate. Spermidine and spermine inhibit (noncompetitively) only at high concentrations (10 mM). Ornithine inhibits at high concentrations (2 mM). Spectral studies have shown that the observed kinetics of competitive inhibition at low concentrations of polyamine changing to noncompetitive inhibition at high polyamine concentrations are due to competition between enzyme and substrate (or inhibitor) for free (non-enzyme bound) pyridoxal phosphate. Noncompetitive inhibition arises through the formation of transient Schiff base complexes between amines and free pyridoxal phosphate. It also appears that the binding of substrate to the active site takes place through Schiff base formation with enzyme bound pyridoxal phosphate.

The Effect of Polyamines on the Photochemical Reactivity of DNA

AbstractThe effects of diaminopentane (cadaverine), diaminoethane and the polyamine, spermine, on the photoreactions of 4,5′, 8‐trimethylpsoralen with DNA were studied. Near ultraviolet (UV) light (360 nm) irradiation of psoralen with DNA results in the formation of psoralen monoadducts with pyrimidine bases and DNA interstrand crosslinks. The polyamines studied reduced the rate of both photoreactions to the same extent by a factor of 1.5–2.0. The magnitude of the effect increased with polyamine concentration. Effectiveness was in the order spermine > cadaverine > diaminoethane. This is also the same order for stabilization of the DNA double‐helix. Under conditions of higher DNA stability (higher ionic strength), higher polyamine concentrations were required to obtain an effect which was smaller than at low ionic strength. It is concluded that the photoreactions of psoralen with pyrimidines in DNA require some distortion of the double‐helix. This distortion is made more difficult in the presence of stabilizing agents like polyamines and therefore the rate of the reaction is reduced. If crosslinking of DNA by psoralen is a two‐step reaction then it must be concluded that the second step does not require further distortion. Consistently with the above, polyamines also reduced the UV‐induced dimerization of thymine in DNA, although to a lesser extent.

Étude au microscope électronique de l'interaction de l'acide désoxyribonucléique et des polyamines

Interactions between polyamines and deoxyribonucleic acid are studied by means of electron microscopy on thin sections and by spreading method. With high concentrations of polyamines (5 × 10 −3 M or more) in aldehydic fixing fluids, DNA molecules become aggregated and consist mainly of coarse fibres which are generally highly interconnected. Low concentrations of polyamines (10 −3 M or less) give pictures which look like that obtained without polyamine, if fixation is followed by post-treatment with uranyl acetate. In such conditions, polyamines combine probably with DNA by intramolecular bonds. These intra- or inter-strand interactions within DNA molecules do not prevent their aggregation. The same result is obtained with calcium. Instead, uranyl cations bind irreversibly to DNA and enable visualization of individual molecules without intermolecular cross-links. With high concentrations, DNA molecules are linked by intermolecular bonds. The significance of the aggregation of DNA molecules by polyamines is discussed at the light of results obtained by biochemical methods.