Trypanosoma cruzi as a model system to study the expression of exogenous genes coding for polyamine biosynthetic enzymes. Induction of DFMO resistance in transgenic parasites.

Trypanosoma cruzi, the etiologic agent of Chagas' disease, is a polyamine auxotroph organism because its genome contains neither ornithine decarboxylase (ODC) nor arginine decarboxylase (ADC) genes, presumably lost during evolution. After transformation with a recombinant plasmid bearing the complete coding region of Crithidia fasciculata ODC gene, the transgenic parasites were able to synthesize putrescine and simultaneously became susceptible to alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. We have studied the emergence of DFMO-resistant T. cruzi after one-step selection of ODC-transformed parasites cultivated in the presence of high levels of the drug (5 mM). Our results have indicated a duplication of the ODC gene copy number in the drug-resistant cell line. The ODC transcripts and the corresponding translation products showed very significant increases (about 7- and 25-fold, respectively) in DFMO-resistant parasites, while the ODC enzymatic activity was 5 times higher than in drug-sensitive T. cruzi. The unequal increases of ODC protein and enzymatic activity in DFMO-resistant protozoa strongly suggest that in addition to gene amplification and enhanced transcription and translation, the assembly of ODC polypeptide chains into dimeric active enzyme molecules might also contribute to regulate the development of DFMO resistance.

Difluoromethylornithine decreases long-lasting protein oxidation induced by neonatal ethanol exposure in the hippocampus of adolescent rats.

BACKGROUND: Ethanol exposure and withdrawal during central nervous system development can cause oxidative stress and produce severe and long-lasting behavioral and morphological alterations in which polyamines seem to play an important role. However, it is not known if early ethanol exposure causes long-lasting protein oxidative damage and if polyamines play a role in such a deleterious effect of ethanol. METHODS: In this study we investigated the effects of early ethanol exposure (6 g/kg/d, by gavage), from postnatal day (PND) 1 to 8, and of the administration of difluoromethylornithine (DFMO, 500 mg/kg, i.p., on PND 8), a polyamine biosynthesis inhibitor, on the extent of oxidative modification of proteins. Indices of oxidative modification of proteins included protein carbonyls, 3-nitrotyrosine (3-NT), and protein bound 4-hydroxynonenal (HNE) in the hippocampus, cerebellum, hypothalamus, striatum, and cerebral cortex of Sprague-Dawley rats at PND 40. RESULTS: Both ethanol and DFMO administration alone increased protein carbonyl immunoreactivity in the hippocampus at PND 40, but the combination of DFMO and ethanol resulted in no effect on protein carbonyl levels. No alterations in the content of protein-bound HNE, 3-NT, or carbonyl were found in any other cerebral structure. CONCLUSIONS: These results suggest that the hippocampus is selectively affected by early ethanol exposure and by polyamine synthesis inhibition. In addition, the results suggest a role for polyamines in the long-lasting increase of protein carbonyls induced by ethanol exposure and withdrawal.

The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus Sclerotinia sclerotiorum.

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40-50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum.

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. Alpha-Difluoromethylornithine (DFMO) and alpha-difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.

Effects of alpha-difluoromethylornithine on the Fas expression and apoptosis in Hep-2 cells

DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.

Effects of alpha-difluoromethylornithine on the Fas expression and apoptosis in Hep-2 cells

DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.(AU)

Effects of alpha-difluoromethylornithine on the Fas expression and apoptosis in Hep-2 cells.

DFMO is an irreversible inhibitor of ornithine decarboxilase (ODC), the key enzyme in mammalian polyamine biosynthesis, and has been shown to induce apoptosis. In this paper, the relation between the effects of DFMO on the polyamine content, apoptotic index and Fas expression in HEP-2 cells was determined. Fas is a type I membrane protein with a molecular mass of 45 kDa, which mediates apoptosis. The results suggest that the treatment with the polyamine inhibitor DFMO induced the expression of the surface antigen Fas, which could be responsible for trigger apoptosis in these cells.

Inhibition of polyamine synthesis arrests trichomonad growth and induces destruction of hydrogenosomes.

Trichomonad parasites such as Tritrichomonas foetus produce large amounts of putrescine (1,4-diaminobutane), which is transported out of the cell via an antiport mechanism which results in the uptake of a molecule of spermine. The importance of putrescine to the survival of the parasite and its role in the biology of T. foetus was investigated by use of the putrescine analogue 1, 4-diamino-2-butanone (DAB). Growth of T. foetus in vitro was significantly inhibited by 20 mM DAB, which was reversed by the addition of exogenous 40 mM putrescine. High-performance liquid chromatography analysis of 20 mM DAB-treated T. foetus revealed that putrescine, spermidine, and spermine levels were reduced by 89, 52, and 43%, respectively, compared to those in control cells. The DAB treatment induced several ultrastructural alterations, which were primarily observed in the redox organelles termed hydrogenosomes. These organelles were progressively degraded, giving rise to large vesicles that displayed material immunoreactive with an antibody to beta-succinyl-coenzyme A synthetase, a hydrogenosomal enzyme. A protective role for polyamines as stabilizing agents in the trichomonad hydrogenosomal membrane is proposed.

The interaction of 2,4-dichlorophenoxyacetic acid, ribosomes and polyamines in Azospirillum brasilense.

2,4-Dichlorophenoxyacetic acid (2,4-D) is an herbicide used extensively in agriculture. We had previously determined that 1 mM 2,4-D could inhibit cell growth, DNA and protein synthesis of Azospirillum brasilense. The present work was designed to determine if these alterations are a consequence of 2,4-D action on polyamine biosynthesis and if the protein synthesis inhibition is a result of ribosomal impairment. In this paper we demonstrate that 2,4-D alters the metabolism of polyamines and, thus, affects protein synthesis at the ribosomal level.