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1.
J Biol Chem ; 296: 100182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33310703

RESUMO

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Adenosina Trifosfatases/genética , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Mitoguazona/farmacologia , Mutação , Sequenciamento Completo do Genoma/métodos
2.
Curr Pharm Des ; 26(8): 838-866, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32039675

RESUMO

In this report, we extend the SAR analysis of a number of lipophilic guanylhydrazone analogues with respect to in vitro growth inhibition of Trypanosoma brucei and Trypanosoma cruzi. Sleeping sickness and Chagas disease, caused by the tropical parasites T. brucei and T. cruzi, constitute a significant socioeconomic burden in low-income countries of sub-Saharan Africa and Latin America, respectively. Drug development is underfunded. Moreover, current treatments are outdated and difficult to administer, while drug resistance is an emerging concern. The synthesis of adamantane-based compounds that have potential as antitrypanosomal agents is extensively reviewed. The critical role of the adamantane ring was further investigated by synthesizing and testing a number of novel lipophilic guanylhydrazones. The introduction of hydrophobic bulky substituents onto the adamantane ring generated the most active analogues, illustrating the synergistic effect of the lipophilic character of the C1 side chain and guanylhydrazone moiety on trypanocidal activity. The n-decyl C1-substituted compound G8 proved to be the most potent adamantane derivative against T. brucei with activity in the nanomolar range (EC50=90 nM). Molecular simulations were also performed to better understand the structure-activity relationships between the studied guanylhydrazone analogues and their potential enzyme target.


Assuntos
Mitoguazona/análogos & derivados , Tripanossomicidas , Trypanosoma brucei brucei , Trypanosoma cruzi , Mitoguazona/farmacologia , Relação Estrutura-Atividade , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos
3.
Int J Dev Biol ; 62(9-10): 647-652, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30378390

RESUMO

The polyamines putrescine, spermidine and spermine are essential polycations involved in the regulation of cellular proliferation. They exert dynamic effects on nucleic acids and macromolecular synthesis in vitro but their specific functions in vivo are poorly understood. Here, we have modulated the spermidine levels either by overexpressing the S-adenosylmethionine decarboxylase (samdc) gene or treating the cells with methylglyoxal-bis (guanylhydrazone) (MGBG), an inhibitor of SAMDC. In Dictyostelium, overexpression of SAMDC slowed cell proliferation, delayed development and arrested cells in the S-phase of the cell cycle. Treatment with MGBG reduced cell proliferation and stimulated development, but in samdcOE cells, it increased cell proliferation suggesting critical levels of spermidine to be important. In samdcOE cells, spermidine levels remained high throughout development but only small changes in the spermine levels were observed. Initial putrescine levels did increase but reverted to wild-type levels after the mound stage. As tight regulation of polyamine homeostasis is required, we identified genes that could be involved in its maintenance. In conclusion, we characterised samdcOE cells and observed the maintenance of polyamine homeostasis during the development of Dictyostelium cells.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Homeostase , Poliaminas/metabolismo , Adenosilmetionina Descarboxilase/antagonistas & inibidores , Animais , Dictyostelium/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Mitoguazona/farmacologia
4.
PLoS One ; 13(3): e0192680, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29538412

RESUMO

Monocyte activation and polarization play essential roles in many chronic inflammatory diseases. An imbalance of M1 and M2 macrophage activation (pro-inflammatory and alternatively activated, respectively) is believed to be a key aspect in the etiology of these diseases, thus a therapeutic approach that regulates macrophage activation could be of broad clinical relevance. Methylglyoxal-bis-guanylhydrazone (MGBG), a regulator of polyamine metabolism, has recently been shown to be concentrated in monocytes and macrophages, and interfere with HIV integration into the DNA of these cells in vitro. RNA expression analysis of monocytes from HIV+ and control donors with or without MGBG treatment revealed the only gene to be consistently down regulated by MGBG to be osteopontin (OPN). The elevated expression of this pro-inflammatory cytokine and monocyte chemoattractant is associated with various chronic inflammatory diseases. We demonstrate that MGBG is a potent inhibitor of secreted OPN (sOPN) in cultured monocytes with 50% inhibition achieved at 0.1 µM of the drug. Furthermore, inhibition of OPN RNA transcription in monocyte cultures occurs at similar concentrations of the drug. During differentiation of monocytes into macrophages in vitro, monocytes express cell surface CD16 and the cells undergo limited DNA synthesis as measured by uptake of BrdU. MGBG inhibited both activities at similar doses to those regulating OPN expression. In addition, monocyte treatment with MGBG inhibited differentiation into both M1 and M2 classes of macrophages at non-toxic doses. The inhibition of differentiation and anti-OPN effects of MGBG were specific for monocytes in that differentiated macrophages were nearly resistant to MGBG activities. Thus MGBG may have potential therapeutic utility in reducing or normalizing OPN levels and regulating monocyte activation in diseases that involve chronic inflammation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Mitoguazona/farmacologia , Monócitos/metabolismo , Osteopontina/biossíntese , Relação Dose-Resposta a Droga , Humanos , Macrófagos/citologia , Monócitos/citologia , Transcrição Gênica
5.
J Neurovirol ; 24(2): 213-219, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29435829

RESUMO

Non-human primate models of AIDS and neuroAIDS are critical to study HIV infection of the CNS, neuropathology, and immune activation and macrophage accumulation that occurs in HAND. SIV, similar to HIV, infects CD4+ T lymphocytes and monocytes/macrophages. Virus enters the CNS early, and macrophage activation correlates with CNS disease, as well as inflammation outside of the CNS. Antiretroviral in HIV+ humans and SIV+ Rhesus macaques results in non-detectable plasma virus, decreased or non-detectable viral RNA or protein in the CNS. But, viral DNA rebounds following therapy interruption, demonstrating the presence of replication competent virus in the CNS within myeloid cells. In this brief review, we discuss our findings using a Rhesus macaque model of SIV-associated CNS infection and pathology, focusing on monocyte/macrophage activation and the link between CNS and cardiac disease. We conclude with recent studies using adjunctive therapy targeting monocytes/macrophages with ART to prevent or diminish CNS pathology that may be associated with HAND.


Assuntos
Antivirais/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Disfunção Cognitiva/tratamento farmacológico , Modelos Animais de Doenças , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Animais , Terapia Antirretroviral de Alta Atividade , Sistema Nervoso Central/virologia , Disfunção Cognitiva/imunologia , Disfunção Cognitiva/fisiopatologia , Disfunção Cognitiva/virologia , Quimioterapia Combinada/métodos , Humanos , Macaca mulatta , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/virologia , Minociclina/farmacologia , Mitoguazona/farmacologia , Natalizumab/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Vírus da Imunodeficiência Símia/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Carga Viral/efeitos dos fármacos , Latência Viral/fisiologia
6.
J Neurovirol ; 23(4): 568-576, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28462488

RESUMO

Peripheral neuropathy (PN) is a major comorbidity of HIV infection that is caused in part by chronic immune activation. HIV-PN is associated with infiltration of monocytes/macrophages to the dorsal root ganglia (DRG) causing neuronal loss and formation of Nageotte nodules. Here, we used an oral form of methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine biosynthesis inhibitor, to specifically reduce activation of myeloid cells. MGBG is selectively taken up by monocyte/macrophages in vitro and inhibits HIV p24 expression and DNA viral integration in macrophages. Here, MGBG was administered to nine SIV-infected, CD8-depleted rhesus macaques at 21 days post-infection (dpi). An additional nine SIV-infected, CD8-depleted rhesus macaques were used as untreated controls. Cell traffic to tissues was measured by in vivo BrdU pulse labeling. MGBG treatment significantly diminished DRG histopathology and reduced the number of CD68+ and CD163+ macrophages in DRG tissue. The number of recently trafficked BrdU+ cells in the DRG was significantly reduced with MGBG treatment. Despite diminished DRG pathology, intraepidermal nerve fiber density (IENFD) did not recover after treatment with MGBG. These data suggest that MGBG alleviated DRG pathology and inflammation.


Assuntos
Inibidores Enzimáticos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Mitoguazona/farmacologia , Monócitos/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Administração Oral , Animais , Linfócitos T CD8-Positivos/virologia , Movimento Celular/efeitos dos fármacos , DNA Viral/genética , Gânglios Espinais/imunologia , Gânglios Espinais/patologia , Gânglios Espinais/virologia , Proteína do Núcleo p24 do HIV/genética , Depleção Linfocítica , Macaca mulatta , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Macrófagos/virologia , Masculino , Monócitos/imunologia , Monócitos/patologia , Monócitos/virologia , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/imunologia , Fibras Nervosas/patologia , Fibras Nervosas/virologia , Doenças do Sistema Nervoso Periférico/imunologia , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/virologia , Poliaminas/antagonistas & inibidores , Poliaminas/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento
7.
J Acquir Immune Defic Syndr ; 74(5): 583-592, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28141779

RESUMO

BACKGROUND: Despite effective combination antiretroviral therapy, HIV-infected individuals develop comorbidities, including cardiovascular disease, where activated macrophages play a key role. To date, few therapies target activated monocytes and macrophages. METHODS: We evaluated a novel oral form of the polyamine biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone (MGBG) on cardiovascular inflammation, carotid artery intima-media thickness (cIMT), and fibrosis in a simian immunodeficiency virus infection model of AIDS. Eleven simian immunodeficiency virus-infected animals received MGBG (30 mg/kg) once daily and 8 received a placebo control both beginning at 21 days postinfection (dpi). Animals were time sacrificed at 49 days post infection (dpi), when their matched placebo controls developed AIDS (63, 70, 77, 80), or at the study end-point (84 dpi). Aorta, carotid artery, and cardiac tissues were analyzed. Quantitative analyses of macrophage populations and T lymphocytes were done and correlated with cIMT and fibrosis. RESULTS: MGBG treatment resulted in 2.19-fold (CD163), 1.86-fold (CD68), 2.31-fold (CD206), and 2.12-fold (MAC387) decreases in macrophages in carotid arteries and significant 2.07-fold (CD163), 1.61-fold (CD68), 1.95-fold (MAC387), and 1.62-fold (CD206) decreases in macrophages in cardiac tissues. cIMT (1.49-fold) and fibrosis (2.05-fold) also were significantly decreased with MGBG treatment. Numbers of macrophage and the degree of fibrosis in treated animals were similar to uninfected animals. A positive correlation between decreased macrophage in the carotid artery and cIMT, and cardiac macrophages and fibrosis was found. CONCLUSIONS: These data demonstrate that directly targeting macrophages with MGBG can reduce cardiovascular inflammation, cIMT, and fibrosis. They suggest that therapies targeting macrophages with HIV could be used in conjunction with combination antiretroviral therapy.


Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Fatores Imunológicos/administração & dosagem , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Mitoguazona/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/complicações , Animais , Artérias Carótidas/patologia , Fibrose/patologia , Fatores Imunológicos/farmacologia , Macaca mulatta , Macrófagos/imunologia , Mitoguazona/farmacologia , Placebos/administração & dosagem , Resultado do Tratamento , Túnica Íntima/patologia
8.
PLoS One ; 11(10): e0165321, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27780273

RESUMO

This study determined if the variation in grain filling parameters between two different spikelet types of rice (Oryza sativa L.) is regulated by the hormonal levels in the grains. Two rice mutants, namely, a large-grain mutant (AZU-M) and a small-grain mutant (ZF802-M), and their respective wild types (AZU-WT and ZF802-WT) were grown in the field. The endosperm cell division rate, filling rate, and hormonal levels: zeatin + zeatin riboside (Z+ZR), indo-3-acetic acid (IAA), polyamines (PAs), and abscisic acid (ABA) were determined. The results showed that there was no significant difference between the filling and endosperm cell division rates. These rates were synchronous between the superior and inferior spikelets for both mutants. However, the abovementioned parameters were significantly different between the two spikelet types for the two wild types. The superior spikelets filled faster and their filling rate was higher compared to the inferior ones. Changes in the concentrations of plant hormones were consistent with the observed endosperm cell division rate and the filling rate for both types of spikelets of mutant and wild type plants. Regression analysis showed a significant positive correlation between cell division and filling rates with the concentrations of the investigated hormones. Exogenous chemical application verified the role of ABA, IAA, and PAs in grain filling. The results indicate that poor filling of inferior spikelets in rice occurs primarily due to the reduced hormone concentrations therein, leading to lower division rate of endosperm cells, fewer endosperm cells, slower filling rate, and smaller grain weight.


Assuntos
Oryza/metabolismo , Reguladores de Crescimento de Plantas/análise , Ácido Abscísico/análise , Ácido Abscísico/isolamento & purificação , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Endosperma/efeitos dos fármacos , Endosperma/metabolismo , Ensaio de Imunoadsorção Enzimática , Mitoguazona/farmacologia , Oryza/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Poliaminas/análise , Poliaminas/isolamento & purificação , Putrescina/farmacologia , Espermidina/farmacologia , Zeatina/análise , Zeatina/isolamento & purificação
9.
Int J Mol Sci ; 16(12): 28534-48, 2015 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-26633377

RESUMO

Tyrosinase catalyzes two distinct sequential reactions in melanin biosynthesis: The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone. Developing functional modulators of tyrosinase is important for therapeutic and cosmetic purposes. Given the abundance of thiourea moiety in known tyrosinase inhibitors, we studied other thiourea-containing drugs as potential tyrosinase inhibitors. The thiourea-containing drugs in clinical use were retrieved and tested for their ability to inhibit tyrosinase. We observed that methimazole, thiouracil, methylthiouracil, propylthiouracil, ambazone, and thioacetazone inhibited mushroom tyrosinase. Except for methimazole, there was limited information regarding the activity of other drugs against tyrosinase. Both thioacetazone and ambazone significantly inhibited tyrosinase, with IC50 of 14 and 15 µM, respectively. Ambazone decreased melanin content without causing cellular toxicity at 20 µM in B16F10 cells. The activity of ambazone was stronger than that of kojic acid both in enzyme and melanin content assays. Kinetics of enzyme inhibition assigned the thiourea-containg drugs as non-competitive inhibitors. The complex models by docking simulation suggested that the intermolecular hydrogen bond via the nitrogen of thiourea and the contacts via thione were equally important for interacting with tyrosinase. These data were consistent with the results of enzyme assays with the analogues of thiourea.


Assuntos
Reposicionamento de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Tioureia/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Cinética , Melaninas/metabolismo , Melanoma Experimental , Camundongos , Mitoguazona/análogos & derivados , Mitoguazona/farmacologia , Modelos Moleculares , Conformação Molecular , Monofenol Mono-Oxigenase/química , Ligação Proteica , Tioureia/análogos & derivados , Tioureia/farmacologia
10.
J Virol ; 89(22): 11176-89, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26223636

RESUMO

UNLABELLED: Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current antiretroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV-infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage-tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages. IMPORTANCE: Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage-targeted therapy for HIV infection.


Assuntos
Adenosilmetionina Descarboxilase/antagonistas & inibidores , Antirretrovirais/farmacologia , HIV-1/efeitos dos fármacos , Macrófagos/virologia , Mitoguazona/farmacologia , Monócitos/virologia , Integração Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Transporte Biológico Ativo , Antígenos CD4/biossíntese , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteína do Núcleo p24 do HIV/biossíntese , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Receptores CCR5/biossíntese
11.
J Plant Physiol ; 171(10): 779-88, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24877669

RESUMO

The possible involvement of polyamines in the salt stress adaptation was investigated in grapevine (Vitis vinifera L.) plantlets focusing on photosynthesis and oxidative metabolism. Salt stress resulted in the deterioration of plant growth and photosynthesis, and treatment of plantlets with methylglyoxal-bis(guanylhydrazone) (MGBG), a S-adenosylmethionine decarboxylase (SAMDC) inhibitor, enhanced the salt stress effect. A decrease in PSII quantum yield (Fv/Fm), effective PSII quantum yield (Y(II)) and coefficient of photochemical quenching (qP) as well as increases in non-photochemical quenching (NPQ) and its coefficient (qN) was observed by these treatments. Salt and/or MGBG treatments also triggered an increase in lipid peroxidation and reactive oxygen species (ROS) accumulation as well as an increase of superoxide dismutase (SOD) and peroxidase (POX) activities, but not ascorbate peroxidase (APX) activity. Salt stress also resulted in an accumulation of oxidized ascorbate (DHA) and a decrease in reduced glutathione. MGBG alone or in combination with salt stress increased monodehydroascorbate reductase (MDHAR), SOD and POX activities and surprisingly no accumulation of DHA was noticed following treatment with MGBG. These salt-induced responses correlated with the maintaining of high level of free and conjugated spermidine and spermine, whereas a reduction of agmatine and putrescine levels was observed, which seemed to be amplified by the MGBG treatment. These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Antioxidantes/metabolismo , Fotossíntese/fisiologia , Poliaminas/metabolismo , Estresse Fisiológico , Vitis/fisiologia , Ácido Ascórbico/metabolismo , Clorofila/metabolismo , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mitoguazona/farmacologia , Estresse Oxidativo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Poliaminas/análise , Poliaminas/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Sódio/farmacologia , Vitis/efeitos dos fármacos , Vitis/enzimologia , Vitis/crescimento & desenvolvimento
12.
Free Radic Biol Med ; 71: 36-48, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24589373

RESUMO

Nitric oxide (NO) and polyamines (PAs) are two kinds of important signal in mediating plant tolerance to abiotic stress. In this study, we observed that both NO and PAs decreased alkaline stress in tomato plants, which may be a result of their role in regulating nutrient balance and reactive oxygen species (ROS), thereby protecting the photosynthetic system from damage. Further investigation indicated that NO and PAs induced accumulation of each other. Furthermore, the function of PAs could be removed by a NO scavenger, cPTIO. On the other hand, application of MGBG, a PA synthesis inhibitor, did little to abolish the function of NO. To further elucidate the mechanism by which NO and PAs alleviate alkaline stress, the expression of several genes associated with abiotic stress was analyzed by qRT-PCR. NO and PAs significantly upregulated ion transporters such as the plasma membrane Na(+)/H(+) antiporter (SlSOS1), vacuolar Na(+)/H(+) exchanger (SlNHX1 and SlNHX2), and Na(+) transporter and signal components including ROS, MAPK, and Ca(2+) signal pathways, as well as several transcription factors. All of these play important roles in plant adaptation to stress conditions.


Assuntos
Antioxidantes/metabolismo , Óxido Nítrico/metabolismo , Raízes de Plantas/metabolismo , Poliaminas/metabolismo , Hidróxido de Sódio/farmacologia , Solanum lycopersicum/metabolismo , Adaptação Fisiológica , Benzoatos/farmacologia , Transporte Biológico , Cálcio/metabolismo , Regulação da Expressão Gênica de Plantas , Hidroponia , Imidazóis/farmacologia , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitoguazona/farmacologia , Óxido Nítrico/antagonistas & inibidores , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Poliaminas/antagonistas & inibidores , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico
13.
Prikl Biokhim Mikrobiol ; 49(2): 124-8, 2013.
Artigo em Russo | MEDLINE | ID: mdl-23795469

RESUMO

A new biotest system was developed based on highly proliferating human cell cultures (lines LNCaP and PC-3). With the help of this system, two known synthetic polyamines--alpha-difluoromethylornithine (DFMO) and methylglioxalbis(guanylhydrason) (MGBG)--as well as four new synthetic analogues difenyl containing amines (DFCA-1-DFCA-4) with molecular weights of 725.5 (DFCA-1), 755.5 (DFCA-2), 655.5 (DFCA-3), and 681.5 Da (DFCA-4) were tested. In this biotest system, DFMO (0.1-400 microM) did not reveal functional activity, whereas for MGBG a cytotoxic effect was registered (100-200 microM). DFCA-1, DFCA-2, and DFCA-4 had a similar effect at concentrations of 10 microM and higher; DFCA-3, at a concentration of 50 microM and higher. Thus, DFCA-1 has a higher level of antiproliferating activity and may be considered as the most potent cytostatic agent.


Assuntos
Compostos de Bifenilo/farmacologia , Citostáticos/farmacologia , Eflornitina/farmacologia , Mitoguazona/farmacologia , Poliaminas/farmacologia , Bioensaio , Compostos de Bifenilo/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citostáticos/síntese química , Eflornitina/síntese química , Humanos , Masculino , Mitoguazona/síntese química , Poliaminas/síntese química , Relação Estrutura-Atividade
14.
Int J Biochem Cell Biol ; 45(6): 1042-50, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23500523

RESUMO

The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27(Kip1) was greatly increased, so its mechanism was studied in detail. Synthesis of p27(Kip1) was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5'-untranslated region (5'-UTR) in p27(Kip1) mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and α-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary.


Assuntos
Poliaminas Biogênicas/metabolismo , Divisão Celular/fisiologia , Fase G1/fisiologia , Fase G2/fisiologia , Fase S/fisiologia , Adenosilmetionina Descarboxilase/antagonistas & inibidores , Adenosilmetionina Descarboxilase/metabolismo , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Células HeLa , Humanos , Mitoguazona/análogos & derivados , Mitoguazona/farmacologia , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Fase S/efeitos dos fármacos
15.
PLoS One ; 7(11): e47101, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23144800

RESUMO

The anticancer activity of DNA intercalators is related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. Polyamines (spermine in particular) are almost exclusively bound to nucleic acids and are involved in many cellular processes that require nucleic acids. Until now, the effects of polyamines on DNA intercalator activities have remained unclear because intercalation is the most important mechanism employed by DNA-binding drugs. Herein, using actinomycin D (ACTD) as a model, we have attempted to elucidate the effects of spermine on the action of ACTD, including its DNA-binding ability, RNA and DNA polymerase interference, and its role in the transcription and replication inhibition of ACTD within cells. We found that spermine interfered with the binding and stabilization of ACTD to DNA. The presence of increasing concentrations of spermine enhanced the transcriptional and replication activities of RNA and DNA polymerases, respectively, in vitro treated with ActD. Moreover, a decrease in intracellular polyamine concentrations stimulated by methylglyoxal-bis(guanylhydrazone) (MGBG) enhanced the ACTD-induced inhibition of c-myc transcription and DNA replication in several cancer cell lines. The results indicated that spermine attenuates ACTD binding to DNA and its inhibition of transcription and DNA replication both in vitro and within cells. Finally, a synergistic antiproliferative effect of MGBG and ACTD was observed in a cell viability assay. Our findings will be of significant relevance to future developments in combination with cancer therapy by enhancing the anticancer activity of DNA interactors through polyamine depletion.


Assuntos
Antineoplásicos/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA/metabolismo , Dactinomicina/farmacologia , Substâncias Intercalantes/farmacologia , Espermina/metabolismo , Bacteriófago T7/enzimologia , Linhagem Celular Tumoral , DNA Polimerase I/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Humanos , Mitoguazona/farmacologia , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Espermina/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos
16.
Planta ; 235(6): 1221-37, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22167259

RESUMO

Exogenous ethylene and some inhibitors of polyamine biosynthesis can induce mature-fruit abscission in olive, which could be associated with decreased nitric oxide production as a signaling molecule. Whether H2O2 also plays a signaling role in mature-fruit abscission is unknown. The possible involvement of H2O2 and polyamine in ethylene-induced mature-fruit abscission was examined in the abscission zone and adjacent cells of two olive cultivars. Endogenous H2O2 showed an increase in the abscission zone during mature-fruit abscission, suggesting that accumulated H2O2 may participate in abscission signaling. On the other hand, we followed the expression of two genes involved in the polyamine biosynthesis pathway during mature-fruit abscission and in response to ethylene or inhibitors of ethylene and polyamine. OeSAMDC1 and OeSPDS1 were expressed differentially within and between the abscission zones of the two cultivars. OeSAMDC1 showed slightly lower expression in association with mature-fruit abscission. Furthermore, our data show that exogenous ethylene or inhibitors of polyamine encourage the free putrescine pool and decrease the soluble-conjugated spermidine, spermine, homospermidine, and cadaverine in the olive abscission zone, while ethylene inhibition by CoCl2 increases these soluble conjugates, but does not affect free putrescine. Although the impact of these treatments on polyamine metabolism depends on the cultivar, the results confirm that the mature-fruit abscission may be accompanied by an inhibition of S-adenosyl methionine decarboxylase activity, and the promotion of putrescine synthesis in olive abscission zone, suggesting that endogenous putrescine may play a complementary role to ethylene in the normal course of mature-fruit abscission.


Assuntos
Vias Biossintéticas/genética , Frutas/crescimento & desenvolvimento , Frutas/genética , Regulação da Expressão Gênica de Plantas , Olea/crescimento & desenvolvimento , Olea/genética , Poliaminas/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Cicloexilaminas/farmacologia , Etilenos/farmacologia , Frutas/citologia , Frutas/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Peróxido de Hidrogênio/metabolismo , Microscopia Confocal , Mitoguazona/farmacologia , Olea/citologia , Olea/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Putrescina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Solubilidade/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
17.
J Plant Physiol ; 167(1): 47-53, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19651461

RESUMO

This study investigated the effects of cinnamic acid (CA) on ribulose-1,5-bisphosphate carboxylase (RuBPC) activity and the endogenous polyamine levels of cowpea leaves. The results show that 0.1 mM CA treatment decreased photosynthetic rate (P(n)) and RuBPC activity, but it did not affect the maximal photochemical efficiency of PSII (F(v)/F(m)), the actual photochemical efficiency of PSII (PhiPSII), intercellular CO(2) concentration (C(i)), and relative chlorophyll content. These suggest that the decrease in P(n) is at least partially attributed to a lowered RuBPC activity. In addition, 0.1 mM CA treatment increased the putrescine (Put) level, but decreased spermidine (Spd) and spermine (Spm) levels, thereby reducing the (Spd+Spm)/Put (PAs) ratio in the leaves. The exogenous application of 1 mM Spd markedly reversed these CA-induced effects for polyamine and partially restored the PAs ratio and RuBPC activity in leaves. Methylglyoxal-bis (guanylhydrazone) (MGBG), which is an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), results in the inability of activated cells to synthesize Spd and exacerbates the negative effects induced by CA. The exogenous application of 1 mM D-arginine (D-Arg), which is an inhibitor of Put biosynthesis, decreased the levels of Put, but increased the PAs ratio and RuBPC activity in leaves. These results suggest that 0.1 mM CA inhibits RuBPC activity by decreasing the levels of endogenous free and perchloric acid soluble (PS) conjugated Spm, as well as the PAs ratio.


Assuntos
Cinamatos/farmacologia , Fabaceae/efeitos dos fármacos , Fabaceae/enzimologia , Ribulose-Bifosfato Carboxilase/antagonistas & inibidores , Espermina/metabolismo , Arginina/farmacologia , Modelos Lineares , Malondialdeído/metabolismo , Mitoguazona/farmacologia , Percloratos/metabolismo , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Ribulose-Bifosfato Carboxilase/metabolismo , Solubilidade/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos
18.
J Cell Physiol ; 220(3): 680-9, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19472210

RESUMO

Cation-Cl- cotransporters (CCCs) belong to a large family of proteins that includes 9 isoforms, two of which have still not been ascribed a transport function (CCC8 and CCC9) while the others are all known to promote Cl(-)-coupled Na+ and/or K+ movement at the cell surface. The CCCs are also included in a larger family termed amino acid-polyamine-organocation carriers (APCs). In contrast to the CCCs, however, polyamine (PA) transporters have thus far been isolated from unicellular species exclusively and do not all belong to the APC family. In this work, we have found that a splice variant of CCC9 (CCC9a) promotes PA-amino acid transport at the surface of HEK-293 cells. We have also found that the influx of PAs in CCC9a-expressing cells is inhibited by pentamidine as well as furosemide, and that it increases further in the presence of specific amino acids but not of Na+, K+, or Cl-. Hence, a group of substrates that are directly transported by CCC9 and the molecular identity of a PA transport system in animal cells may have been uncovered for the first time. These findings are of special interest given that intracellular PAs play a key role in cell proliferation.


Assuntos
Aminoácidos/metabolismo , Membrana Celular/metabolismo , Poliaminas/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Transporte Biológico , Membrana Celular/efeitos dos fármacos , Cloretos/metabolismo , Furosemida/farmacologia , Células HT29 , Humanos , Cinética , Mitoguazona/farmacologia , Paraquat/farmacologia , Pentamidina/farmacologia , Potássio/metabolismo , Isoformas de Proteínas , Sódio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio , Simportadores de Cloreto de Sódio-Potássio/genética , Transfecção
19.
J Plant Physiol ; 166(2): 213-8, 2009 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-18456370

RESUMO

Seed germination is sensitive to glucose (Glc), nitric oxide (NO) and polyamine (PA). To elucidate whether cross-talk among Glc, NO and PAs occurs in mediation of seed germination, effects of Glc, NO and spermine on seed germination of Lotus japonicus were studied. Glc retarded seed germination in a concentration-dependent manner. NO donor sodium nitroprusside (SNP) alleviated Glc-induced inhibition of seed germination, whereas the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) diminished the SNP-dependent alleviation of seed germination. These observations indicate that Glc may inhibit seed germination by interacting with NO signaling pathways. Exogenous spermine enhanced and the inhibitor of the spermine synthase, methylglyoxal-bis-guanyl hydrazone (MGBG), inhibited seed germination, respectively. Like SNP, spermine alleviated the Glc-induced inhibition of seed germination, whereas MGBG exaggerated the Glc-induced inhibition of seed germination. These results suggest that Glc may inhibit the spermine synthesis, leading to reductions in seed germination. NO scavenger and spermine synthase inhibitor diminished the SNP-induced alleviation of Glc-induced inhibition of seed germination. These findings reveal that both NO and spermine participate in the Glc-induced inhibition of seed germination in L. japonicus.


Assuntos
Germinação/efeitos dos fármacos , Glucose/farmacologia , Lotus/fisiologia , Óxido Nítrico/farmacologia , Sementes/efeitos dos fármacos , Sementes/fisiologia , Espermina/farmacologia , Benzoatos/farmacologia , Imidazóis/farmacologia , Lotus/efeitos dos fármacos , Mitoguazona/farmacologia , Nitratos/farmacologia , Nitritos/farmacologia , Nitroprussiato/farmacologia
20.
Acta Biol Hung ; 59(1): 93-102, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18401948

RESUMO

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.


Assuntos
Meristema/efeitos dos fármacos , Mitose/efeitos dos fármacos , Cebolas/efeitos dos fármacos , Poliaminas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Cicloeximida/farmacologia , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Meristema/citologia , Mitoguazona/farmacologia , Mitose/fisiologia , Cebolas/fisiologia , Putrescina/farmacologia , Espermidina/farmacologia , Espermina/farmacologia
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