Polyamines modulate mouse sperm motility.

Polyamines are polycationic molecules which contains two or more amino groups (-NH3+) highly charged at physiological pH, and among them we found spermine, spermidine, putrescine, and cadaverine. They interact with proteins, nucleic acids, modulate Ca2+, K+, and Na+ channels, and protect sperm from oxidative stress. In this work, we evaluate the effect of spermine, spermidine, and putrescine on the total, progressive and kinematic parameters of motility, capacitation, acrosome reaction, also in presence and absence of the dbcAMP, an analogue of the cAMP, and the IBMX, a phosphodiesterase inhibitor. In addition, we evaluated the intracellular concentrations of cAMP [cAMP]i, and performed an in silico analysis between polyamines and the sAC from mouse to predict the possible interaction among them. Our results showed that all polyamines decrease drastically the total, progressive and the kinetic parameters of sperm motility, decrease the capacitation, and only spermidine and putrescine impeded the acquisition of acrosome reaction. Moreover, the effect of polyamines was attenuated but not countered by the addition of db-cAMP and IBMX, suggesting a possible inhibition of the sAC. Also, the presence of polyamines induced a decrease of the [cAMP]i, and the in silico analysis predicted a strong interaction among polyamines and the sAC. Overall, the evidence suggests that probably the polyamines interact and inhibit the activity of the sAC.

Putrescine supplementation shifts macrophage L-arginine metabolism related-genes reducing Leishmania amazonensis infection.

Leishmania is a protozoan that causes leishmaniasis, a neglected tropical disease with clinical manifestations classified as cutaneous, mucocutaneous, and visceral leishmaniasis. In the infection context, the parasite can modulate macrophage gene expression affecting the microbicidal activity and immune response. The metabolism of L-arginine into polyamines putrescine, spermidine, and spermine reduces nitric oxide (NO) production, favoring Leishmania survival. Here, we investigate the effect of supplementation with L-arginine and polyamines in infection of murine BALB/c macrophages by L. amazonensis and in the transcriptional regulation of genes involved in arginine metabolism and proinflammatory response. We showed a reduction in the percentage of infected macrophages upon putrescine supplementation compared to L-arginine, spermidine, and spermine supplementation. Unexpectedly, deprivation of L-arginine increased nitric oxide synthase (Nos2) gene expression without changes in NO production. Putrescine supplementation increased transcript levels of polyamine metabolism-related genes Arg2, ornithine decarboxylase (Odc1), Spermidine synthase (SpdS), and Spermine synthase (SpmS), but reduced Arg1 in L. amazonensis infected macrophages, while spermidine and spermine promoted opposite effects. Putrescine increased Nos2 expression without leading to NO production, while L-arginine plus spermine led to NO production in uninfected macrophages, suggesting that polyamines can induce NO production. Besides, L-arginine supplementation reduced Il-1b during infection, and L-arginine or L-arginine plus putrescine increased Mcp1 at 24h of infection, suggesting that polyamines availability can interfere with cytokine/chemokine production. Our data showed that putrescine shifts L-arginine-metabolism related-genes on BALB/c macrophages and affects infection by L. amazonensis.

Effects of putrescine injection in broiler breeder eggs.

The aim of this study was to evaluate the effects of increasing doses of putrescine injected in ovo on hatchability, intestinal morphology and pre-starter performance of broilers. For this purpose, 720 eggs from broiler breeders were separated into a negative control (no injection) and injection treatments with increasing doses of putrescine (0.05; 0.1; 0.15 and 0.2%), totalling five treatments of 144 eggs each. Eggs were distributed in a completely randomized design inside the setter and the injection of solutions occurred at 17 days of incubation. After hatch, 330 birds were housed in mixed lots following the original treatments, totalling 5 treatments of 6 replicates with 11 birds each. Six birds per treatment were weighed and euthanized by cervical dislocation to collect the liver, intestine and breast 24 hr after injection, at hatch and 24 hr after hatch. At 2 days of age, intestines were collected from 4 animals per treatment to analyse histomorphology. The effects of putrescine levels were evaluated by polynomial regression models, ANOVA and Tukey test at 5% probability. The hatchability decreased linearly in response to increased doses of putrescine. The percentage of residual yolk was lower in animals that received putrescine compared to the control. After injection, the percentage of breast increased linearly, and the percentage of intestine had a quadratic response to increased doses of putrescine. However, 24 hr after hatch, the percentage of intestine linearly decreased, and the percentage of liver linearly increased in response to increased doses of putrescine. Villus height increased quadratically, crypt depth decreased linearly, and goblet cells increased linearly in response to the putrescine dose. FI and BWG were not affected in the pre-starter phase; however, FCR increased in response to increased levels of putrescine. Due to putrescine effects on embryos, it is recommended that the doses injected in ovo not exceed 0.1%.

Proteomic profile approach of effect of putrescine depletion over Trichomonas vaginalis.

Infection with Trichomonas vaginalis produces a malodorous seropurulent vaginal discharge due to several chemicals, including polyamines. The presence of 1,4-diamino-2-butanone (DAB) reduces the amount of intracellular putrescine by 90%, preventing the cotransport of exogenous spermine. DAB-treated parasites present morphological changes, which are restored by adding exogenous putrescine into the culture medium. However, the effect of polyamines over the trichomonad proteomic profile is unknown. In this study, we used a proteomic approach to analyze the polyamine-depletion and restoration effect by exogenous putrescine on T. vaginalis proteome. In the presence of inhibitor DAB, we obtained 369 spots in polyamine-depleted condition and observed 499 spots in the normal culture media. With DAB treatment, the intensity of 43 spots was increased but was found to be reduced in 39 spots, as compared to normal conditions. Interestingly, in DAB-treated parasites restored with a medium with added exogenous putrescine, 472 spots were found, of which 33 were upregulated and 63 were downregulated in protein intensity. Some of these downregulated proteins in DAB-treated parasites are involved in several cellular pathways such as glycolysis, glycolytic fermentation, arginine dihydrolase pathway, redox homeostasis, host cell binding mediated by carbohydrate, chaperone function, and cytoskeletal remodeling. Interestingly, the intensity of some of the proteins was restored by adding exogenous putrescine. In conclusion, the presence of DAB altered the proteomic profile of T. vaginalis, resulting in a decrease in the intensity of 130 proteins and an increase in the intensity of 43 proteins that was restored by the addition of putrescine.

Polyamines regulate phosphorylation-dephosphorylation kinetics in a crustacean gill (Na+, K+)-ATPase.

Aiming to clarify the mechanism of inhibition of (Na+, K+)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na+, K+)-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s-1) in adults than in juveniles (0.053 ± 0.003 s-1) for spermidine, but similar to juveniles (0.059 ± 0.004 s-1) for putrescine. Maximum phosphointermediate formation for the (Na+, K+)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na+, K+)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.

Putrescine treatment reverses α-tocopherol-induced desynchronization of polyamine and retinoid metabolism during rat liver regeneration.

BACKGROUND: The pre-treatment with α-tocopherol inhibits progression of rat liver proliferation induced by partial hepatectomy (PH), by decreasing and/or desynchronizing cyclin D1 expression and activation into the nucleus, activation and nuclear translocation of STAT-1 and -3 proteins and altering retinoid metabolism. Interactions between retinoic acid and polyamines have been reported in the PH-induced rat liver regeneration. Therefore, we evaluated the effect of low dosage of α-tocopherol on PH-induced changes in polyamine metabolism. METHODS: This study evaluated the participation of polyamine synthesis and metabolism during α-tocopherol-induced inhibition of rat liver regeneration. In PH-rats (Wistar) treated with α-tocopherol and putrescine, parameters indicative of cell proliferation, lipid peroxidation, ornithine decarboxylase expression (ODC), and polyamine levels, were determined. RESULTS: Pre-treatment with α-tocopherol to PH-animals exerted an antioxidant effect, shifting earlier the increased ODC activity and expression, temporally affecting polyamine synthesis and ornithine metabolism. Whereas administration of putrescine induced minor changes in PH-rats, the concomitant treatment actually counteracted most of adverse actions exerted by α-tocopherol on the remnant liver, restituting its proliferative potential, without changing its antioxidant effect. Putrescine administration to these rats was also associated with lower ODC expression and activity in the proliferating liver, but the temporally shifting in the amount of liver polyamines induced by α-tocopherol, was also "synchronized" by the putrescine administration. The latter is supported by the fact that a close relationship was observed between fluctuations of polyamines and retinoids. CONCLUSIONS: Putrescine counteracted most adverse actions exerted by α-tocopherol on rat liver regeneration, restoring liver proliferative potential and restituting the decreased retinoid levels induced by α-tocopherol. Therefore interactions between polyamines and retinol, mediated by the oxidant status, should be taken into consideration in the development of new therapeutic strategies for pathologies occurring with liver cell proliferation.

Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

Cytotoxicity of 1,4-diamino-2-butanone, a putrescine analogue, to RKO cells: mechanism and redox imbalance.

α-Aminocarbonyl metabolites (e.g., 5-aminolevulinic acid and aminoacetone) and the wide spectrum microbicide 1,4-diamino-2-butanone (DAB) have been shown to exhibit pro-oxidant properties. In vitro, these compounds undergo phosphate-catalyzed enolization at physiological pH and subsequent superoxide radical-propagated aerobic oxidation, yielding a reactive α-oxoaldehyde and H2O2. DAB cytotoxicity to pathogenic microorganisms has been attributed to the inhibition of polyamine biosynthesis. However, the role played in cell death by reactive DAB oxidation products is still poorly understood. This work aims to clarify the mechanism of DAB-promoted pro-oxidant action on mammalian cells. DAB (0.05-10 mM) treatment of RKO cells derived from human colon carcinoma led to a decrease in cell viability (IC50 ca. 0.3 mM DAB, 24 h incubation). Pre-addition of either catalase (5 µM) or aminoguanidine (20 mM) was observed to partially inhibit the toxic effects of DAB to the cells, while N-acetyl-L-cysteine (NAC, 5 mM) or reduced glutathione (GSH, 5 mM) provided almost complete protection against DAB. Changes in redox balance and stress response pathways were indicated by the increased expression of HO-1, NQO1 and xCT. Moreover, the observation of caspase 3 and PARP cleavage products is consistent with DAB-triggered apoptosis in RKO cells, which was corroborated by the partial protection afforded by the pan-caspase inhibitor z-VAD-FMK. Finally, DAB treatment disrupted the cell cycle in response to increased p53 and activation of ATM. Altogether, these data support the hypothesis that DAB exerts cytotoxicity via a mechanism involving not only polyamine biosynthesis but also by DAB oxidation products.

1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells.

The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other α-aminocarbonyl metabolites, DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H(2)O(2), NH(4)(+) ion, and a highly toxic α-oxoaldehyde. In vitro, DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC(50)c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 µM) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 µM buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells.

Change in lipoperoxidation but not in scavenging enzymes activity during polyamine embryoprotection in rat embryo cultured in hyperglycemic media.

DM1 complicated with pregnancy is the cause of neonatal malformations and low-for-gestational-age neonates. With the use of the whole-embryo culture system, it has been demonstrated that high glucose causes embryo dysmorphogenesis. Previously, our group has found that spermidine or spermine addition reverts almost fully the severity and frequency of dysmorphogenesis, whereas the effect of arginine and putrescine it is only partial. A hypothesis for polyamine mechanism is the amelioration of oxidative stress caused by high glucose. The purpose of this work was to evaluate the effect of polyamines over the activity of scavenging enzymes and lipoperoxidation in whole-embryo rat in culture. Post-implantation (gestational day 10.5) rat embryos were cultured for 24 h in normal medium or hyperglycemic medium, alone or supplemented with L-arginine or polyamine. Embryos were recovered and visualized, and morphologic parameters were registered. Cultured embryos were homogenized, and superoxide dismutase and glutathione-reductase activities, as well as lipoperoxidation, were measured. The activity of superoxide dismutase and glutathione peroxidase were not affected by the treatment, but lipoperoxidation was increased in embryos cultured in hyperglycemic medium; spermidine or spermine supplementation restore lipoperoxidation to near-normal values, and putrescine and L-arginine reverts only partially the glucose effect. Taken together, these results pointed out that spermidine and spermine embryoprotection could be mediated by direct antioxidant activity. However, further studies are needed to support this hypothesis.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation.

The α-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37°C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other α-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml-1) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(•), and those with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(•) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 µM) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity.

The putrescine analogue 1,4-diamino-2-butanone affects polyamine synthesis, transport, ultrastructure and intracellular survival in Leishmania amazonensis.

Polyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC(50) 144 microM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H(3)]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P<0.05) lower than controls, presumably due to interference with putrescine uptake and/or synthesis. These results suggest that putrescine may be involved in leishmanial survival, possibly by maintaining the parasite's mitochondrial function. The use of analogues to interfere with polyamine/diamine synthesis and transport may shed light on its function in intracellular parasite survival and lead to identification of new targets for leishmaniasis chemotherapy.

Effects of a putrescine analog on Giardia lamblia.

The protozoan Giardia lamblia is the most frequent intestinal parasite of first-world countries and a major cause of waterborne disorder often referred to as traveler's diarrhea. We have previously noticed that the putrescine analog 1,4-diamino-2-butanone (DAB) remarkably inhibits the growth of anaerobic trichomonad and Trypanosoma cruzi parasites. Here, we examined the role of polyamines in Giardia cells using this putrescine analog. DAB impaired parasite proliferation dose-dependently. The analog induced increased flagella numbers and sometimes four ventral disks as well as asymmetrical division, indicating truncated or deregulated cytokinesis. Electron microscopy analysis revealed that DAB also triggered the encystment process. Oxidative stress was evaluated by measuring lipid peroxidation by thiobarbituric acid reactive substances (TBARS) detection. Trophozoites incubated either with 1 mM of DAB or putrescine for 18 h displayed increased lipoperoxide levels. Addition of 200 microM aminoguanidine, a polyamine/diamine oxidase inhibitor, partially reverted the DAB, but not the putrescine effects, indicating that the DAB effects are due, at least in part, to DAB oxidation end products. These data indicate that polyamines play a role in Giardia cell division, differentiation, and antioxidant defenses.

Putrescine produces antidepressant-like effects in the forced swimming test and in the tail suspension test in mice.

Putrescine, a polyamine present at high concentrations in the mammalian brain, was suggested to play a role in the modulation of depression. Thus, this study investigated the effect of putrescine in the mouse forced swimming test (FST) and in the tail suspension test (TST), two models predictive of antidepressant activity. Putrescine significantly reduced the immobility time both in the FST and in the TST (dose range of 1-10 mg/kg, i.p.), without changing locomotion in an open-field. I.c.v. injection of putrescine (0.1-10 nmol/site) also reduced the immobility time in the FST and in the TST. The pretreatment of mice with arcaine (1 mg/kg, i.p., an antagonist of the polyamine-site of NMDA receptor) completely blocked the anti-immobility effect of putrescine (10 mg/kg, i.p.). A subeffective dose of putrescine (0.1 mg/kg, i.p.) produced a synergistic antidepressant-like effect with agmatine (0.001 mg/kg, i.p.) in the FST. Moreover, a subeffective dose of putrescine (0.01 nmol/site, i.c.v.) produced a synergistic antidepressant-like effect with arcaine (50 microg/site, i.c.v.). The results indicate that putrescine produces antidepressant-like effects in the FST that seems to be mediated through its interaction with the polyamine-site of NMDA receptors.

Polyamines as a defense mechanism against lipoperoxidation in Trypanosoma cruzi.

The polyamines, spermine and spermidine--organic polycations that are absolutely required for eukaryotic cell growth--are shown here to function in Trypanosoma cruzi epimastigotes, as protectors of membrane lipoperoxidation by reactive oxygen species generated either by H2O2/Fe2+ or nifurtimox. In vitro, spermine and spermidine inhibited lipoperoxidation in a dose dependent manner. Spermine was more efficient than spermidine in its inhibitory effect. Lipid peroxidation induced by H2O2 showed an IC50 of 0.55 mM for spermine and 0.9 mM for spermidine while an IC50 of 0.8 mM for spermine and 1.5 mM for spermidine were observed when lipoperoxidation was elicited by nifurtimox. Likewise in vivo, both exogenously added spermine and spermidine or endogenous increase of spermine levels induced by phorbol ester, protected against lipoperoxidation and decreased citotoxicity provoked by nifurtimox. Putrescine and cadaverine, also present in T. cruzi had no effect at all. None of the polyamines had any effect neither on the scavenging of superoxide anion nor on the regulation of antioxidant enzymes such as superoxide dismutase and peroxidases involved in H2O2 detoxification. Here we point out that spermine, by acting as a protector of membrane lipoperoxidation might contribute to survival of T. cruzi continuously exposed to oxidative stress.

Polyamines inhibit both platelet aggregation and glycoprotein IIb/IIIa activation.

Platelet aggregation is inhibited by the polyamines putrescine, spermidine, and spermine. To date, the mechanism of action has not been clearly identified. Evidence suggests that polyamines may interact with the fibrinogen receptor (GP IIb/IIIa), interfering with platelet-platelet attachment. The effect of polyamines on human platelet aggregation and GP IIb/IIIa activation was evaluated. For the aggregation experiments, platelets were obtained from heparin- or citrate-collected blood. Our results indicate that the polyamines putrescine, spermidine, and spermine cause a dose-dependent inhibition of ADP- or collagen-mediated platelet aggregation with an order of potency spermine>spermidine>putrescine. In addition, spermine arrests or inhibits thrombin-, epinephrine-, arachidonate-, or ristocetin-induced platelet aggregation. Expression of platelet membrane glycoproteins IIb, IIIa, and IX is not reduced by polyamines. However, spermine inhibits the ADP- or thrombin-induced activation of GP IIb/IIIa. It is concluded that the final step in aggregation, common to all agonists, ie, fibrinogen binding to GP IIb/IIIa, is inhibited by spermine through inhibition of the agonist-induced activation of GP IIb/IIIa that precedes fibrinogen-ligand binding.

Specificity of DNA methylation changes during fungal dimorphism and its relationship to polyamines.

We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.

Modulation by polyamines of gamma-glutamyl transpeptidase activity and lactate production in cultured Sertoli cells from immature and adult regressed golden hamster.

Polyamines are involved in cellular growth and differentiation. To analyze a possible role of polyamines on the regulation of Sertoli cell function, we studied the effect of putrescine, spermidine, and spermine on gamma-glutamyl transpeptidase (gamma-GTP) activity and lactate production on Sertoli cell cultures obtained from immature and adult-regressed golden hamsters. Sertoli cells were cultured for 7 days. The 72 hour conditioned media obtained on day 6 were used to evaluate lactate levels. Gamma glutamyl transpeptidase activity was determined in the cells harvested on day 7. Cultured Sertoli cells isolated from immature and adult-regressed golden hamsters exhibited a clear morphological response to follicle-stimulating hormone (FSH) and to spermine. Gamma glutamyl transpeptidase activity increased in response to FSH in a dose-dependent manner. Dose-dependent stimulation of lactate production by FSH was also observed. For each functional parameter, a similar ED50 value of FSH stimulation was observed in both groups of animals. Spermine increased basal and FSH-stimulated gamma-GTP activity in immature and adult-regressed Sertoli cell cultures. A stimulatory effect of spermidine and putrescine on gamma-GTP activity was exclusively observed in adult-regressed Sertoli cell cultures. In Sertoli cells obtained from immature hamsters, spermine exerted a stimulatory effect on basal and FSH-stimulated lactate production. These results suggest that, in addition to the known effects of hormones and paracrine factors, polyamines may influence the functionality of Sertoli cells.

Effect of infiltrated polyamines on polygalacturonase activity and chilling injury responses in zucchini squash (Cucurbita pepo L.).

The effect of exogenous polyamines on electrolyte leakage, chilling index, polygalacturonase activity (PG), ethylene production, and firmness in zucchini squash fruits stored for 12 days at 2 degrees C or 10 degrees C, 85-90% RH was evaluated. Fruits were infiltrated with putrescine (PUT) spermidine (SPD) and spermine (SPM) at 0.1, 0.25, 0.5, 2.0, and 4.0 mM. All polyamines exerted a protective effect on cell and organelle membranes. The most effective was SPD, which reduced electrolyte leakage between 62% and 82%, compared to control fruits stored at 2 degrees C. At 10 degrees C they did not exhibit chilling injury (CI) symptoms, while at 2 degrees C SPM (0.5 mM) and SPD (0.5 mM) diminished them 92% and 100%, respectively; which extended storage life for 8-10 days at 2 degrees C. High concentrations of polyamines (>2.0 mM) caused the appearance of CI symptoms. PG activity diminished proportionally to the concentration of polyamine except for the concentration at 4.0 mM. No significant changes were observed in ethylene production.