RESUMO
In yeast, Ca(2+) and long chain alkylguanidines interact with mitochondria modulating the opening of the yeast mitochondrial unspecific channel. Mammalians possess a similar structure, the mitochondrial permeability transition pore. The composition of these pores is under debate. Among other components, the voltage-dependent anion channel has been proposed as a component of either pore. In yeast from an industrial strain, octylguanidine and calcium closed the yeast mitochondrial unspecific channel. Here, the effects of the cations Ca(2+) or octylguanidine and the voltage-dependent anion channel effector decavanadate were evaluated in yeast mitochondria from either a wild type or a voltage-dependent anion channel deletion laboratory strain. It was observed that in the absence of voltage-dependent anion channel, the yeast mitochondrial unspecific channel was desensitized to Ca(2+), octylguanidine or decavanadate but remained sensitive to phosphate. It is thus suggested that in yeast mitochondria, the voltage-dependent anion channel has a cation binding site where Ca(2+) and octylguanidine interact, conferring cation sensitivity to the yeast mitochondrial unspecific channel.
Assuntos
Cálcio/metabolismo , Guanidinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Cálcio/química , Cátions Bivalentes/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Deleção de Genes , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Oxigênio/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sensibilidade e Especificidade , Vanadatos/farmacologia , Canais de Ânion Dependentes de Voltagem/genéticaRESUMO
A role for sperm-specific proteins during the early embryonic development has been suggested by a number of recent studies. However, little is known about the participation of transcription factors in that stage. Here, we show that the signal transducer and activator of transcription 1 (Stat1), but not Stat4, was phosphorylated in response to capacitation and the acrosomal reaction (AR). Moreover, Stat1 phosphorylation correlated with changes in its localization: during capacitation, Stat1 moved from the cytoplasm to the theca/flagellum fraction. During AR, Stat1 phosphorylation increased again. In addition, blocking protein kinase A (PKA) and PKC during capacitation abolished both phosphorylation and migration of Stat1. Our results show tight spatio-temporal rearrangements of Stat1, suggesting that after fertilization Stat1 participates in the first rounds of transcription within the male pronucleus.
Assuntos
Reação Acrossômica/fisiologia , Fator de Transcrição STAT1/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/metabolismo , Ativação Transcricional , Animais , Transporte Biológico , Western Blotting/métodos , Cálcio/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Ácido Egtázico/farmacologia , Eletroforese em Gel de Poliacrilamida , Genisteína/farmacologia , Cobaias , Indóis/farmacologia , Isoflavonas/farmacologia , Isoquinolinas/farmacologia , Masculino , Maleimidas/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Fator de Transcrição STAT1/análise , Fator de Transcrição STAT4/análise , Fator de Transcrição STAT4/metabolismo , Cauda do Espermatozoide/metabolismo , Sulfonamidas/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
In rat hepatocytes, the role of cAMP and Ca(2+) as secondary messengers in the ureagenic response to stimulation of specific adenosine receptor subtypes was explored. Analyzed receptor subtypes were: A(1), A(2A), A(2B) and A(3). Each receptor subtype was stimulated with a specific agonist while blocking all other receptor subtypes with a battery of specific antagonists. For the A(1) and A(3) adenosine receptor subtypes, the secondary messenger was the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)). Accordingly, the A(1) or A(3)-mediated increase in [Ca(2+)](cyt) and in ureagenic activity were both inhibited by chelating Ca(2+) with either EGTA or BAPTA-AM. Also, Gd(3+) blocked both the increase in [Ca(2+)](cyt) and ureagenesis, suggesting that a Ca(2+) channel may be involved in the response to both A(1) and A(3). A partial effect was observed with the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. The concentration of cyclic AMP ([cAMP]) increased in response to stimulation of either the A(2A) or the A(2B) adenosine receptor subtypes, while it decreased slightly in response to stimulation of either A(1) or A(3). The stimulation of either the A(2A) or A(2B) adenosine receptor subtypes resulted in an increase in [cAMP] and an ureagenic response which were not sensitive to EGTA, BAPTA-AM, Gd(3+) or to thapsigargin. In addition, the adenylyl cyclase inhibitor MDL12,330A blocked the ureagenic response to A(2A) and A(2B), but not the response to either A(1) or A(3). Our results indicate that in the ureagenic liver response to adenosine, the secondary messenger for both, the A(1) and A(3) adenosine receptor subtypes is [Ca(2+)](cyt), while the message from the A(2A) and A(2B) adenosine receptor subtypes is relayed by [cAMP].
Assuntos
Hepatócitos/metabolismo , Receptores Purinérgicos P1/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Ureia/metabolismo , Adenosina/metabolismo , Inibidores de Adenilil Ciclases , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Quelantes/farmacologia , AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Hepatócitos/efeitos dos fármacos , Iminas/farmacologia , Masculino , Antagonistas de Receptores Purinérgicos P1 , Ratos , Ratos Wistar , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
In mammals, biotin, well known for its role as the cofactor of carboxylases, also controls the expression not only of proteins involved in this function, but also of a large number and variety of other different proteins. As a first step towards looking for a rationale for these phenomena, we intend to compare these regulatory functions of biotin between the rat and the much less evolutionized eukaryote, Saccharomyces cerevisiae. Thus far, we have measured growth in yeast cultured on different concentrations of biotin to choose the experimental conditions to be used (2, 200 and 2000 microM) and have found that a band corresponding to the biotinylated S. cerevisiae Arc1p protein appears at streptavidin Western blots at a biotin concentration above 2000 muM, its density increasing with higher biotin amounts. We will now study changes in yeast transcriptome with these varying concentrations and compare them with changes observed in the rat.
Assuntos
Biotina/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Biotina/metabolismo , Biotinilação , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismoRESUMO
The oregano spice includes various plant species. The most common are the genus Origanum, native of Europe, and the Lippia, native of Mexico. Among the species of Origanum. their most important components are the limonene, gamma-cariofilene, rho-cymenene, canfor, linalol, alpha-pinene, carvacrol and thymol. In the genus Lippia, the same compounds can be found. The oregano composition depends on the specie, climate, altitude, time of recollection and the stage of growth. Some of the properties of this plant's extracts are being currently studied due to the growing interest for substituting synthetic additives commonly found in foods. Oregano has a good antioxidant capacity and also presents antimicrobial activity against pathogenic microorganisms like Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, among others. These are all characteristics of interest for the food industry because they may enhance the safety and stability of foods. There are also some reports regarding the antimutagenic and anticarcinogenic effect of oregano; representing an alternative for the potential treatment and/or prevention of certain chronic ailments, like cancer.
Assuntos
Origanum/química , Lippia/química , Lippia/classificação , Lippia/metabolismo , Origanum/classificação , Origanum/metabolismoRESUMO
El orégano comprende varias especies de plantas que son utilizadas con fines culinarios, siendo las más comúnes el Origanum vulgare, nativo de Europa, y el Lippia graveolens, originario de México. Entre las especies de Origanum se encuentran como componentes principales el limoneno, el b-cariofileno, el r-cimeno, el canfor, el linalol, el a-pineno, el carvacrol y el timol. En el género Lippia pueden encontrarse estos mismos compuestos. Su contenido depende de la especie, el clima, la altitud, la época de recolección y el estado de crecimiento.Algunas propiedades de los extractos del orégano han sido estudiadas debido al creciente interés por sustituir los aditivos sintéticos en los alimentos. El orégano tiene una buena capacidad antioxidante y antimicrobiana contra microorganismos patógenos como Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, entre otros. Estas características son muy importantes para la industria alimentaria ya que pueden favorecer la inocuidad y estabilidad de los alimentos como también protegerlos contra alteraciones lipídicas. Existen además algunos informes sobre el efecto antimutagénico y anticarcinogénico del orégano sugiriendo que representan una alternativa potencial para el tratamiento y/o prevención de trastornos crónicos como el cáncer
Assuntos
Antioxidantes , Lippia mexicana , Odorantes , Óleos Voláteis , Timol , Ciências da NutriçãoRESUMO
Stress resistance is essential for survival. The mechanisms of molecule stabilization during stress are of interest for biotechnology, where many enzymes and other biomolecules are increasingly used at high temperatures and/or salt concentrations. Diverse organisms, exhibit rapid synthesis and accumulation of the disaccharide trehalose in response to stress. Trehalose is also rapidly hydrolyzed as soon as stress ends. In isolated enzymes, trehalose stabilizes both, structure and activity. In contrast, at optimal assay conditions, trehalose inhibits enzyme activity. A general mechanism underlying the trehalose effects observed at all temperatures probably is the trehalose-mediated increase in solution viscosity that leads to protein domain motion inhibition. This may be analyzed using Kramer's theory. The role of viscosity in the effects of trehalose is analyzed in examples from the literature and in studies on the plasma membrane H(+)-ATPase from Kluyveromyces lactis. In the cell, it may be proposed that the large concentration of trehalose reached during stress stabilizes structures through viscosity. However, once stress ends trehalose has to be rapidly hydrolyzed in order to avoid the viscosity-mediated inhibition of enzymes.
Assuntos
Enzimas/metabolismo , Trealose/metabolismo , Estresse Oxidativo , Dobramento de Proteína , ViscosidadeRESUMO
The K+ uptake pathways in yeast mitochondria are still undefined. Nonetheless, the K+-mediated mitochondrial swelling observed in the absence of phosphate (PO4) and in the presence of a respiratory substrate has led to propose that large K+ movements occur in yeast mitochondria. Thus, the uptake of K+ by isolated yeast mitochondria was evaluated. Two parallel experiments were conducted to evaluate K+ transport; these were mitochondrial swelling and the uptake of the radioactive K+ analog 86Rb+. The opening of the yeast mitochondrial unspecific channel (YMUC) was regulated by different PO4 concentrations. The high protein concentrations used to measure 86Rb+ uptake resulted in a slight stabilization of the transmembrane potential at 0.4 mM PO4 but not at 0 or 4 mM PO4. At 4 mM PO4 swelling was inhibited while, in contrast, 86Rb+ uptake was still observed. The results suggest that an energy-dependent K+ uptake mechanism was unmasked when the YMUC was closed. To further analyze the properties of this K+ uptake system, the Mg2+ and quinine sensitivity of both swelling and 86Rb+ uptake were evaluated. Under the conditions where the unspecific pore was closed, K+ transport sensitivity to Mg2+ and quinine increased. In addition, when Zn2+ was added as an antiport inhibitor, uptake of 86Rb+ increased. It is suggested that in yeast mitochondria, the K+ concentration is highly regulated by the equilibrium of uptake and exit of this cation through two specific transporters.