Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement.

The deleterious effects of H202 on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H2O2 resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H2O2 on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H2O2 on respiratory complexes was almost completely recovered by beta-mercaptoethanol treatment. H2O2 treatment resulted in full resistance to Qo site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Qo) of complex III is the target for H2O2. H2O2 did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H2O2 addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.

In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction.

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.

In yeast, Ca2+ and octylguanidine interact with porin (VDAC) preventing the mitochondrial permeability transition.

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.

Fluorescence quenching by nucleotides of the plasma membrane H+-ATPase from Kluyveromyces lactis.

The yeast plasma membrane H+-ATPase isolation procedure was improved; a highly pure enzyme (90-95%) was obtained after centrifugation on a trehalose concentration gradient. H+-ATPase kinetics was slightly cooperative: Hill number = 1.5, S0.5 = 800 microM ATP, and turnover number = 36 s-1. In contrast to those of other P-type ATPases, H+-ATPase fluorescence was highly sensitive to nucleotide binding; the fluorescence decreased 60% in the presence of both 5 mM ADP and AMP-PNP. Fluorescence titration with nucleotides allowed calculation of dissociation constants (Kd) from the binding site; Kd values for ATP and ADP were 700 and 800 microM, respectively. On the basis of amino acid sequence and homology model analysis, we propose that binding of the nucleotide to the N-domain is coupled to the movement of a loop beta structure and to the exposure of the Trp505 residue located in the loop. The recombinant N-domain also displayed a large hyperbolic fluorescence quenching when ATP binds; however, it displayed a higher affinity for ATP (Kd = 100 microM). We propose for P-type ATPases that structural movements during nucleotide binding could be followed if a Trp residue is properly located in the N-domain. Further, we propose the use of trehalose in enzyme purification protocols to increase the purity and quality of the isolated protein and to perform structural studies.

F-actin involvement in guinea pig sperm motility.

Sperm motility is a must for natural fertilization to occur. During their travel through the epididymis, mammalian spermatozoa gradually acquire the ability to move. This is accomplished through a sliding movement of the outer doublet microtubules of the axoneme which is energized by the dynein ATPase. Within its complex structure, the mammalian sperm flagellum contains F-actin and thus, we decided to test in the guinea pig sperm flagellum the role of F-actin in motility. During maturation, capacitation, and the acrosome reaction, a gradual decrease of the relative concentration of F-actin was observed. Motility increased as spermatozoa became able to fertilize. Gelsolin, phalloidin, and KI inhibited sperm motility. Gelsolin canceled sperm motility within 20 min of treatment while 0.6 M KI had immediate effects. Phalloidin diminished hyperactive sperm motility slightly. All three compounds significantly increased the relative concentration of F-actin. Latrunculins are conventional drugs that destabilize the F-actin cytoskeleton. Latrunculin A (LAT A) did not affect sperm motility; but significantly increased F-actin relative concentration. The results suggested that in guinea pig spermatozoa, randomly severing F-actin filaments inhibits flagellar motility; while end filament alteration does not. Thus, specific filament regions seem to be important for sperm motility.

In rat hepatocytes, different adenosine receptor subtypes use different secondary messengers to increase the rate of ureagenesis.

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].

In guinea pig spermatozoa, the procaine-promoted synchronous acrosome reaction results in highly fertile cells exhibiting normal F-actin distribution.

In guinea pig spermatozoa, procaine induces Ca(2+) independent hyperactivated motility suggestive of sperm capacitation. Nonetheless, in the presence of high extracellular Ca(2+), procaine increases cytoplasmic Ca(2+). We analyze the procaine effect on the acrosome reaction (AR) processes in guinea pig spermatozoa. Results indicated that: (i) in spermatozoa pre-incubated 5-30 min in MCM-PLG medium, procaine produced synchronous AR, (ii) the acrosome-reacted sperm number increased with the capacitation period before procaine treatment and with procaine concentration, (iii) acrosome reaction was blocked when Ca(2+) was omitted, (iv) plasma membrane-outer acrosomal membrane fusion started within 2 min after procaine treatment, (v) in acrosome-reacted spermatozoa, actin polymerization occurred and F-actin was located in the equatorial and post-acrosomal regions and (vi) procaine treatment resulted in highly fertile acrosome-reacted spermatozoa. This is the first report indicating that procaine promotes synchronic AR in mammalian spermatozoa. If procaine promotes premature AR of spermatozoa in vivo, it might be a factor for infertility in patients exposed to this local anesthetic.

Effects of biotin on growth and protein biotinylation in Saccharomyces cerevisiae.

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.

Selective herbicide activity of 2,5-di(benzylamine)-p-benzoquinone against the monocot weed Echinochloa crusgalli. An in vivo analysis of photosynthesis and growth.

Six 2,5-diamino-p-benzoquinone derivatives previously characterized as photosystem I electron acceptors were tested for their postemergence herbicide activity. By induction kinetics of chlorophyll a fluorescence performed in vivo it was determined that 2,5-di(benzylamine)-p-benzoquinone diverted electrons at the reducing side of the chloroplast photosystem I. This derivative decreased the efficiency of photosystem II as evidenced by the decrease in the F(v)/F(m) change in Echinochloa crusgalli leaf disks. In addition, 2,5-di(benzylamine)-p-benzoquinone was a CO(2) assimilation inhibitor to Rubisco: the A/C(i) curve analysis indicates that 2,5-di(benzylamine)-p-benzoquinone affected both the carboxylation reaction itself and the regeneration of RuBP. 2,5-di(benzylamine)-p-benzoquinone did not exhibit any effect on the dicot plants Phaseolus vulgaris and Physalis ixocarpa or the monocot Zea mays. These species may have metabolized the herbicide to an inactive compound. Thus, 2,5-di(benzylamine)-p-benzoquinone was found to be a selective herbicide against the monocot weed E. crusgalli.

[Oregano: properties, composition and biological activity]. / El orégano: propiedades, composición y actividad biológica de sus componentes.

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.

El orégano: propiedades, composición y actividad biológica de sus componentes / Oregano: properties, composition and biological activity

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

Trehalose-enzyme interactions result in structure stabilization and activity inhibition. The role of viscosity.

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.

Measuring Solution Viscosity and its Effect on Enzyme Activity.

In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increased by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the H(+)-ATPase from the plasma membrane of Kluyveromyces lactis. A direct correlation was found between viscosity (eta) and the inhibition of the maximum rate of catalysis (V(max)). The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination of enzyme kinetics and the application of Kramers' equation to evaluate the effect of viscosity on the rate of ATP hydrolysis by the H(+)-ATPase.

In Saccharomyces cerevisiae, cations control the fate of the energy derived from oxidative metabolism through the opening and closing of the yeast mitochondrial unselective channel.

The yeast mitochondrial unspecific channel (YMUC) sensitivity to inorganic (Ca2+ or Mg2+) or organic (hexyl or octyl-guanidine) cations was measured. The rate of oxygen consumption in State 3 and State 4, the transmembrane potential (deltapsi), mitochondrial swelling, and the polyethylene-glycol mediated recontraction were used to follow opening of the YMUC. Addition of 0.4 mM PO4 did not close the YMUC, although it did enhance the sensitivity to Ca2+ (I50 decreased from 50 to 0.3 mM) and Mg2+ (I50 decreased from 5 to 0.83 mM Mg2+). The Ca2+ concentration needed to close the YMUC was higher than the concentrations usually observed in the cell. Nonetheless, Mg2+, Ca2+, and PO4 exhibited additive effects. These cations did not inhibit contraction of preswollen mitochondria, suggesting that the YMUC/cation interaction was labile. Octyl-guanidine (OG-I50 7.5 microM) was the only cation which inhibited mitochondrial recontraction, probably as a result of membrane binding stabilization through its hydrophobic tail. The PO4-dependent, Ca(2+)/Mg(2+)-mediated closure of the YMUC may be a means to control the proportion of oxidative energy producing ATP or being lost as heat.

Closure of the yeast mitochondria unspecific channel (YMUC) unmasks a Mg2+ and quinine sensitive K+ uptake pathway in Saccharomyces cerevisiae.

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.

Trehalose-mediated inhibition of the plasma membrane H+-ATPase from Kluyveromyces lactis: dependence on viscosity and temperature.

The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures. At 20 degrees C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased V(max) and increased S(0.5) (substrate concentration when initial velocity equals 0.5 V(max)), mainly at high trehalose concentrations (0.6 to 0.8 M). The quotient V(max)/S(0.5) decreased from 5.76 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the absence of trehalose to 1.63 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the presence of 0.8 M trehalose. The decrease in V(max) was linearly dependent on solution viscosity (eta), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramer's theory for reactions in solution. In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted. In the absence of trehalose, increasing the temperature up to 40 degrees C resulted in an exponential increase in V(max) and a decrease in enzyme cooperativity (n), while S(0.5) was not modified. As temperature increased, the effect of trehalose on V(max) decreased to become negligible at 40 degrees C, in good correlation with the temperature-mediated decrease in viscosity. The trehalose-mediated increase in S(0.5) was similar at all temperatures tested, and thus, trehalose effects on V(max)/S(0.5) were always observed. Trehalose increased the activation energy for ATP hydrolysis. Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.