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La oxidasa alterna (AOX) es una metaloproteína monotópica que se encuentra unida a la cara interna de la membrana interna mitocondrial. Esta enzima cataliza la transferencia de electrones del ubiquinol al oxígeno con formación de agua como uno de los productos. Cuando la vía citocrómica se inhibe por la presencia de cianuro u otros agentes xenobióticos, la AOX permite el flujo de electrones del NADH al oxígeno y, por tanto, la actividad catabólica y anabólica del ciclo de Krebs. La AOX se encuentra en plantas, hongos, algunos protistas y metazoarios primitivos y se le ha atribuido la capacidad de conferir osmoprotección y resistencia al estrés oxidativo en las células. En esta revisión, se analiza la estructura de la enzima, se aborda su participación en la respuesta ante el estrés osmótico y oxidativo en hongos, su influencia en la síntesis de ATP y la regulación de su expresión genética.

The F

Lactate is considered to be a waste metabolite produced during muscle fatigue. In contrast with this simplistic point of view, in this review we provide evidence of the multiple and complex functions of this metabolite. We show that: 1) lactate is the final product of the glycolysis regardless the oxygen concentration in the cell 2) lactate is part of two types of shuttle, one that functions in the intermembrane space of the mitochondrion, and another intercellular, which is responsible for feeding lactate to certain cell types, such as neurons or heart muscle, 3) in sperm,lactate is transported directly to the mitochondrial matrix and there it is oxidized to produce pyruvate and NADH, 4) in the liver, lactate participates in the oxidation of ethanol through the generation of hydrogen peroxide, 5) Depending on the cell line, lactate can function as anti-inflammatory agent (endocrine) and/or a regulator of gene expression. Keywords: Lactic fermentation; hypoxia; muscle fatigue; gene regulator

Medical education faces new challenges in the development and acquisition of competencies in medical students, and although there are teaching strategies that can be helpful to reach this goal, PBL is one of the best strategies. In order to take advantage of PBL, it is important to understand the method and the various ways in which it has been implemented, as well as to identify the difficulties and disadvantages of PBL. The objective of this article is to review the PBL method, its advantages and disadvantages, and some frequent mistakes in its implementation, emphasizing the Mexican context. Key words: Medical education; PBL; didactic; competences

This paper considers the adoption of computational analysis within research designs modeled after the extended case method. Echoing calls to augment the power of contemporary researchers through the adoption of computational text analysis methods, we offer a framework for thinking about how such techniques can be integrated into quasi-ethnographic workflows to address structural sociological claims. We focus, in particular, on how this adoption of novel forms of evidence impacts corpus design and interpretation (which we tie to matters of casing), theoretical elaboration (which we associate to moving empirical claims across scales), and verification (which we see as a process of reflexive scaffolding of theoretical claims). We provide an example of the use of this framework through a study of the marketization of social scientific knowledge in the United Kingdom.

It is becoming increasingly common to hear life scientists say that high quality life science research relies upon high quality laboratory animal care. However, the idea that animal care is a crucial part of scientific knowledge production is at odds with previous social science and historical scholarship regarding laboratory animals. How are we to understand this discrepancy? To begin to address this question, this paper seeks to disentangle the values of scientists in identifying animal care as important to the production of high quality scientific research. To do this, we conducted a survey of scientists working in the United Kingdom who use animals in their research. The survey found that being British is associated with thinking that animal care is a crucial part of conducting high quality science. To understand this finding, we draw upon the concept of ‘civic epistemologies’ (Jasanoff 2005; Prainsack 2006) and argue that ‘animals’ and ‘care’ in Britain may converge in taken‐for‐granted assumptions about what constitutes good scientific knowledge. These ideas travel through things like state regulations or the editorial policies of science journals, but do not necessarily carry the embodied civic epistemology of ‘animals’ and ‘science’ from which such modes of regulating laboratory animal welfare comes.

An oft stated corrollary, sometimes taken as its definition, of the Efficient Markets Hypothesis is that in an efficient market it should not be possible to systematically make excess or abnormal returns. This begs the question of excess or abnormal relative to what? Traditional benchmarks either fail to distinguish between trading returns and market returns, or are dependent on an associated asset pricing model, thus leading to the joint-hypothesis problem. In this paper we discuss a purely empirical measure - Excess Trading Returns - derived from the difference in profits associated with an agent portfolio where one or more trades were executed relative to a Buy-and-Hold portfolio where they were not, the Buy-and-Hold benchmark being dynamic and/or unique to the agent. With this measure in hand we introduce the relative inefficiency associated with a pair of agents, agent groups or trading strategies and from this define an Inefficiency Matrix that can provide a complete empirical characterization of the inefficiencies inherent in an entire market.

La obesidad es un problema de salud global, asociada con enfermedades cardiovasculares. El análisis de la relación existente entre el elevado consumo de glucosa y la concomitante formación de depósitos de grasa, descrita por el ciclo de Randle, permitirá desarrollar una comprensión de los procesos metabólicos involucrados en el balance entre la formación y la degradación de los lípidos. Esta revisión tiene como objetivo, proporcionar una actualización del ciclo y de sus diferentes niveles de regulación, así como la participación de mTORC1 y la cinasa dependiente de AMP (AMPK) durante el estado postprandial y de ayuno.

Las levaduras son organismos microscópicos que están distribuidos en toda la Tierra, de modo que algunas han adaptado su metabolismo para proliferar en ambientes extremos. Las levaduras que habitan en la Antártica son un grupo de microorganismos adaptados al frío que han sido poco estudiadas. En esta revisión se describen algunas de las adaptaciones metabólicas que les permiten habitar en ambientes extremos, por ejemplo, el de la Antártica. También se abordan las consideraciones relevantes para saber si una levadura es extremófila, así como los criterios utilizados para clasificar a las levaduras por crecimiento y temperatura. Además, se explica el papel de las vías de biosíntesis de carotenoides y lípidos que están involucradas en contrarrestar a las especies reactivas de oxígeno generadas por estrés oxidante en levaduras pigmentadas y oleaginosas del género Rhodotorula. La revisión también considera aspectos de investigación básica y la importancia de las levaduras oleaginosas de la Antártica para el desarrollo de algunas aplicaciones biotecnológicas.

El presente trabajo de investigacion, se ha pensado a partir de un grupo de actores desmovilizados del conflicto armado que se encuentran purgando sus penas en la Carcel la Picota de Bogota, con quienes se busca identificar las principales problematicas que van en contra de la Seguridad Humana. Para tal fin, se ha pensado el desarrollo de una investigacion cualitativa, de tipo etnografico y corte hermeneutico, en la cual se ha seleccionado como tecnica e instrumento de recoleccion de la informacion la entrevista semiestructurada y grupos focales; de igual manera, una vez recolectada la informacion, esta sera sistematizada y analizada a traves de la triangulacion de la informacion, para asi de esta manera, poder determinar, cuales son las principales problematicas sociales que afectan la Seguridad Humana, de los actores desmovilizados de grupos al margen de la ley, ubicados en la carcel La Picota de Bogota.

"Obesity is a growing global health concern, closely related to cardiovascular diseases. Understanding the correlation between excessive sugar consumption and the formation of fat deposits, described in the Randle cycle, will allow us to have a better grasp on metabolic processes that disrupt the balance between fat formation and degradation processes. The goal of this review is to expand and update the information about the Randle cycle and describe their different levels of regulation. In addition, the participation of mTORC1 and the AMP dependent Kinase (AMPK) during the postprandial and fasting states is described."

Type 2 alternative NADH dehydrogenases (NDH‐2) participate indirectly in the generation of the electrochemical proton gradient by transferring electrons from NADH and NADPH into the ubiquinone pool. Due to their structural simplicity, alternative NADH dehydrogenases have been proposed as useful tools for gene therapy of cells with defects in the respiratory complex I. In this work, we report the presence of three open reading frames, which correspond to NDH‐2 genes in the genome of Ustilago maydis. These three genes were constitutively transcribed in cells cultured in YPD and minimal medium with glucose, ethanol, or lactate as carbon sources. Proteomic analysis showed that only two of the three NDH‐2 were associated with isolated mitochondria in all culture media. Oxygen consumption by permeabilized cells using NADH or NADPH was different for each condition, opening the possibility of posttranslational regulation. We confirmed the presence of both external and internal NADH dehydrogenases, as well as an external NADPH dehydrogenase insensitive to calcium. Higher oxygen consumption rates were observed during the exponential growth phase, suggesting that the activity of NADH and NADPH dehydrogenases is coupled to the dynamics of cell growth.

Las transformaciones epistemológicas desarrolladas sobre los procesos de desmovilización en Colombia retan a la escuela a abrir la discusión teórica sobre el actual tránsito de las lógicas de seguridad humana —ancladas en las tendencias jerarquizadas del bienestar y el asistencialismo— hacia nuevas alternativas de búsqueda en torno al desarrollo humano y el empoderamiento de los sujetos, desde la potencialización tanto de las necesidades fundamentales como de las capacidades humanas. Esto con el fin de gestar procesos de educación ciudadana en los que el desmovilizado adquiera un rol protagónico y activo. La presente investigación, desarrollada desde un enfoque cualitativo, problematiza desde la hermenéutica la discusión teórica a partir del análisis documental que reconstruye el devenir histórico de la desmovilización y la voz de aquellos que han acompañado estos procesos, lo cual resulta crucial en la construcción del nuevo país.

Ustilago maydis is an aerobic basidiomycete that fully depends on oxidative phosphorylation for its supply of ATP, pointing to mitochondria as a key player in the energy metabolism of this organism. Mitochondrial F1F0-ATP synthase occurs in supramolecular structures. In this work, we isolated the monomer (640kDa) and the dimer (1280kDa) and characterized their subunit composition and kinetics of ATP hydrolysis. Mass spectrometry revealed that dimerizing subunits e and g were present in the dimer but not in the monomer. Analysis of the ATPase activity showed that both oligomers had Michaelis-Menten kinetics, but the dimer was 7 times more active than the monomer, while affinities were similar. The dimer was more sensitive to oligomycin inhibition, with a Ki of 24nM, while the monomer had a Ki of 169nM. The results suggest that the interphase between the monomers in the dimer state affects the catalytic efficiency of the enzyme and its sensitivity to inhibitors.

This paper explores patents for monetary objects as mechanisms for exploring the future of money and its role in society. By looking at families of patents around monetary objects, the paper highlights the ontological, pecuniary, and pedagogical expectations about the use of money in nineteenth century American societies.

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, U. maydis mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in U. maydis under diverse energy conditions. Supercomplexes were obtained by solubilization of U. maydis mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I1:IV1), 1400 kDa (I1:III2), 1600 kDa (I1:III2:IV1), and 1800 kDa (I1:III2:IV2). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.

Mitochondrial aldehyde dehydrogenase (ALDH2) has been proposed as a key enzyme in cardioprotection during ischemia-reperfusion processes. This proposal led to the search for activators of ALDH2 with the aim to develop cardioprotective drugs. Alda-1 was the first activator of ALDH2 identified and its cardioprotective effect has been extensively proven in vivo; however, the mechanism of activation is not fully understood. A crystallographic study showed that Alda-1 binds to the entrance of the aldehyde-binding site; therefore, Alda-1 should in essence be an inhibitor. In the present study, kinetic experiments were performed to characterize the effect of Alda-1 on the properties of ALDH2 (kinetic parameters, determination of the rate-limiting step, reactivity of the catalytic cysteine) and on the kinetic mechanism (type of kinetics, sequence of substrates entering, and products release). The results showed that Alda-1 dramatically modifies the properties of ALDH2, the Km for NAD+ decreased by 2.4-fold, and the catalytic efficiency increased 4.4-fold; however, the Km for the aldehyde increased 8.6-fold, thus, diminishing the catalytic efficiency. The alterations in these parameters resulted in a complex behavior, where Alda-1 acts as inhibitor at low concentrations of aldehyde and as an activator at high concentrations. Additionally, the binding of Alda-1 to ALDH2 made the deacylation less limiting and diminished the pKa of the catalytic cysteine. Finally, NADH inhibition patterns indicated that Alda-1 induced a change in the sequence of substrates entry and products release, in agreement with the proposal of both substrates entering ALDH2 by the NAD+ entrance site.

During the lactation period, rat pups are fed by the dam, and the patterns of mother-pup interaction change during this period. Additionally, there are changes in feeding; first, mother´s milk is the only food needed for sustenance, and later, it is combined with solid food and water. GH serum concentrations depend on both maternal-pup interaction and energy metabolism. In the artificial rearing (AR) procedure, pups are deprived of mother-pup interaction, and the feeding pattern is controlled. This rearing paradigm has been used in rats to analyze the effects of maternal deprivation on social behavior. In the present study, we analyzed the variation in GH, acylated ghrelin and IGF-1 serum concentrations throughout the lactation period in AR pups. At pnd7, the maternal rearing (MR) pups responded to a 4 h fast with a drop in GH serum concentration, which is a well-known response to maternal deprivation. GH serum levels in the AR pups did not change, suggesting an adaptation phenomenon. A dopamine inhibitory effect of GH secretion was observed in pnd7 cultured somatotropes, suggesting dopamine regulation of GH secretion at this age. Acylated ghrelin serum levels in the AR pups showed an inverted pattern compared to that in the MR pups, which was related to the artificial feeding pattern. IGF-1 serum levels were lower in the AR pups than in MR pups, which was associated with hepatic GH resistance and with low Igf1 mRNA expression at pnd7. Interestingly, at pnd14, both pup groups showed high hepatic Igf1 mRNA expression but low IGF-1 serum levels, and this was inverted at pnd21. However, serum glucose levels were lower in the AR pups at pnd14 but reached the same levels as the MR pups at pnd21. Moreover, hepatomegaly and higher hepatic GH-receptor levels were observed in the AR pups at pnd21, which was in agreement with an absence of a solid food meal. During AR, the pups lost the maternal interaction-stimulated GH secretion, which correlated with lower IGF-1 serum levels during the first week of postnatal development. Later, the AR pups exhibited hepatic responses, in order to satisfy the metabolic demand for the normal weaning, with low carbohydrates levels in their meal.

Saccharomyces cerevisiae arose from an interspecies hybridization (allopolyploidiza-tion), followed by Whole Genome Duplication. Diversification analysis of ScAlt1/ScAlt2 indicated that while ScAlt1 is an alanine transaminase, ScAlt2 lost this activity, constituting an example in which one of the members of the gene pair lacks the apparent ancestral physiological role. This paper analyzes structural organization and pyridoxal phosphate (PLP) binding properties of ScAlt1 and ScAlt2 indicating functional diversification could have determined loss of ScAlt2 alanine transaminase activity and thus its role in alanine metabolism. It was found that ScAlt1 and ScAlt2 are dimeric enzymes harboring 67% identity and intact conservation of the catalytic residues, with very similar structures. However, tertiary structure analysis indicated that ScAlt2 has a more open conformation than that of ScAlt1 so that under physiological conditions, while PLP interaction with ScAlt1 allows the formation of two tautomeric PLP isomers (enolimine and ketoenamine) ScAlt2 preferentially forms the ketoenamine PLP tautomer, indicating a modified polarity of the active sites which affect the interaction of PLP with these proteins, that could result in lack of alanine transaminase activity in ScAlt2. The fact that ScAlt2 forms a catalytically active Schiff base with PLP and its position in an independent clade in “sensu strictu” yeasts suggests this protein has a yet undiscovered physiological function.

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in lipid droplets (LDs). LD index is obtained without lipid extraction. It allows measuring the LDs content in high-throughput experiments under different conditions such as growth in rich or nitrogen depleted media. Albeit the method was described for the first time to study the lipid droplet metabolism in Saccharomyces cerevisiae, it was successfully applied to the basidiomycete Ustilago maydis. Interestingly, and because LDs are organelles phylogenetically conserved in eukaryotic cells, the method can be applied to a large variety of cells, from yeast to mammalian cells. The LD index is based on the liquid fluorescence recovery assay (LFR) of the BODIPY 493/503 under quenching conditions, by the addition of cells fixed with formaldehyde. Potassium iodine is used as a fluorescence quencher. The ratio between the fluorescence and the optical density slopes is named LD index. Slopes are calculated from the straight lines obtained when BODIPY fluorescence and optical density at 600 nm (OD600) are plotted against sample addition. Optimal data quality is reflected by correlation coefficients equal or above 0.9 (r ≥ 0.9). Multiple samples can be read simultaneously as it can be implemented in a microplate. Since BODIPY 493/503 is a lipophilic fluorescent dye that partitions into the lipid droplets, it can be used in many types of cells that accumulate LDs.

A kinetic study of thioredoxin-glutathione reductase (TGR) fromTaenia crassicepsmetacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproducedin silico.

Ustilago maydis is an aerobic basidiomycete that fully depends on oxidative phosphorylation for its supply of ATP, pointing to mitochondria as a key player in the energy metabolism of this organism. Mitochondrial F

The mitochondrial alternative oxidase is an important enzyme that allows respiratory activity and the functioning of the Krebs cycle upon disturbance of the respiration chain. It works as a security valve in transferring excessive electrons to oxygen, thereby preventing potential damage by the generation of harmful radicals. A clear biological function, besides the stress response, has so far convincingly only been shown for plants that use the alternative oxidase to generate heat to distribute volatiles. In fungi it was described that the alternative oxidase is needed for pathogenicity. Here, we investigate expression and function of the alternative oxidase at different stages of the life cycle of the corn pathogen Ustilago maydis (Aox1). Interestingly, expression of Aox1 is specifically induced during the stationary phase suggesting a role at high cell density when nutrients become limiting. Studying deletion strains as well as overexpressing strains revealed that Aox1 is dispensable for normal growth, for cell morphology, for response to temperature stress as well as for filamentous growth and plant pathogenicity. However, during conditions eliciting respiratory stress yeast-like growth as well as hyphal growth is strongly affected. We conclude that Aox1 is dispensable for the normal biology of the fungus but specifically needed to cope with respiratory stress.

When cultured in medium limited of nitrogen sources, the phytopathogen Ustilago maydis produces two amphipathic glycolipids: Ustilagic acid (UA) and Mannosylerythritol lipid (MEL), which in addition to the hydrophilic moiety, contain dior tri-hydroxylated C16 fatty acids (UA), or C8 and C16 saturated fatty acids (MEL). We compared the growth and morphology of cells in YPD and in minimum media containing glucose and nitrogen sources such as nitrate or urea and those deprived of nitrogen. Nitrogen-starved cells showed a dramatic accumulation of internal lipids identified as lipid droplets when stained with the hydrophobic probe BODIPY; these lipid droplets were enriched in unsaturated fatty acids. Fatty acids in YPD or medium containing nitrate as nitrogen source showed a combination of saturated/unsaturated lipids, but when urea was the nitrogen source, cells only contained saturated fatty acids. The glycolipid profiles produced in the presence or absence of nitrogen showed preferences towards the production of one kind of glycolipid: cells in media containing nitrate or urea produced different proportions of UA/MEL, but under nitrogen starvation cells contained only UA. The emulsification capacity of the glycolipids produced in media with or without nitrogen was similar (72% - 76%). HPLC of the glycolipids allowed the separation of fractions with different emulsifying characteristics. Our results indicate that U. maydis accumulates lipid droplets when deprived of nitrogen source and confirm that UA is not under nitrogen control, but rather that MEL and lipid droplets are produced and oppositely regulated by nitrogen.

During the interphase the nuclear DNA of metazoan cells is organized in supercoiled loops anchored to constituents of a nuclear substructure or compartment known as the nuclear matrix. The stable interactions between DNA and the nuclear matrix (NM) correspond to a set of topological relationships that define a nuclear higher-order structure (NHOS). Current evidence suggests that the NHOS is cell-type-specific. Biophysical evidence and theoretical models suggest that thermodynamic and structural constraints drive the actualization of DNA-NM interactions. However, if the topological relationships between DNA and the NM were the subject of any biological constraint with functional significance then they must be adaptive and thus be positively selected by natural selection and they should be reasonably conserved, at least within closely related species. We carried out a coarse-grained, comparative evaluation of the DNA-NM topological relationships in primary hepatocytes from two closely related mammals: rat and mouse, by determining the relative position to the NM of a limited set of target sequences corresponding to highly-conserved genomic regions that also represent a sample of distinct chromosome territories within the interphase nucleus. Our results indicate that the pattern of topological relationships between DNA and the NM is not conserved between the hepatocytes of the two closely related species, suggesting that the NHOS, like the karyotype, is species-specific.

Mitochondrial complexes I, III2, and IV from human cytotrophoblast and syncytiotrophoblast associate to form supercomplexes or respirasomes, with the following stoichiometries: I1:(III2)1 and I1:(III2)1–2:IV1–4. The content of respirasomes was similar in both cell types after isolating mitochondria. However, syncytiotrophoblast mitochondria possess low levels of dimeric complex V and do not have orthodox cristae morphology. In contrast, cytotrophoblast mitochondria show normal cristae morphology and a higher content of ATP synthase dimer. Consistent with the dimerizing role of the ATPase inhibitory protein (IF1) (Garcia, J. J., Morales-Rios, E., Cortes-Hernandez, P., and Rodriguez-Zavala, J. S. (2006) Biochemistry 45, 12695–12703), higher relative amounts of IF1 were observed in cytotrophoblast when compared with syncytiotrophoblast mitochondria. Therefore, there is a correlation between dimerization of complex V, IF1 expression, and the morphology of mitochondrial cristae in human placental mitochondria. The possible relationship between cristae architecture and the physiological function of the syncytiotrophoblast mitochondria is discussed.

Phosphoglycerate kinase (EC 2.7.2.3) catalyzes reversible phosphoryl transfer from 1,3-bisphosphoglycerate to ADP to synthesize 3-phosphoglycerate and ATP during glycolysis. Phosphoglycerate kinases from several sources can use GDP/GTP as alternative substrates to ADP/ATP; however, the maximal velocities (Vm) reached with the guanine nucleotides are ∼ 50% of those displayed with the adenine nucleotides. By contrast, Entamoeba histolytica phosphoglycerate kinase (EC 2.7.2.10) is the only reported phosphoglycerate kinase displaying higher activity with GDP/GTP and lower affinities for the adenine nucleotides. To elucidate the molecular basis of the Entamoeba histolytica phosphoglycerate kinase selectivity for GDP/GTP, a conformational analysis was carried out on a homology model based on crystallographic structures of yeast and pig phosphoglycerate kinases. Some amino acid residues involved in the purine ring binding site not previously described were detected. Accordingly, Y239, E309 and V311 were replaced by site-directed mutagenesis in the Entamoeba histolytica phosphoglycerate kinase gene for the corresponding amino acid residues present in the adenine nucleotide-dependent phosphoglycerate kinases and the recombinant proteins were purified. Kinetic analysis of the enzymes showed that the single mutants Y239F, E309Q, E309M and V311L increased their catalytic efficiencies (Vm/Km) with ADP/ATP as a result of both, increased Vm and decreased Km values. Furthermore, a higher catalytic efficiency in the double mutant Y239F/E309M was achieved, which was mainly due to an increased affinity for ADP/ATP with a concomitant diminished affinity for GDP/GTP. The main Entamoeba histolytica phosphoglycerate kinase amino acid residues involved in the selectivity for guanine nucleotides were thus identified.

The effects of octyl gallate on Ustilago maydis yeast cells were analysed in relation to its capacity to oxidize compounds (pro-oxidant actions). All phenolic compounds tested inhibited the alternative oxidase (AOX). However, only octyl gallate induced a morphological change in yeast cells and collapsed the mitochondrial membrane potential. In contrast to octyl gallate, propyl gallate and nordihydroguaiaretic acid caused only a negligible cell change and the membrane potential was not affected. Our findings show that structurally related phenolic compounds do not necessarily exert similar actions on target cells. Preincubation of U. maydis cells with trolox inhibited the change to pseudohyphal growth produced by octyl gallate. These results suggest that in addition to the inhibitory action of octyl gallate on the AOX, this compound induces a switch from yeast to a mycelium, probably through the formation of lipid peroxides.

Of the four transmembrane helices (M4, M5, M6, and M8) that pack together to form the ion-binding sites of P(2)-type ATPases, M8 has until now received the least attention. The present study has used alanine-scanning mutagenesis to map structure-function relationships throughout M8 of the yeast plasma-membrane H(+)-ATPase. Mutant forms of the ATPase were expressed in secretory vesicles and at the plasma membrane for measurements of ATP hydrolysis and ATP-dependent H(+) pumping. In secretory vesicles, Ala substitutions at a cluster of four positions near the extracytoplasmic end of M8 led to partial uncoupling of H(+) transport from ATP hydrolysis, while substitution of Ser-800 (close to the middle of M8) by Ala increased the apparent stoichiometry of H(+) transport. A similar increase has previously been reported following the substitution of Glu-803 by Gln (Petrov, V. et al., J. Biol. Chem. 275:15709-15718, 2000) at a position known to contribute directly to Ca(2+) binding in the Ca(2+)-ATPase of sarcoplasmic reticulum (Toyoshima, C., et al., Nature 405: 647-655, 2000). Four other mutations in M8 interfered with H(+)-ATPase folding and trafficking to the plasma membrane; based on homology modeling, they occupy positions that appear important for the proper bundling of M8 with M5, M6, M7, and M10. Taken together, these results point to a key role for M8 in the biogenesis, stability, and physiological functioning of the H(+)-ATPase.