The gene encoding spermidine synthase was cloned from the human malaria parasite Plasmodium falciparum. Northern and Western blot analyses revealed a stage specific expression during the erythrocytic schizogony with the maximal amount of transcript and protein in mature trophozoites. Immunofluorescence assays (IFAs) suggest a cytoplasmatic localisation of the spermidine synthase in P. falciparum. The spermidine synthase polypeptide of 321 amino acids has a molecular mass of 36.6kDa and contains an N-terminal extension of unknown function that, similarly, is also found in certain plants but not in animal or bacterial orthologues. Omitting the first 29 amino acids, a truncated form of P. falciparum spermidine synthase has been recombinantly expressed in Escherichia coli. The enzyme catalyses the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) onto putrescine with Km values of 35 and 52microM, respectively. In contrast to mammalian spermidine synthases, spermidine can replace to some extent putrescine as the aminopropyl acceptor. Hence, P. falciparum spermidine synthase has the capacity to catalyse the formation of spermine that is found in small amounts in the erythrocytic stages of the parasite. Among the spermidine synthase inhibitors tested against P. falciparum spermidine synthase, trans-4-methylcyclohexylamine (4MCHA) was found to be most potent with a Ki value of 0.18microM. In contrast to the situation in mammals, where inhibition of spermidine synthase has no or only little effect on cell proliferation, 4MCHA was an efficient inhibitor of P. falciparum cell growth in vitro with an IC50 of 35microM, indicating that P. falciparum spermidine synthase represents a putative drug target.

Our previous results from a limited number of cell lines have suggested that the bis(ethyl)polyamine analogues exert a phenotype-specific response in human lung cancer cells. In the present study, we have extended this work to analyze the response of the 4 major forms of human lung cancer to the polyamine analogue N1,N12-bis(ethyl)spermine (BESpm). The results suggest that non-small cell phenotypes are much more sensitive to the cytotoxic effects of BESpm than the small cell lung carcinoma phenotype. Further, there appears to be a positive association between the level of induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in response to the analogue and the kinetic response of cells. Specifically, cells in which SSAT activity is highly induced by BESpm are killed by the compound. Although induction of SSAT appears to occur at both the level of increased steady-state mRNA and enzyme activity, SSAT activity appears to be a better indicator of cell sensitivity to BESpm than SSAT mRNA levels. These results have significance both for the potential use of polyamine analogues in treating specific forms of human lung cancer and for understanding the regulation of SSAT at the molecular level.

Ischemia-reperfusion injury (IRI) in liver and other organs is manifested as an injury phase followed by recovery and resolution. Control of cell growth and proliferation is essential for recovery from the injury. We examined the expression of three related regulators of cell cycle progression in liver IRI: spermidine/spermine N-acetyltransferase (SSAT), p21 (a cyclin-dependent kinase inhibitor), and stathmin. Mice were subjected to hepatic IRI, and liver tissues were harvested at timed intervals. The expression of SSAT, the rate-limiting enzyme in the polyamine catabolic pathway, had increased fivefold 6 h after IRI and correlated with increased putrescine levels in the liver, consistent with increased SSAT enzymatic activity in IRI. The expression of p21, which is transactivated by p53, was undetectable in sham-operated animals but was heavily induced at 12 and 24 h of reperfusion and declined to undetectable baseline levels at 72 h of reperfusion. The interaction of the polyamine pathway with the p53-p21 pathway was shown in vitro, where activation of SSAT with polyamine analog or the addition of putrescine to cultured hepatocytes induced the expression of p53 and p21 and decreased cell viability. The expression of stathmin, which is under negative transcriptional regulation by p21 and controls cell proliferation and progression through mitosis, remained undetectable at 6, 12, and 24 h of reperfusion and was progressively and heavily induced at 48 and 72 h of reperfusion. Double-immunofluorescence labeling with antibodies against stathmin and PCNA, a marker of cell proliferation, demonstrated colocalization of stathmin and PCNA at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation. The distinct and sequential upregulation of SSAT, p21, and stathmin, along with biochemical activation of the polyamine catabolic pathway in IRI in vivo and the demonstration of p53-p21 upregulation by SSAT and putrescine in vitro, points to the important role of regulators of cell growth and cell cycle progression in the pathophysiology and/or recovery in liver IRI. The data further suggest that SSAT may play a role in the initiation of injury, whereas p21 and stathmin may be involved in the resolution and recovery after liver IRI.

Ornithine decarboxylase (ODC) plays a key role in the biosynthesis of polyamines, which are necessary for cell growth and differentiation. ODC expression is very tightly controlled in all normal cells; however, regulation of its expression is altered in many tumor cells resulting in much higher basal levels of ODC in tumors. To investigate the potential role of ODC overexpression in epidermal tumorigenesis, we constructed a replication-defective retroviral vector to overexpress a truncated ODC protein in epidermal cells. Stable viral infection of mouse epidermal cells dramatically increases not only the basal ODC activity but also the basal putrescine and spermidine levels. In all infected epidermal cells with high polyamine levels, DNA synthesis is increased as measured by [3H]thymidine incorporation into DNA as well as increased bromodeoxyuridine staining in the nuclei of ODC-infected epidermal cells. ODC viral infection of nontumorigenic BK-1 epidermal cells and primary cultures of mouse keratinocytes and fibroblasts from newborn mouse skin yields no tumors when injected s.c. into athymic nude mice or when transplanted as skin grafts onto nude mice. Epidermal cell lines SP-1 and 308 (which possess an activated rasHa gene) are not tumorigenic when injected s.c. into nude mice. However, following infection with the ODC virus, they form tumors filled with keratin and papilloma-like projections of hyperplastic epidermal cells displaying dysplasia and many mitotic figures. These data indicate that ODC overexpression by itself is not sufficient to induce tumors in normal cells but that increased expression of ODC enhances tumor development in initiated premalignant epidermal cells.

The objective of this study was to elucidate the role and mechanism of nitric oxide (NO) synthase (NOS) in modulating the growth of the Caco-2 human colon carcinoma cell line. The two novel observations reported here are, first, that NG-hydroxy-L-arginine (NOHA) inhibits Caco-2 tumor cell proliferation, likely by inhibiting arginase activity, and, second, that NO causes cytostasis by mechanisms that might involve inhibition of ornithine decarboxylase (ODC) activity. Both arginase and ODC are enzymes involved in the conversion of arginine to polyamines required for cell proliferation. Cell growth was monitored by cell count, cell protein analysis, and DNA synthesis. NOHA (1-30 microM) and NO in the form of DETA/NO (1-30 microM) inhibited cell proliferation by 30-85%. The cytostatic effect of NOHA was prevented by addition of excess ornithine, putrescine, spermidine, or spermine to cell cultures, whereas the cytostatic effect of NO (DETA/NO) and alpha-difluoromethylornithine (ODC inhibitor) was unaffected by ornithine but was prevented by putrescine, spermidine, or spermine. The cytostatic effect of NOHA appeared to be independent of its conversion to NO, and the effect of NO appeared to be independent of cGMP. NOHA inhibited urea production by Caco-2 cells and inhibited arginase catalytic activity (85% at 3 microM), whereas NO (DEA/NO and SNAP) inhibited ODC activity >>/=60% at 30 microM) without affecting arginase activity. Coculture of Caco-2 cells with lipopolysaccharide/cytokine-activated rat aortic endothelial cells markedly slowed Caco-2 cell proliferation, and this was blocked by NOS inhibitors. These observations that NOHA and NO may inhibit sequential steps in the arginine-polyamine pathway suggest a novel biological role for NOS in the inhibition of cell proliferation of certain tumor cells and possibly other cell types.

Reducing the concentration of polyamines (spermine, spermidine, and putrescine) in the body pool may slow the cancer process. Because dietary spermine, spermidine, and putrescine contribute to the body pool of polyamines, quantifying them in the diet is important. Limited information about polyamine content of food is available, especially for diets in the United States. This brief report describes the development of a polyamine database linked to the Fred Hutchinson Cancer Center food frequency questionnaire (FFQ). Values for spermine, spermidine, and putrescine were calculated and reported per serving size (nmol/serving). Of the foods from the database that were evaluated, fresh and frozen corn contain the highest levels of putrescine (560,000 nmol/serving and 902,880 nmol/serving) and spermidine (137,682 nmol/serving and 221,111 nmol/serving), and green pea soup contains the highest concentration of spermine (36,988 nmol/serving). The polyamine database and FFQ were tested with a convenience sample (n=165). Average daily polyamine intakes from the sample were: 159,133 nmol/day putrescine, 54,697 nmol/day spermidine, and 35,698 nmol/day spermine. Orange and grapefruit juices contributed the greatest amount of putrescine (44,441 nmol/day) to the diet. Green peas contributed the greatest amount of spermidine (3,283 nmol/day) and ground meat contributed the greatest amount of spermine (2,186 nmol/day). Development of this database linked to an FFQ provides a means of estimating polyamine intake and contributes to investigations relating polyamines to cancer.

The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The Kt values for uptake activities of putrescine, spermidine and spermine were 3.7 microM, 0.38 microM and 0.23 microM in that order. The uptake activity was inhibited by carbonyl cyanide m-chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM K+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 microM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane-potential-dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.

Ischemia-reperfusion injury (IRI) is the major cause of acute renal failure in native and allograft kidneys. Identifying the molecules and pathways involved in the pathophysiology of renal IRI will yield valuable new diagnostic and therapeutic information. To identify differentially regulated genes in renal IRI, RNA from rat kidneys subjected to an established renal IRI protocol (bilateral occlusion of renal pedicles for 30 min followed by reperfusion) and time-matched kidneys from sham-operated animals was subjected to suppression subtractive hybridization. The level of spermidine/spermine N(1)-acetyltransferase (SSAT) mRNA, an essential enzyme for the catabolism of polyamines, increased in renal IRI. SSAT expression was found throughout normal kidney tubules, as detected by nephron segment RT-PCR. Northern blots demonstrated that the mRNA levels of SSAT are increased by greater than threefold in the renal cortex and by fivefold in the renal medulla at 12 h and returned to baseline at 48 h after ischemia. The increase in SSAT mRNA was paralleled by an increase in SSAT protein levels as determined by Western blot analysis. The concentration of putrescine in the kidney increased by approximately 4- and approximately 7.5-fold at 12 and 24 h of reperfusion, respectively, consistent with increased functional activity of SSAT. To assess the specificity of SSAT for tubular injury, a model of acute renal failure from Na(+) depletion (without tubular injury) was studied; SSAT mRNA levels remained unchanged in rats subjected to Na(+) depletion. To distinguish SSAT increases from the effects of tubular injury vs. uremic toxins, SSAT was increased in cis-platinum-treated animals before the onset of renal failure. The expression of SSAT mRNA and protein increased by approximately 3.5- and >10-fold, respectively, in renal tubule epithelial cells subjected to ATP depletion and metabolic poisoning (an in vitro model of kidney IRI). Our results suggest that SSAT is likely a new marker of tubular cell injury that distinguishes acute prerenal from intrarenal failure.

We measured the elastic response of single DNA molecules at various concentrations of the trivalent cation, spermidine. When added spermidine caused the DNA to collapse, the force-extension curves showed either plateaus or stick-release patterns depending on the concentration. The periodic stick-release response determines a characteristic length, which may reflect toroidal supercoiling. At high concentrations of spermidine, we observed the reelongation of single molecules of collapsed DNA. Thus condensation occurs between lower and upper critical concentrations, verifying that the transition is reentrant as theoretically predicted.

Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS-deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS-deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS-deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis. We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens.

Recent studies have implicated alterations in the expression of polyamine-related genes in the brains of suicide completers including widespread downregulation of spermidine/spermine N1-acetyltransferase, the key enzyme in polyamine catabolism, suggesting compensatory mechanisms attempting to increase brain levels of polyamines. Given the complexity of the polyamine system, quantification of the levels of the polyamines is an essential step in understanding the downstream effects of dysregulated gene expression. We developed a method using high-resolution capillary gas chromatography (GC) in combination with mass spectrometry (MS) for quantitation of polyamines from post-mortem brain tissue, which allowed us to accurately measure spermidine and putrescine concentrations in post-mortem brain tissues. Using this method, we analyzed putrescine and spermidine levels in a total of 126 samples from Brodmann areas 4, 8/9, and 11, from 42 subjects, comprising 16 suicide completers with major depression, 13 non-depressed suicide completers, and 13 control subjects. Both putrescine and spermidine levels fell within the expected nanomolar ranges and were significantly elevated in the brain of suicide completers with a history of major depression as compared with controls. These results were not accounted by possible confounders. This is the first GC-MS study to analyze the expression of putrescine and spermidine from post-mortem brain tissue and confirms the hypothesis raised by previous studies indicating alterations in putrescine and spermidine levels in suicide/major depression.

BACKGROUND

Many mammalian cells possess an active polyamine uptake system but little is known about the molecular mechanism of this transporter. The fate of polyamines taken up from the medium and the relationship to polyamine homeostasis remains to be fully established. The aim of this study was to develop a range of modified polyamines, particularly ligands incorporating a fluorophore, to explore the structural tolerances of the polyamine transport system and to probe the intracellular location of polyamines acquired from the medium.

RESULTS

We synthesised a wide range of polyamine analogues incorporating cytotoxic agents, fluorescent chromophores and bulky substituents. All of these analogues have been shown to be good competitive inhibitors of spermidine uptake in a range of mammalian cells. Direct evidence for uptake of the fluorescent polyamine analogues and their subcellular distribution was obtained from confocal laser scanning fluorescence microscopy, which showed that they accumulated in granular structures within the cytoplasm and not in the nucleus. We demonstrated that their uptake is through the polyamine transport system by showing that pretreatment with DFMO, a potent inhibitor of polyamine biosynthesis, led to enhanced uptake, and cells deficient in the polyamine transport system did not accumulate these polyamine analogues.

CONCLUSIONS

The polyamine transport system has a surprisingly broad structural tolerance. Fluorophore-containing polyamine analogues derived from the extracellular pool are located in granular structures within the cytoplasm and not to any great extent in the nuclei of mammalian cells. These observations might be consistent with a mechanism involving receptor-mediated endocytosis, and the granular 'structures' seen might reflect polyamine compartmentalisation within vesicles.

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.

Mutant Gy male mice, which have previously been described as having disruption of the phosphate-regulating Phex gene and a spermine synthase gene [Meyer, Henley, Meyer, Morgan, McDonald, Mills and Price (1998) Genomics, 48, 289-295; Lorenz, Francis, Gempel, Böddrich, Josten, Schmahl and Schmidt (1998) Hum. Mol. Genet. 7, 541-547], as well as mutant Hyp male mice, which have disruption of the Phex gene only, were examined along with their respective normal male littermates. Biochemical analyses of extracts of brains, hearts and livers of 5-week-old mice showed that Gy males lacked any significant spermine synthase activity as well as spermine content. Organs of Gy males had a higher spermidine content. This was caused not only by the lack of conversion of spermidine into spermine, but also because of compensatory increases in the activities of other polyamine biosynthetic enzymes. Gy males were half the body weight of their normal male littermates at weaning age. Hyp males, however, were no different in size when compared with their controls. High mortality of Gy males occurs by weaning age and this mortality was shown to be largely post-natal. Embryonic fibroblasts were isolated from Gy males and their normal male littermates and were similarly shown to lack any significant spermine synthase activity as well as spermine content. The lack of spermine, however, had no significant effect on the growth of immortalized fibroblasts or of primary fibroblast cultures. Similarly, there was no difference in the time of senescence of primary fibroblast cultures from Gy males compared with cultures derived from normal male littermates. However, the lack of spermine did increase the sensitivity of immortalized fibroblasts to killing by the chloroethylating agent 1, 3-bis(2-chloroethyl)-N-nitrosourea. Therefore both the Gy male mice and derived embryonic fibroblasts provide valuable models to study the importance of spermine and spermine synthase, without the use of inhibitors which may have additional side effects.

To further explore the biochemical basis of Cd toxicity in developing wheat seedlings, we studied the possible role of nitric oxide (NO) and polyamines as signaling molecules involved in metal-induced root growth inhibition. When used at 0.1 mM, sodium nitroprusside, a NO-releasing compound, inhibited root growth to a similar extent as Cd and enhanced the polyamine contents as Cd also did. Putrescine and spermidine treatments caused significant decreases in root growth with spermine giving the greatest level of inhibition (77% reduction). The simultaneous addition of Cd and inhibitors of putrescine biosynthesis (DFMA and DFMO) prevented increases in putrescine levels but did not restore normal root growth. NO content, as evidenced by the fluorescent probe DAF-FM diacetate, was found to be significantly increased in the roots of both Cd and polyamine treated plants, especially in those exposed to spermine. The effect was specific for NO since the NO scavenger cPTIO almost suppressed the fluorescent signal. Concerning the oxidative status of the root system, only Cd and spermine enhanced lipid peroxidation in roots. At the same time, all treatments led to a significant increase in levels of the non-enzymatic antioxidant defense glutathione. Our results strongly suggest that Cd and spermine treatments induce NO formation in wheat roots which, in turn, is involved in root growth inhibition.

Analogues of S-adenosylmethionine that were designed as inhibitors of S-adenosylmethionine decarboxylase were tested for their abilities to inhibit the purified enzyme from rat prostate. The most potent inhibitors were 5'-deoxy-5'-[N-methyl-N-[2-(aminooxy)ethyl]amino]adenosine (MAOEA) and 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine (MHZPA), which had I50 values of 400 nM and 70 nM, respectively, when added directly to the assay medium under standard conditions. These compounds were irreversible inactivators of the enzyme, and more than 95% of the activity was lost within 15 min of exposure to 5 microM MAOEA or 0.5 microM MHZPA. Both inhibitors led to a large reduction in the content of decarboxylated S-adenosylmethionine in L1210 cells and to a substantial decrease in the production of 5'-(methylthio)adenosine by these cells. These results are consistent with their bringing about an inhibition of S-adenosylmethionine decarboxylase activity in the cell which leads to a reduction in the synthesis of spermidine and spermine. Analysis of the polyamine content in L1210 cells exposed to 100 microM MAOEA or 50 microM MHZPA showed that this was the case and that putrescine levels were greatly increased while spermidine and spermine content declined. The combined application of 100 microM MAOEA and 5 mM alpha-(difluoromethyl)ornithine (an ornithine decarboxylase inhibitor) to L1210 cells completely prevented the synthesis of putrescine, spermidine, and spermine for up to 48 h. The reduction in polyamine content brought about by MHZPA or MAOEA could be partially prevented by the addition of decarboxylated S-adenosylmethionine to the culture medium. These inhibitors also brought about an inhibition of cell growth which could be reversed by the addition of spermidine.(ABSTRACT TRUNCATED AT 250 WORDS)

The rates of synthesis and turnover of the rare amino acid hypusine [N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid] in protein were studied in relationship to polyamine metabolism and growth rates in rat hepatoma tissue-culture (HTC) cells. Hypusine is selectively formed in the eukaryotic translation initiation factor eIF-4D, by a post-translational mechanism involving spermidine [Cooper, Park, Folk, Safer & Braverman (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1854-1857]. The half-life of the hypusine-containing protein was longer than 24 h. In cells whose intracellular spermidine pools had been initially depleted, by using DL-alpha-difluoromethylornithine (DFMO), maximum synthesis rates of hypusine in protein were 5-10 times higher, on restoration of endogenous spermidine contents by exogenous addition, than those observed in untreated exponential-phase cultures. In cells pretreated with DFMO, the rate of hypusine synthesis was constant for up to 1 h after the addition of 5 microM-spermidine, whereas endogenous spermidine contents varied from less than 1 to more than 10 nmol/mg of protein. However, the overall amount of hypusine formed, during the first 1 h after the addition of various concentrations of spermidine (0.05-10 microM) to the culture medium, was markedly dependent on the final endogenous spermidine content achieved at the end of the 1 h measurement interval. Early in exponential-phase growth, protein-bound hypusine was synthesized at a rate of 1-2 pmol/h per mg of protein. This rate decreased to less than 0.5 pmol/h per mg of protein when cell growth rates decreased as cultures reached high cell densities. Analysis of the polyamine substrate specificity for hypusine formation showed that N1-acetylspermidine did not compete with spermidine in the reaction, nor did N1-(buta-2,3-dienyl)-N2-methylbutane-1,4-diamine, and irreversible inhibitor of polyamine oxidase, block the reaction. On the basis of comparative radiolabelling experiments, spermine was either a poor substrate, or not a substrate, for hypusine formation. These results confirm that spermidine is the likely precursor of the aminohydroxybutyl moiety of hypusine, and show that overall hypusine formation, but not necessarily the synthesis rate, is dependent on the endogenous spermidine concentration, especially under conditions where spermidine concentrations are initially low, as is the case after DFMO treatment, and then increase.

The effects of NaCl (260 mM) and sorbitol (360 mM) isoosmotic stresses on polyamine titers in lupin (Lupinus luteus L. var. Ventus) in relation to organ-specific responses were investigated. Analysis showed that during the first few hours (4 h) of salt and osmotic stress higher amounts of putrescine (Put) and spermidine (Spd) were accumulated in the roots and leaves of lupin seedlings. After exposing the plants to a longer duration (24 h) of exposure to NaCl, the level of free Put decreased in roots and cotyledons by about 48% and 54%, respectively, and increased in hypocotyls and leaves by about 27% and 73%, respectively. The Level of free Spd also decreased in roots by about 50%, in contrast to the increase of Spd observed in hypocotyls and leaves by about 50% and 70%, respectively. The effect of non-ionic stress on the level of Put and Spd in studied organs of lupin was similar to that of NaCl. Free spermine was at an undetectable level in examined organs. However, in the roots of lupin growing for 24 h in the presence of NaCl and/or sorbitol, the activity of arginine decarboxylase (ADC) (EC 4.1.1.19) increased by about 66% and 80%, respectively. ADC activity in leaves was similar to that observed in the control. Additionally, in the roots and leaves of lupin growing under the stress condition (NaCl or sorbitol), a higher level of polyamines (PAs) bound to microsomal membranes was observed. It is probable that PAs bound to microsomal membranes prevent stress-induced damage. We conclude that both stresses induce biosynthesis of Put and other PAs in the roots, as well as Put accumulation in the leaves, and this may indicate translocation of Put from the roots to the shoot. The possible role of PAs in adaptive mechanisms to stress is discussed.

Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G. In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.

The effect of the 5'-untranslated region of ornithine decarboxylase (ODC) mRNA and of spermidine on the translation of ODC mRNA was studied in a rabbit reticulocyte cell-free system. The ODC mRNAs, possessing different sizes of the 5'-untranslated region and named mODC241, mODC188, mODC85, and mODC24 according to the number of nucleotides in the 5'-untranslated region, were synthesized by in vitro transcription techniques through the use of plasmids containing various sizes of mouse ODC cDNA. As the size of the 5'-untranslated region of ODC mRNA increased, the efficiency of the translation decreased and the degree of stimulation of the translation by 0.2 mM spermidine increased. The inhibition of ODC mRNA translation by high concentrations of spermidine (0.6-1 mM) also increased with an increase in the size of the 5'-untranslated region. The spermidine effects were increased greatly when the size of the 5'-untranslated region of ODC mRNA was increased from 85 to 188 nucleotides. These results suggest that the nucleotides 70-220 upstream from the initiator AUG are involved in the decrease in translational efficiency. In addition, at least the nucleotides in the 70-170 region upstream from the initiator AUG are important in the strong stimulation of ODC synthesis by low spermidine concentrations and in the inhibition of ODC synthesis at high spermidine concentrations.

The goal of the current study was to examine whether polyamines are involved in the regulation of transcription and posttranscription of the protooncogenes c-myc and c-jun in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis, for 4 or 6 days not only almost completely depleted total (whole) cellular and nuclear polyamines but also significantly decreased expression of the protooncogenes c-myc and c-jun in IEC-6 cells. Using nuclear run-on transcription assay, we demonstrated that the basal rate of transcription of c-myc was decreased by 55% at 4 days and by 60% at 6 days in the DFMO-treated cells. The c-jun transcription in DFMO-treated cells was decreased by 75% at 4 days and 85% at 6 days. The transcription rates of c-myc and c-jun were dramatically stimulated by 5% dialyzed fetal bovine serum (dFBS) in normal quiescent cells. However, polyamine depletion significantly prevented the increased transcription of these two genes in the DFMO-treated cells exposed to 5% dFBS. Furthermore, direct administration of spermidine to isolated nuclei from polyamine-deficient (caused by DFMO) cells resulted in a 2- to 2.5-fold increase in c-myc and c-jun transcription. There were no significant changes in the half-lives of c-myc and c-jun mRNAs between the controls and the DFMO-treated cells. These results indicate that 1) polyamines are required for the transcription of the protooncogenes c-myc and c-jun in IEC-6 cells and 2) depletion of intracellular polyamines has no effect on posttranscriptional regulation of c-myc and c-jun mRNAs. These findings suggest that polyamines play an important role in the regulation of the transcription of protooncogenes, and this may be one mechanism by which polyamines modulate mucosal cell division.

Activator protein 1 (AP-1) is a group of dimeric transcription factors composed of protooncogene (Jun and Fos) subunits that bind to a common DNA site, the AP-1 binding site. The proteins of c-Jun, JunB, and Fos are essential for initiation of the cell cycle. Conversely, the activation of the junD gene slows cell growth in some cell types. The current study tests the hypothesis that polyamines influence cell growth by altering the balance of positive and negative Jun/AP-1 activities in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor for polyamine synthesis, for 4 and 6 days completely depleted cellular polyamine levels, while AP-1 binding activity was significantly increased. Spermidine, when given together with DFMO, restored AP-1 binding activity toward normal. The increased AP-1 complexes in polyamine-deficient cells were dramatically supershifted by the anti-JunD antibody but not by antibodies against c-Jun, JunB, or Fos proteins. There were significant increases in JunD mRNA and protein in DFMO-treated cells, although expression of the c-fos, c-jun, and junB genes decreased. The increase in JunD/AP-1 activity in DFMO-treated cells was associated with a significant decrease in cell division. Exposure of control quiescent cells to 5% dialyzed serum increased c-Jun/AP-1 but not JunD/AP-1 activities. DFMO prevented the stimulation of c-Jun/AP-1 activity induced by 5% dialyzed serum. These results indicate that 1) polyamine depletion is associated with an increase in AP-1 binding activity and 2) the increase in AP-1 activity in the DFMO-treated cells was primarily contributed by an increase in the JunD/AP-1. These findings suggest that polyamines regulate cell growth at least partially by modulating the balance of positive and negative Jun/AP-1 activities in the intestinal mucosa.