Energy metabolism in human pluripotent stem cells and their differentiated counterparts

Background: Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells. Methodology/Principal Findings: We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism. Conclusions/Findings: Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH). © 2011 Varum et al

Choice of parents for dry bean (Phaseolus vulgaris L.) breeding. I. Interactions of mean components by generation and by location.

Four dry bean cultivars (Phaseolus vulgaris L.) adapted to the southern region of the state of Minas Gerais, Brazil, were crossed with five exotic cultivars according to a partial diallel design. The nine parents and twenty hybrid population, were tested in six experiment in fully randomized blocks. In one of the experiments, the segregation populations were in the F2 generation. Trials were held at two locations in the state of Minas Gerais and at one location in the state of Goias in 1986. All twenty nine-genetic materials showed heterogeneity concerning grain yield in all trials, with cultivars having a greater effect (additive gene effect) than heterosis (dominance effect), especially in the more advanced generations. No interactions between cultivar x generation effects and cultivar heterosis x generation effects were observed at any location. However, cultivar and cultivar heterosis x locations were significant, suggesting that the choice of segregating populations for genetic improvement of commons beans should be based on the performance of the populations at the locations where they will be used, but disregarding generation. In this way, because of larger quantities of seed, the material can be evaluated in the F4 generation. Despite components of means x location interactions, the population ESAL 501 x A 354 was the most promissing for selection based on grain yield because of the general combining ability of its parents and of grain quality

Selection of common bean segregating populations using genetic and phenotypic parameters and RAPD markers.

In an attempt to identify strategies for choosing common bean segregating populations, the diallel parameters, the contribution of homozygous (m+a?) and heterozygous (d) loci, the heritability (h2a), the genetic distance (RAPD), and the selection potential (Z) were estimated in a study carried out in two stages. In the first, 30 hybrid combinations (partial diallel) were assessed in the F2 and F3 generations. Increasing grain yield was due dominance effect (d) although, both GCA and SCA were significant. d varied greatly and gave an information like SCA, however h2a alone was not indicative of success in a breeding program. In the second stage, five populations with lesser d and five with higher were selected, and twenty-seven families were taken from each, which were assessed with the parents and seven controls. The estimated parameters were used to check those from the first stage. There was partial agreement among the estimates and the percentages of lines superior to Pérola cultivar, which suggests that two or more estimates are needed to identify the promising segregating populations

THE MOLECULAR MECHANISMS OF IRON AND FERRITIN METABOLISM IN NORMAL AND NEOPLASTIC CELLS

Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse

UVB irradiation as a tool to assess ROS-induced damage in human spermatozoa

One of the consequences of oxygen metabolism is the production of reactive oxygen species (ROS) which in a situation of imbalance with antioxidants can damage several biomolecules, compromise cell function and even lead to cellular death. The particularities of the sperm cell make it particularly vulnerable to ROS attack compromising its functionality, mirrored in terms of fertility outcome and making the study of the origin of sperm ROS, as well as the alterations they cause very important. In the present work, we used UVB irradiation, an easy experimental approach known as a potent inducer of ROS formation, to better understand the origin of ROS damage without any confounding effects that usually exist in disease models in which ROS are reported to play a role. To address these issues we evaluated sperm mitochondrial ROS production using the Mitosox Red Probe, mitochondrial membrane potential using the JC-1 probe, lipid peroxidation through BODIPY probe and vitality using PI. We observed that UVB irradiation leads to an increase in sperm mitochondrial ROS production and lipid peroxidation that occur previously to an observable mitochondrial dysfunction. We concluded that sperm UVB irradiation appears to be a good and easily manipulated in vitro model system to study mitochondria-induced oxidative stress in spermatozoa and its consequences, which may be relevant in terms of dissecting the action pathways of many other pathologies, drugs and contaminants, including endocrine disruptors.S.A. is the recipient of a FCT fellowship (SFRH/BPD/ 63190/2009). Centre for Neuroscience and Cell Biology (CNC) funding is supported by FCT (PEst-C/SAU/LA0001/2011)

Progresso genético após quatro ciclos de seleção recorrente no melhoramento do feijoeiro.

seleção recorrente na cultura do feijoeiro visando a obtenção de novas linhagens com grãos tipo carioca iniciou-se na Universidade Federal de Lavras (UFLA) em 1990. A cada ciclo, após a seleção para recombinação, a avaliação das melhores famílias é continuada, de modo que o processo é dinâmico gerando sempre novas linhagens. Nesse trabalho é estimado o progresso com a seleção recorrente, utilizando as cinco melhores linhagens identificadas em cada ciclo seletivo

Seleção recorrente no melhoramento do feijoeiro de grãos tipo carioca.

Está sendo conduzido um programa de seleção recorrente visando a obtenção de linhagens com grãos tipo carioca, de porte ereto e mais produtivas e resistentes à antracnose, mancha angular e fusário, que as disponíveis atualmente para as regiões Sul e Alto Paranaíba

Prediction of seed-yield potential of common bean populations.

Earliest possible prediction of seed-yield potential of autogamous crop populations increases breeding program efficiency by saving time and resources. Alternatives for obtaining seed-yield predictions were compared by evaluating four common-bean populations in F1 and F2 generations together with the parents. Mean components (m + a' and d) and variances were estimated. The potential of each population was predicted by using both these and the Jinks and Pooni (1976) procedure, which allows probability estimation of each population of originating lines surpassing a determined standard. Estimate efficiency was determined by evaluating performances of 62 F5:7 families from each population. Mean component m + a' estimates obtained for the F1 and F2 generations proved efficient in predicting seed yield of F7 generation lines as did d for estimate variance among F7 generation families. In addition, the Jinks and Pooni (1976) procedure proved efficient in early prediction of common bean population genetic potentials, especially when using the m + a' estimate

Role of a Transbilayer pH Gradient in the Membrane Fusion Activity of the Influenza Virus Hemagglutinin: Use of the R18 Assay to Monitor Membrane Merging

It had been suggested that influenza virus-mediated membrane fusion might be dependent on a pH gradient across a target membrane. We have designed experiments in which this issue could be addressed. Two populations of liposomes were prepared, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.4 (physiological cytosol pH) or at pH 5.0 (endosomal pH at which influenza virus displays maximal fusion activity). By monitoring fusion using the R18 assay, we found that the internal pH of the target liposomes did not influence membrane merging as mediated by the influenza virus hemagglutinin, thus demonstrating that a transmembrane pH gradient is not required in this fusion process