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1.
J Biol Chem ; 285(3): 1733-42, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19897478

RESUMO

Mitochondrial biogenesis is a complex process. It necessitates the participation of both the nuclear and the mitochondrial genomes. This process is highly regulated, and mitochondrial content within a cell varies according to energy demand. In the yeast Saccharomyces cerevisiae, the cAMP pathway is involved in the regulation of mitochondrial biogenesis. An overactivation of this pathway leads to an increase in mitochondrial enzymatic content. Of the three yeast cAMP protein kinases, we have previously shown that Tpk3p is the one involved in the regulation of mitochondrial biogenesis. In this paper, we investigated the molecular mechanisms that govern this process. We show that in the absence of Tpk3p, mitochondria produce large amounts of reactive oxygen species that signal to the HAP2/3/4/5 nuclear transcription factors involved in mitochondrial biogenesis. We establish that an increase in mitochondrial reactive oxygen species production down-regulates mitochondrial biogenesis. It is the first time that a redox sensitivity of the transcription factors involved in yeast mitochondrial biogenesis is shown. Such a process could be seen as a mitochondria quality control process.


Assuntos
Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , AMP Cíclico/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
2.
Vet Parasitol ; 233: 32-38, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-28043385

RESUMO

Trypanosoma congolense and T. vivax are the main causative agents of animal African trypanosomosis (AAT), a disease which hinders livestock production throughout sub-Saharan Africa and in some parts of South America. Although two trypanocidal drugs are currently available, the level of treatment is low due to the difficulty in diagnosing the disease in the field. The major clinical signs of AAT such as anaemia, weight loss, and infertility, are common to several other endemic livestock diseases. Current diagnostic methods, based on the visualization of the parasite in the blood, or on the detection of its DNA or the antibodies it triggers in the host, are not suitable for direct use in the field as they require specialized equipment and personnel. Thus, we developed a quick-format diagnostic test (15min) based on the recombinant TcoCB and TvGM6 antigens for detection of T. congolense and T. vivax, respectively, aimed at providing farmers and veterinarians in the field with the means to conduct a quick diagnosis. The specificity and sensitivity of the test were evaluated using sera from experimentally infected cattle, and fresh blood when possible. The prototype, which includes both antigens, shows a specificity of 95.9 (95% C.I., 90.4%-100%) and a sensitivity of 92.0% (95% C.I., 85.9%-98.1%) for T. congolense and 98.2% (95% C.I., 94.7%-100%) for T. vivax. The high levels of sensitivity and specificity of this rapid test, the possibility of using directly whole blood, and the ease of interpreting the result, all contribute to make of this test a valuable candidate to contribute to the control of AAT in the field. However, further tests with more representative, numerous and fresh reference samples are necessary in order to compare this test with the ELISA, the current gold standard serological test for trypanosomosis.


Assuntos
Doenças dos Bovinos/diagnóstico , Testes Sorológicos/veterinária , Tripanossomíase Africana/veterinária , África Subsaariana , Animais , Antígenos de Protozoários/metabolismo , Bovinos , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Testes Sorológicos/normas , Fatores de Tempo , Tripanossomíase Africana/diagnóstico
3.
Biochim Biophys Acta ; 1706(1-2): 117-25, 2005 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-15620372

RESUMO

During aerobic cell growth, mitochondria must meet energy demand either by adjusting cellular mitochondrial content or by adjusting ATP production flux, allowing a constant growth yield. On respiratory substrate, the Ras/cAMP pathway has been shown to be involved in this process in the yeast Saccharomyces cerevisiae. We show that of the three cAMP protein kinase catalytic subunits, Tpk3p is the one specifically involved in the regulation of cellular mitochondrial content when energy demand decreases. In decreased energy demand, the Deltatpk3 mitochondrial enzymatic content decreases leading to a subsequent decrease in the cellular growth rate. Moreover, enzymatic content decreases in the Deltatpk3 isolated mitochondria, suggesting that the amount of cellular mitochondria is not affected, but rather that the mitochondria are modified. Our study points to an important decrease in the cytochrome c content in the Deltatpk3 mitochondria, which leads to a decrease in the slipping process at the level of cytochrome-c-oxidase.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocromos c/metabolismo , Proteínas Fúngicas/metabolismo , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Proteínas ras/metabolismo , Eletroforese em Gel de Poliacrilamida , Medições Luminescentes , Mitocôndrias/fisiologia , Consumo de Oxigênio/fisiologia , Força Próton-Motriz , Saccharomyces cerevisiae/crescimento & desenvolvimento
4.
J Physiol Sci ; 64(1): 13-20, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24043354

RESUMO

Knockout of Kv1.3 improves glucose homeostasis and confers resistance to obesity. Additionally, Kv1.3 inhibition enhances glucose uptake. This is thought to occur through calcium release. Kv1.3 inhibition in T-lymphocytes alters mitochondrial membrane potential, and, as many agents that induce Ca(2+) release or inhibit mitochondrial function activate AMPK, we hypothesised that Kv1.3 inhibition would activate AMPK and increase glucose uptake. We screened cultured muscle with a range of Kv1.3 inhibitors for their ability to alter glucose uptake. Only Psora4 increased glucose uptake in C2C12 myotubes. None of the inhibitors had any impact on L6 myotubes. Magratoxin activated AMPK in C2C12 myotubes and only Pap1 activated AMPK in the SOL. Kv1.3 inhibitors did not alter cellular respiration, indicating a lack of effect on mitochondrial function. In conclusion, AMPK does not mediate the effects of Kv1.3 inhibitors and they display differential effects in different skeletal muscle cell lines without impairing mitochondrial function.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/efeitos dos fármacos , Músculo Esquelético/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Ficusina/farmacologia , Técnicas In Vitro , Camundongos , Mitocôndrias Musculares/efeitos dos fármacos , Modelos Animais , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Proteínas Associadas a Pancreatite , Ratos , Venenos de Escorpião/farmacologia
5.
PLoS One ; 8(10): e78565, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24205263

RESUMO

BACKGROUND: Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km(2) of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases. METHODOLOGY/PRINCIPLE FINDINGS: the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4). CONCLUSION/SIGNIFICANCE: this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.


Assuntos
Trypanosoma vivax/imunologia , Tripanossomíase Bovina/diagnóstico , Glicoproteínas Variantes de Superfície de Trypanosoma/sangue , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Trypanosoma vivax/genética , Tripanossomíase Bovina/sangue , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia
6.
Science ; 336(6083): 918-22, 2012 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-22517326

RESUMO

Salicylate, a plant product, has been in medicinal use since ancient times. More recently, it has been replaced by synthetic derivatives such as aspirin and salsalate, both of which are rapidly broken down to salicylate in vivo. At concentrations reached in plasma after administration of salsalate or of aspirin at high doses, salicylate activates adenosine monophosphate-activated protein kinase (AMPK), a central regulator of cell growth and metabolism. Salicylate binds at the same site as the synthetic activator A-769662 to cause allosteric activation and inhibition of dephosphorylation of the activating phosphorylation site, threonine-172. In AMPK knockout mice, effects of salicylate to increase fat utilization and to lower plasma fatty acids in vivo were lost. Our results suggest that AMPK activation could explain some beneficial effects of salsalate and aspirin in humans.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Salicilatos/metabolismo , Salicilatos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Substituição de Aminoácidos , Animais , Aspirina/farmacologia , Sítios de Ligação , Compostos de Bifenilo , Metabolismo dos Carboidratos/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Células HEK293 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Mutação , Consumo de Oxigênio/efeitos dos fármacos , Fosforilação , Pironas/farmacologia , Ratos , Salicilatos/sangue , Tiofenos/farmacologia
7.
Cell Metab ; 11(6): 554-65, 2010 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-20519126

RESUMO

A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) gamma2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca(2+) ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca(2+), and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Dinitrofenóis/farmacologia , Metabolismo Energético , Ativação Enzimática , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Naftalimidas/farmacologia , Oligomicinas/farmacologia , Fosforilação , Subunidades Proteicas/metabolismo
8.
J Biol Chem ; 281(37): 26779-84, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16849319

RESUMO

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes taking place during net biomass formation and cell property maintenance. A crucial parameter for growth description is its yield, i.e. the efficiency of the transformation from substrate consumption to biomass formation. Using numerous yeast strains growing on different respiratory media, we have shown that the growth yield is identical regardless of the strain, growth phase, and respiratory substrate used. This homeostasis is the consequence of a strict linear relationship between growth and respiratory rates. Moreover, in all conditions tested, the oxygen consumption rate was strictly controlled by the cellular content of respiratory chain compounds in such a way that, in vivo, the steady state of oxidative phosphorylation was kept constant. Thus, the growth yield homeostasis depends on the tight adjustment of the cellular content of respiratory chain compounds to the growth rate. Any process leading to a defect in this adjustment allows an energy waste and consequently an energy yield decrease.


Assuntos
Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , AMP Cíclico/metabolismo , Citocromos/metabolismo , Homeostase , Mutação , Oxigênio/metabolismo , Consumo de Oxigênio , Saccharomyces cerevisiae/metabolismo , Termodinâmica
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