RESUMO
Trypanosomatids present a unique mechanism for detoxification of peroxides that is dependent on trypanothione (bisglutathionylspermidine). Ornithine decarboxylase (ODC) and γ-glutamylcysteine synthetase (GSH1) produce molecules that are direct precursors of trypanothione. In this study, Leishmania guyanensis odc and gsh1 overexpressor cell lines were generated to investigate the contribution of these genes to the trivalent antimony (SbIII)-resistance phenotype. The ODC- or GSH1-overexpressors parasites presented an increase of two and four-fold in SbIII-resistance index, respectively, when compared with the wild-type line. Pharmacological inhibition of ODC and GSH1 with the specific inhibitors α-difluoromethylornithine (DFMO) and buthionine sulfoximine (BSO), respectively, increased the antileishmanial effect of SbIII in all cell lines. However, the ODC- and GSH1-overexpressor were still more resistant to SbIII than the parental cell line. Together, our data shows that modulation of ODC and GSH1 levels and activity is sufficient to affect L. guyanensis susceptibility to SbIII, and confirms a role of these genes in the SbIII-resistance phenotype.
Assuntos
Antimônio/farmacologia , Glutamato-Cisteína Ligase/metabolismo , Leishmania guyanensis/efeitos dos fármacos , Leishmania guyanensis/enzimologia , Ornitina Descarboxilase/metabolismo , Animais , Western Blotting , Butionina Sulfoximina/farmacologia , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Concentração Inibidora 50 , Leishmaniose Mucocutânea/tratamento farmacológico , Doenças Negligenciadas/tratamento farmacológico , Doenças Negligenciadas/parasitologia , Inibidores da Ornitina Descarboxilase/farmacologia , Coelhos , Proteínas Recombinantes/metabolismoRESUMO
Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.
Assuntos
Autofagia/efeitos dos fármacos , Poliaminas/farmacologia , Trypanosoma cruzi/patogenicidade , Animais , Proteína 5 Relacionada à Autofagia , Células CHO , Cricetinae , Cricetulus , Eflornitina/farmacologia , Embrião de Mamíferos/citologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Espermidina/farmacologia , Fatores de Tempo , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Polyamines (putrescine, spermidine and spermine) are aliphatic amines that are produced by the action of ornithine decarboxylase (ODC) in a rate-limiting and protein kinase C (PKC)-regulated step. Because high levels of polyamines are found in the synovial fluid of arthritic patients, the aim of the present study was to identify the role of peripherally produced polyamines in a model of inflammatory pain induced by adjuvant. The subcutaneous injection of Complete Freund's adjuvant (CFA, 50 µL/paw) caused the development of mechanical allodynia and edema. Moreover, it increased ODC expression and activity and PKC activation. Administration of the selective ODC inhibitor DFMO (10 µmol/paw) attenuated the development of allodynia and edema and decreased ODC activity in both control and CFA-treated animals. Furthermore, administration of the PKC inhibitor GF109203X (1 nmol/paw) reduced allodynia and ODC activity in animals injected with CFA. A subcutaneous injection of putrescine (10 µmol/paw), spermidine (3-10 µmol/paw) or spermine (0.3-3 µmol/paw) into the rat paw also caused mechanical allodynia and edema. The present results suggest that endogenously synthesized polyamines are involved in the development of nociception and edema caused by an adjuvant. Moreover, polyamine production in inflammatory sites seems to be related to an increase in ODC activity stimulated by PKC activation. Thus, controlling polyamine synthesis and action could be a method of controlling inflammatory pain.
Assuntos
Inflamação/metabolismo , Dor/induzido quimicamente , Poliaminas/efeitos adversos , Animais , Eflornitina/farmacologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Medição da Dor , Ratos , Ratos WistarRESUMO
Ovarian sizes (length and width) were measured in young females of Anastrepha fraterculus (Wiedemann) (Diptera, Tephritidae) subjected or not to the inhibitor alpha-difluormethylornithine (alpha-DFMO). The most effective concentration of alpha-DMFO used was 50 mM and the ovarian measurements (length and width) of the treated females were smaller than those of females not treated with alpha-DMFO. These data may suggest some relationship between ornithine decarboxylase (ODC) and sexual maturation in A. fraterculus.
Assuntos
Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores da Ornitina Descarboxilase , Ovário/efeitos dos fármacos , Tephritidae/efeitos dos fármacos , Animais , Feminino , Tamanho do Órgão/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Tephritidae/anatomia & histologia , Tephritidae/enzimologiaRESUMO
Ovarian sizes (length and width) were measured in young females of Anastrepha fraterculus (Wiedemann) (Diptera, Tephritidae) subjected or not to the inhibitor α -difluormethylornithine (α -DFMO). The most effective concentration of α -DMFO used was 50 mM and the ovarian measurements (length and width) of the treated females were smaller than those of females not treated with α -DMFO. These data may suggest some relationship between ornithine decarboxylase (ODC) and sexual maturation in A. fraterculus.
As dimensões dos ovários (comprimento e largura) foram mensuradas em fêmeas jovens da Anastrepha fraterculus (Wiedemann) (Diptera, Tephritidae) submetidas ou não ao inibidor α -difluormetilornitina (α -DFMO). A concentração mais efetiva de α -DMFO utilizada foi 50 mM e as medidas (comprimento e largura) das fêmeas tratadas com o inibidor foram menores que as fêmeas não tratadas com inibidor α -DMFO. Estes dados podem sugerir uma relação entre ornitina descarboxilase (ODC) e maturação sexual em A. fraterculus.
Assuntos
Animais , Feminino , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Ornitina Descarboxilase/antagonistas & inibidores , Ovário/efeitos dos fármacos , Tephritidae/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Tephritidae/anatomia & histologia , Tephritidae/enzimologiaRESUMO
Neonatal ethanol (EtOH) exposure is associated with central nervous system dysfunction and neurotoxicity in rats. Increases in polyamine levels have been implicated as one underlying mechanism for some of EtOH's effects on the developing brain. In this study we addressed whether the inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) could reduce behavioral deficits induced by early EtOH exposure. Male and female rat pups received ethanol (6 g/kg/day EtOH i.g.), or isocaloric maltose (control) from postnatal days (PND) 1-8. On PND 8, animals were injected with either saline or DFMO (500 mg/kg, s.c.) immediately following the final neonatal treatment. Subjects were tested for isolation-induced ultrasonic vocalizations (USV) on PND 16; spontaneous activity in an open field apparatus on PND 20 and 21; and balance on PND 31. Animals exposed to EtOH neonatally displayed an increased latency to the first USV and reduced frequencies of USV, hyperactivity and preference for the center of the open field and poorer balance relative to controls. DFMO minimized these deficits in latency to the first USV and balance. These data provide further support that polyamines play a role in some of the functional deficits associated with EtOH exposure during early development and that reducing polyamine activity can improve outcome.
Assuntos
Animais Recém-Nascidos/fisiologia , Depressores do Sistema Nervoso Central/toxicidade , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Etanol/toxicidade , Equilíbrio Postural/efeitos dos fármacos , Isolamento Social , Vocalização Animal/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Masculino , Inibidores da Ornitina Descarboxilase , Ratos , Ratos Sprague-DawleyRESUMO
A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. Alpha-difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.
Assuntos
Ascomicetos/metabolismo , Poliaminas/metabolismo , Adenosilmetionina Descarboxilase/metabolismo , Ascomicetos/efeitos dos fármacos , Ascomicetos/enzimologia , Ascomicetos/crescimento & desenvolvimento , Cicloexilaminas/farmacologia , Eflornitina/farmacologia , Estruturas Fúngicas/efeitos dos fármacos , Estruturas Fúngicas/enzimologia , Estruturas Fúngicas/crescimento & desenvolvimento , Estruturas Fúngicas/metabolismo , Helianthus/microbiologia , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Ornitina Descarboxilase/metabolismo , Doenças das Plantas/microbiologia , Poliaminas/antagonistas & inibidoresRESUMO
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.
Assuntos
Poliaminas Biogênicas/biossíntese , Eflornitina/farmacologia , Expressão Gênica , Ornitina Descarboxilase/genética , Trypanosoma cruzi/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Northern Blotting , Southern Blotting , Primers do DNA , Inibidores da Ornitina Descarboxilase , Reação em Cadeia da Polimerase , Trypanosoma cruzi/enzimologiaRESUMO
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.
Assuntos
Animais Recém-Nascidos/fisiologia , Depressores do Sistema Nervoso Central/toxicidade , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Etanol/toxicidade , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Aldeídos/metabolismo , Animais , Poliaminas Biogênicas/biossíntese , Química Encefálica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Inibidores da Ornitina Descarboxilase , Oxirredução , Ratos , Ratos Sprague-Dawley , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Hexachlorobenzene (HCB) is a lipophilic chemical compound that is widely distributed in the environment. HCB is known to cause liver tumors in experimental animals. In the present study the in vivo effect of HCB treatment on ornithine decarboxylase (ODC) and protein tyrosine kinase (PTK) activities, free polyamine content, and c-Myc, c-Fos, and c-Jun protein levels in rat liver were investigated. HCB (1000 mg/kg body weight) increased hepatic immunodetectable c-Myc, c-Fos, and c-Jun levels after 6 h, and ODC activity and spermine and putrescine content after 18 and 24 h, while maximum stimulation of PTK activity occurred at 12 h. PTK and ODC activities varied in a dose-dependent manner. The time-course of c-Myc, c-Fos, and c-Jun protein levels was different for each proto-oncogene. They were all elevated at the second day of treatment, while only c-Fos and c-Jun remained elevated after 10 days of HCB exposure. These data jointly suggest that the increase in ODC activity may be the consequence of proto-oncogene induction. The alterations in PTK activity suggest that the growth factor signal transduction pathway may be involved in the regulation of the proto-oncogene levels or/and ODC activity. The decrease in PTK activity after the first day, even in the presence of alpha-D-Difluoromethylornithine (DFMO), an inhibitor of ODC activity, suggests that it is not regulated by polyamines. These results may be relevant to the early molecular events involved in HCB tumor promoter activity in rat liver.