The condensation of DNA induced by spermine and spermidine is investigated by equilibrium titrations and stopped-flow and field-jump experiments using scattered light detection. The spermine concentration required for the cooperative condensation process is measured at different DNA concentrations; these data are used to evaluate both the condensation threshold degree of spermine binding and the binding constant of spermine according to an excluded-site model. Stopped-flow measurements of the spermine-induced condensation demonstrate the existence of two processes: (1) A "fast" reaction is observed in the millisecond time range, when the reactant concentrations are around 1 microM; it is associated with a characteristic induction period and is assigned to the intramolecular condensation reaction. (2) A slow reaction with time constants of, e.g., 100 s strongly dependent upon both spermine and DNA concentrations is assigned to an intermolecular DNA association. The unusual time course of the intramolecular condensation reaction with the induction period provides evidence for a "threshold kinetics". During the induction period, spermine molecules are bound to DNA, but the degree of binding remains below the threshold value. As soon as the degree of ligand binding arrives at the threshold, the DNA is condensed in a relatively fast reaction. Model calculations of the spermine binding kinetics according to an excluded-site model demonstrate that the spermine molecules bound to DNA are mobile along the double helix. A comparison of the experimental data with the results of Monte Carlo simulations suggests a rate constant of approximately 200 s-1 for spermine movement by one nucleotide residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Four cDNAs for spermidine synthase (SPDS), which converts the diamine putrescine to the higher polyamine spermidine using decarboxylated S-adenosylmethionine as the co-factor, were isolated from Nicotiana sylvestris, Hyoscyamus niger, and Arabidopsis thaliana. When the N.sylvestris SPDS cDNA was expressed in a SPDS-deficient E. coli mutant, the recombinant protein showed high SPDS activity, but did not have any spermine synthase activity. The plant SPDSs have molecular masses of about 34 kDa, possess the co-factor binding motifs which have been proposed for S-adenosylmethionine, and are more homologous in amino acid sequence to tobacco putrescine N-methyltransferase (PMT) than to SPDSs from mammals and E. coli. The SPDS gene is expressed in root, stem, and leaf in N.sylvestris, whereas the PMT gene is expressed only in root. The potential evolution of plant SPDS and PMT, and their evolutionary relationships with animal SPDS are discussed.

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.

Antizyme (AZ) is known to be a regulator of polyamine metabolism that inhibits ornithine decarboxylase activity and polyamine transport, thus restricting polyamine levels. Transgenic mice with AZ expression targeted to the basal cell layer of the forestomach epithelium by the keratin 5 promoter were used to investigate whether AZ overexpression inhibited uncontrolled cell proliferation in zinc-deficient (ZD) mice and reduced their susceptibility to forestomach carcinogenesis by N-nitrosomethylbenzylamine (NMBA). Four-week-old keratin 5/AZ and wild-type (Wt) littermates were placed on ZD or zinc-sufficient (ZS) diets to form four groups: ZD:AZ, ZD:Wt, ZS:AZ, and ZS:Wt. After 5 weeks, 27-45 mice in each group were treated twice with NMBA and sacrificed 14 weeks later. Independent of zinc intake, AZ mice had significantly lower forestomach tumor incidence and tumor multiplicity than respective Wt littermates (P < 0.001): 21% of ZD:AZ versus 76% of ZD:Wt mice and 3% of ZS:AZ versus 33% of ZS:Wt mice developed tumors. Spermidine content was reduced in NMBA-treated ZD:AZ forestomachs. Zinc deficiency increased the forestomach cell proliferation in Wt mice, but this effect was blocked by AZ. Conversely, apoptosis was substantially higher in control and NMBA-treated ZD:AZ than respective ZD:Wt forestomachs. The restored ZD:AZ forestomach epithelium displayed strong expression of Bax, a proapoptotic protein, and weak staining of cyclin D1 and its catalytic partner Cdk4, key regulatory proteins controlling G(1) to S progression. In contrast, proliferative ZD:Wt forestomach showed strong expression of Bcl-2, an antiapoptotic protein, and overexpression of cyclin D1/Cdk4. Treatment of ZD:Wt mice with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, had similar results to AZ in reducing tumor incidence, spermidine content, decreasing cell proliferation, and increasing apoptosis. These results demonstrate that AZ may act as a tumor suppressor gene stimulating apoptosis and restraining cell proliferation, thereby inhibiting forestomach tumor development. Although effects of AZ on functions other than polyamine metabolism are possible, alterations in polyamines are the most likely explanation for the reduction in tumors, supporting the use of strategies to modulate polyamine levels for cancer chemoprevention in individuals at high risk of developing malignancies of the gastrointestinal tract.

To directly evaluate the role of increased ornithine decarboxylase (ODC) and polyamines in mouse skin carcinogenesis, we used bovine keratin 5 (K5) and keratin 6 (K6) promoter elements to direct the expression of antizyme (AZ) to specific skin cell populations. AZ is a multifunctional regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. K5-AZ mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 0 and 24 h exhibit increases in epidermal and dermal ODC activity that are reduced in magnitude. K6-AZ mice treated similarly do not show any increased ODC activity or protein after a second application due to TPA-induced expression of AZ protein. Epidermal and dermal polyamine content, particularly spermidine, is reduced in untreated K5-AZ mice and TPA-treated K5-AZ and K6-AZ mice. Susceptibility to 7,12-dimethylbenz(a)anthracene/TPA carcinogenesis was also investigated for two K6-AZ transgenic lines [K6-AZ(52) and K6-AZ(18)] and a single K5-AZ line. K6-AZ(52) mice had a substantial delay in tumor onset and a >80% reduction in tumor multiplicity compared with normal littermates. K6-AZ(18) and K5-AZ mice also developed fewer papillomas than littermate controls (35% and 50%, respectively), and the combination of these lines to produce double transgenic animals yielded an additive decrease (70%) in tumor multiplicity. These mice demonstrate for the first time that AZ suppresses tumor growth in an animal cancer model and provide a valuable model system to evaluate the role of ODC and polyamines in skin tumorigenesis.

Two cationic lipids, bis-guanidinium-spermidine-cholesterol (BGSC) and bis-guanidinium-trencholesterol (BGTC)-cholesterol derivatives bearing two guanidinium groups-have been synthesized and tested as artificial vectors for gene transfer. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pKa features of the guanidinium functions for binding DNA via its phosphate groups. Reagent BGTC is very efficient for transfection into a variety of mammalian cell lines when used as a micellar solution. In addition, both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine. These results reveal the usefulness of cholesterol derivatives bearing guanidinium groups for gene transfer.

Calf liver contains two nuclear N-acetyltransferases which are separated by chromatography on hydroxylapatite. Both acetyltransferase A and acetyltransferase B will transfer acetate from acetyl-CoA to either histone or spermidine. The same protein catalyzes the reaction with both substrates; this is shown by a constant ratio of spermidine to histone activity over a 5,000-fold purification and identical heat denaturation kinetics for both spermidine and histone acetyltransferase activity with each enzyme. Histone is preferentially acetylated when both acceptors are present. Both enzymes preferentially acetylate polyamines (spermidine, spermine, and diaminodipropylamine) to diamines. Acetyltransferase A acetylates histones in the order: whole histone greater than H4 greater than H2A greater than H3 greater than H2B greater than H1; acetyltransferase B in the order: whole histone greater than H4 = H3 greater than H2A greater than H2B greater than H1. Michaelis constants are 2 X 10(-4)M for spermidine and 9 X 10(-6)M for acetyl-CoA. Acetyltransferase A has a molecular weight of 150,000; acetyltransferase B 175,000. Both enzymes are strongly inhibited by p-chloromercuribenzoate and weakly inhibited by EDTA.

The naturally occurring polyamines putrescine, spermidine, and spermine are involved in signal transduction. This has been demonstrated by using inhibitors for polyamine biosynthesis (such as alpha-difluoromethylornithine) or adding polyamines to cultured cells. Different polyamines, preferentially activated protein kinases (tyrosine kinases and MAP kinases), stimulated the expression of nuclear protooncogenes (myc, jun, and fos).

The number of alveolar macrophages is decreased during Pneumocystis pneumonia (Pcp), partly because of activation of apoptosis in these cells. This apoptosis occurs in both rat and mouse models of Pcp. Bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals were found to contain high levels of polyamines, including spermidine, N1-acetylspermine, and N1-acetylspermidine. These BAL fluids and exogenous polyamines were able to induce apoptosis in alveolar macrophages. Apoptosis of alveolar macrophages during infection, after incubation with BAL fluids from Pneumocystis-infected animals, or after incubation with polyamines was marked by an increase in intracellular reactive oxygen species, activation of caspases-3 and -9, DNA fragmentation, and leakage of mitochondrial cytochrome c into the cytoplasm. When polyamines were depleted from the BAL fluids of infected animals, the ability of these BAL fluids to induce apoptosis was lost. Interestingly, the apoptosis inducing activity of the polyamine-depleted BAL fluids was restored when polyamines were added back. The results of this study suggested that Pneumocystis infection results in accumulation of high levels of polyamines in the lung. These polyamines activate apoptosis of alveolar macrophages, perhaps because of the ROS that are produced during polyamine metabolism.

We have investigated the interactions of polyamines and the N-methyl-D-aspartate (NMDA) receptor antagonist ifenprodil with the binding of [3H]MK801 to the NMDA receptor. Spermine and spermidine but not putrescine substantially increase [3H]MK801 binding to well washed rat brain membranes in the absence or presence of saturating concentrations of glutamate and glycine. Spermine also increased the association and dissociation of [3H]MK801 from its binding site, suggesting that polyamines activate the NMDA receptor in a similar manner to glycine. Ifenprodil inhibited the binding of [3H]MK801 in a biphasic fashion. The high affinity phase of binding (Ki of approximately 15 nM) accounted for 50-60% of total [3H]MK801 binding in the nominal absence of glutamate, glycine, and polyamines or in the presence of 100 microM glutamate. This fraction was reduced to 20% by the addition of 30 microM glycine and could be abolished by the addition of 50 microM spermine. However, ifenprodil apparently did not act by binding to the polyamine recognition site. The low affinity phase (Ki of 20-40 microM) was insensitive to the presence of positive modulators and may represent binding to the Zn2+ regulatory site. Ifenprodil decreased NMDA and glycine-induced Ca2+ influx into cultured rat brain neurons. The potency of ifenprodil suggests that spermine may activate NMDA receptors in vivo. These data indicate that ifenprodil may bind to the NMDA receptor in a state-dependent fashion and preferentially stabilize an inactivated form of the channel.

The discriminative stimulus properties of compounds that interact with the NMDA receptor complex were investigated in rats trained to discriminate ethanol from saline. Male Wistar rats were trained in a two-lever operant drug discrimination paradigm to make differential responses [fixed ratio 10 (FR10)] for food after ethanol (1 g/kg i.p.; 12% v/v ethanol solution) and saline vehicle injections. Drug effects were assessed by means of generalization and antagonism tests. In the generalization tests, the noncompetitive NMDA antagonists acting at the ion channel dizocilpine, memantine, phencyclidine (PCP) and the sigma1 receptor agonists (+)-pentazocine and (+)-N-allyl-normetazocine (NANM) dose-dependently generalized for ethanol, whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) antagonist GYKI 52466, the glycine antagonists L-701,324 and MRZ 2/502, the polyamine site antagonist arcaine and the polyamine site ligand spermidine, did not. Our results show that the noncompetitive NMDA antagonists fully substitute dose-dependently for ethanol in a drug-discrimination task. The ethanol-like discriminative stimulus effects of PCP, pentazocine and NANM, which are also sigma receptor ligands, are likely to be attributed to their activity at NMDA receptors. We therefore assume that some of the acute effects of ethanol are mediated via NMDA receptor antagonism at the PCP binding site.

Molecular, biochemical and genetic characterization of ornithine decarboxylase, S -adenosylmethionine decarboxylase and spermidine synthase establishes that these polyamine-biosynthetic enzymes are essential for growth and survival of the agents that cause African sleeping sickness, Chagas' disease, leishmaniasis and malaria. These enzymes exhibit features that differ significantly between the parasites and the human host. Therefore it is conceivable that exploitation of such differences can lead to the design of new inhibitors that will selectively kill the parasites while exerting minimal, or at least tolerable, effects on the parasite-infected patient.

The effect of NaCl on the endogenous levels of diamine, putrescine and polyamines, spermidine and spermine, was studied in the shoot system of salt-tolerant and salt-sensitive lines of rice (Oryza sativa L.) cultivars during three growth stages. Salt stress increased the levels of diamine and polyamine in varying degrees among nine rice cultivars investigated. Salt tolerant AU1, Co43, and CSC1 were effective in maintaining high concentrations of spermidine and spermine, while the content of putrescine was not significantly altered in all the growth stages when plants were exposed to salinity. The salt sensitivity in rice was associated with excessive accumulation of putrescine and with low levels of spermidine and spermine in the shoot system of salt-sensitive cultivars Co36, CSC2, GR3, IR20, TKM4, and TKM9 under saline condition. One of the possible mechanisms of saline resistance was observed to be due to the highly increased polyamines against the low increase in diamines. Alternatively, the salt sensitivity could be due to high increase of diamines and an incapacity to maintain high levels of polyamines.

The enzymes glutathione-S-transferases (GSTs, E.C.2.5.1.18) have been associated with detoxification of xenobiotics, limiting oxidative damage and other stress responses in plants. In this study, we report the isolation of a mustard gene, BjGSTF2, homologous to the phi class GSTs and changes in plant growth in vivo and shoot regeneration in vitro were related to GST expression. GST transcripts accumulated differentially in mustard organs, where transcript was most abundant in root. Tissues incubated at high temperature or in the presence of exogenous H2O2, HgCl2, 1-aminocyclopropane-1-carboxylate, salicylic acid and paraquat upregulated GST expression, whereas spermidine was inhibitory. To investigate the in vivo function of GST, transgenic Arabidopsis thalianaplants expressing sense (GST-S6), antisense (GST-A4) and double-stranded BjGSTF2 (GST-DS1) RNAs were generated. GST-S6 was shown to flower two days earlier and was relatively more tolerant to HgCl2 and paraquat, whereas GST-DS1 with least stress tolerance flowered one week later compared to WT and GST-A4. In shoot regeneration response, tissues originated from GST-S6 were highly regenerative, whereas no shoot regeneration was observed in GST-DS1 tissues after 30 days of culture. Results of this study provide the evidence showing that GST plays a role in plant growth and development in vivo and shoot regeneration in vitro.

BACKGROUND

alpha-Difluoromethylornithine (DFMO) is a potent inhibitor of carcinogenesis in experimental animal models. In these animal models, DFMO has been especially active in preventing carcinogen-induced epithelial cancers, including those of the skin, colon, breast, and urinary bladder. Although DFMO is known to exert its diverse biological effects by suppressing intracellular pools of the polyamines putrescine and spermidine, the precise mechanism by which polyamine depletion, induced by DFMO, suppresses carcinogenesis is unknown.

OBJECTIVE

The specific aim of our study was to determine the lowest dose of DFMO that would deplete target tissue (colorectal mucosa) levels of these polyamines in humans who had undergone prior removal of colon polyps while producing minimal toxic effects.

METHODS

A dose de-escalation chemoprevention trial of DFMO was conducted in 111 patients (36 female and 75 male) who were in generally good health, aged 39-79, and who had undergone colonoscopy for surgical removal of an adenomatous colon polyp greater than 3 mm within 5 years prior to entering the study. Groups of patients (12-20 patients per group) were orally treated with single, daily doses of DFMO ranging from 3.0 to 0.1 g/m2 for 4 weeks (28 days). Prior to initiation of DFMO treatment and at the end of treatment, six colorectal biopsy specimens were collected from each patient, along with serum samples. All biopsies were performed between 9 AM and noon to avoid possible effects of diurnal variations in laboratory end points. Samples for analysis of plasma DFMO levels were also collected during this time period on the day after the last day of drug administration.

RESULTS

DFMO caused a decrease in both putrescine content and the ratio of spermidine to spermine for all dose groups down to 0.25 g/m2. Both putrescine content and the ratio of spermidine to spermine and changes in these parameters as a function of DFMO treatment decreased as a function of donor age. None of the 30 patients receiving either 0.25 or 0.5 g/m2 experienced any clinical ototoxicity in this trial.

CONCLUSIONS

DFMO is both safe and effective in reducing colorectal mucosal polyamine contents when it is administered orally to patients at doses as low as 0.25 g/m2 for 28 days. No ototoxicity was observed at doses up to twice this amount.

CONCLUSIONS

If DFMO is also found to be effective in suppressing polyamine contents in other target tissues, it may be useful in preventing a wide range of human epithelial cancers, including those of the prostate and breast.

The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because cellular proliferation requires elevated levels of polyamines. A byproduct of the synthesis of spermidine and spermine is 5'-methylthioadenosine (MTA). In humans MTA is processed by 5'-methylthioadenosine phosphorylase (MTAP) so that significant amounts of MTA do not accumulate. Products of the MTAP reaction (adenine and 5-methylthio-alpha-D-ribose-1-phosphate) are recycled to S-adenosylmethionine, the precursor for polyamine synthesis. Potent inhibitors of MTAP might allow the build-up of sufficient levels of MTA to generate feedback inhibition of polyamine biosynthesis and/or reduce S-adenosylmethionine levels. We recently reported the design and synthesis of a family of potent transition state analogue inhibitors of MTAP. We now report the synthesis of a second generation of stable transition state analogues with increased distance between the ribooxocarbenium ion and purine mimics. These compounds are potent inhibitors with equilibrium dissociation constants as low as 10 pM. The first and second generation inhibitors represent synthetic approaches to mimic early and late features of a dissociative transition state.

The purpose of this study was to examine whether luminal polyamines can substitute for tissue polyamines in the healing process of gastric mucosal stress ulcers. Rats were fasted 22 h, placed in restraint cages, and immersed in water to the xiphoid process for 6 h. Animals were killed either immediately or at 4, 12, or 24 h after the period of stress. Stress significantly increased ornithine decarboxylase (ODC) activity and tissue polyamine content. Mucosal polyamine levels peaked 4 h after stress and remained significantly elevated for 12 h. The healing process, which was significant by 12 h, was inhibited by DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC. DFMO totally prevented the marked increases in ODC and polyamine levels that usually followed stress. Oral administration of polyamines, putrescine, cadaverine, spermidine, or spermine, immediately after stress increased the normal rate of healing and prevented the inhibition of repair caused by DFMO. Spermidine or spermine accelerated healing better than putrescine or cadaverine. The delayed recovery of mucosal DNA, RNA, and protein content after stress in the DFMO-treated rats was also significantly prevented by exogenous polyamines. The reduced amounts of gastric mucosal spermidine and spermine in rats treated with DFMO returned toward control levels after administration of exogenous spermidine (100 mg/kg). These results show that 1) increased levels of polyamines provided by ODC are absolutely required for normal healing of gastric mucosal stress ulcers, 2) the polyamines are active from the luminal side, and 3) polyamines accelerate healing at least partly through a mechanism involving cell renewal.

BACKGROUND

Spermidine, a naturally occurring polyamine, displays a wide variety of internal biological activities including cell growth and proliferation. However, the molecular mechanisms responsible for its anti-inflammatory activity have not yet been elucidated.

METHODS

The anti-inflammatory properties of spermidine were studied using lipopolysaccharide (LPS)-stimulated murine BV2 microglia model. As inflammatory parameters, the production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were evaluated. We also examined the spermidine's effect on the activity of nuclear factor-kappaB (NF-κB), and the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways.

RESULTS

Pretreatment with spermidine prior to LPS treatment significantly inhibited excessive production of NO and PGE2 in a dose-dependent manner, and was associated with down-regulation of expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Spermidine treatment also attenuated the production of pro-inflammatory cytokines, including IL-6 and TNF-α, by suppressing their mRNA expressions. The mechanism underlying spermidine-mediated attenuation of inflammation in BV2 cells appeared to involve the suppression of translocation of NF-κB p65 subunit into the nucleus, and the phosphorylation of Akt and MAPKs.

CONCLUSIONS

The results indicate that spermidine appears to inhibit inflammation stimulated by LPS by blocking the NF-κB, PI3K/Akt and MAPKs signaling pathways in microglia.

The nuclear protein c-Myc is a transcription factor involved in the control of cell cycle. Our previous studies indicated that cellular polyamines are absolutely required for cell proliferation in crypts of small intestinal mucosa and that polyamines have the ability to stimulate expression of the c-myc gene. The current study went further to determine whether induced nuclear c-Myc plays a role in stimulation of cell proliferation by polyamines in intestinal crypt cells (IEC-6 line). Exposure of normal quiescent cells after 24-h serum deprivation to 5% dialyzed fetal bovine serum (dFBS) increased both cellular polyamines and expression of the c-myc gene. Increased c-Myc protein formed heterodimers with its binding partner, Max, and specifically bound to the Myc/Max binding site, which was associated with an increase in DNA synthesis. Depletion of cellular polyamines by pretreatment with alpha-difluoromethylornithine (DFMO) prevented increases in c-myc expression and DNA synthesis induced by 5% dFBS. c-Myc gene transcription and cell proliferation decreased in polyamine-deficient cells, whereas the natural polyamine spermidine given together with DFMO maintained c-myc gene expression and cell growth at normal levels. Disruption of c-myc expression using specific c-myc antisense oligomers not only inhibited normal cell growth (without DFMO) but also prevented the restoration of cell proliferation by spermidine in polyamine-deficient cells. Ectopic expression of wild-type c-myc by recombinant adenoviral vector containing c-myc cDNA increased cell growth. These results indicate that polyamine-induced nuclear c-Myc interacts with Max, binds to the specific DNA sequence, and plays an important role in stimulation of normal intestinal epithelial cell proliferation.

Polyamines are endogenous immunomodulatory molecules. Recent studies revealed that polyamines suppress the production of proinflammatory cytokines and nitric oxide. In the present study, we investigated the effect of the polyamines spermine, spermidine, and putrescine on the production of interleukin (IL)-12 p40, IL-10, and interferon (IFN-gamma) in mouse peritoneal macrophages and spleen cell suspensions. Spermine, but not spermidine or putrescine, suppressed, in a concentration-dependent manner, the production of IL-12 p40 by lipopolysaccharide (LPS)-stimulated macrophages. The effect of spermine was post-transcriptional, because steady-state levels of messenger ribonucleic acid (mRNAs) for IL-12 (p35 and p40) were not affected. In contrast to its inhibitory effect on IL-12 p40, spermine (0.3-3 microM) augmented IL-10 production. The down-regulation of IL-12 p40 by spermine was independent of enhancement of IL-10 by this agent, for spermine retained its ability to suppress IL-12 production in peritoneal macrophages obtained from IL-10-deficient mice. The alterations in cytokine production by spermine did not involve an effect on early intracellular pathways of LPS signal transduction, including the p38 or p42/44 mitogen-activated protein kinases, or the c-jun terminal kinase. In spleen cell suspensions, spermine suppressed the release of IFN-gamma induced either by LPS or anti-CD3 antibody. In summary, spermine exerts anti-inflammatory effects by suppressing IL-12 and IFN-gamma and by augmenting the production of IL-10.

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-l-methionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the tolerance of high-temperature stress in tomato, SAMDC cDNA isolated from Saccharomyces cerevisiae was introduced into tomato genome by means of Agrobacterium tumefaciens through leaf disc transformation. Transgene and expression was confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing yeast SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wild-type plants under high temperature stress, and enhanced antioxidant enzyme activity and the protection of membrane lipid peroxidation was also observed. This subsequently improved the efficiency of CO(2) assimilation and protected the plants from high temperature stress, which indicated that the transgenic tomato presented an enhanced tolerance to high temperature stress (38 degrees C) compared with wild-type plants. Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating high temperature-tolerant germplasm.

Cardiovascular diseases are the most prominent maladies in aging societies. Indeed, aging promotes the structural and functional declines of both the heart and the blood circulation system. In this review, we revise the contribution of known longevity pathways to cardiovascular health and delineate the possibilities to interfere with them. In particular, we evaluate autophagy, the intracellular catabolic recycling system associated with life- and health-span extension. We present genetic models, pharmacological interventions, and dietary strategies that block, reduce, or enhance autophagy upon age-related cardiovascular deterioration. Caloric restriction or caloric restriction mimetics like metformin, spermidine, and rapamycin (all of which trigger autophagy) are among the most promising cardioprotective interventions during aging. We conclude that autophagy is a fundamental process to ensure cardiac and vascular health during aging and outline its putative therapeutic importance.

Translation initiation factor eIF-5A (previously named eIF-4D) is an essential and highly conserved protein in eukaryotic cells that promotes formation of the first peptide bond. One of its lysine residues is post-translationally modified by spermidine to form hypusine, a unique residue required for eIF-5A activity. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B. The two genes are regulated reciprocally by oxygen, where under aerobic conditions TIF51A is expressed and TIF51B is repressed, and under anaerobic conditions the opposite occurs. In order to study the products of the two genes individually, yeast strains were constructed that express either TIF51A or TIF51B under control of a galactose promoter. Each gene gives rise to two isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic), both of which carry the hypusine modification. Expression of either TIF51A or TIF51B promotes growth under both aerobic and anaerobic conditions, indicating that the two gene products function indistinguishably. The human cDNA encoding eIF-5A also was expressed in yeast, and the plasmid shuffle technique was used to demonstrate that the human protein can substitute for the homologous yeast protein in vivo. These results indicate that human and yeast eIF-5A are not only conserved at the sequence level but are functionally interchangeable in vivo.

Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of protein-polyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary amine-containing labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using (31)P NMR, we confirmed that EDAC-mediated reactions between primary amines and polyphosphate result in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores, and immobilized onto solid supports, that immobilized polyphosphate can be readily used to quantify protein binding affinities, that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting, and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles.