Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Bioassay for detection of transgenic soybean seeds tolerant to glyphosate

Glyphosate is a systemic, nonselective, postemergence herbicide that inhibits growth of both weeds and crop plants. Once inside the plant, glyphosate interferes with biosynthesis of aromatic amino acids phenylalanine, tyrosine, and tryptophan, by inhibiting the activity of 5enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme of the shikimate pathway. The objective of this work was to develop a simple, effective and inexpensible method for identification of transgenic soybean tolerant to glyphosate. This technique consisted in germinating soybean seeds in filter paper moistened with 100 to 200 muM of glyphosate. Transgenic soybean seeds tolerant to glyphosate germinated normally in this solution and, between 7 and 10 days, started to develop a primary root system. However non-transgenic seeds stopped primary root growth and emission of secondary roots.

Growth and energy metabolism of Nile tilapia juveniles fed glycerol

The objective of this work was to evaluate the effect of inclusion of dietary glycerol in replacement to starch on the growth and energy metabolism of Nile tilapia juveniles. The experiment was carried out in a completely randomized design with four treatments (0, 5, 10, and 15% purified glycerol) and six replicates. Pelleted, isonitrogenous, and isocaloric diets were provided for 60 days. Growth performance parameters and muscle glucose and protein concentrations were not affected by dietary glycerol levels. The treatment with 15% glycerol presented higher levels of muscle and liver triglycerides. A quadratic effect of treatments on muscle and liver triglyceride concentrations was observed. The treatment with 0% glycerol presented higher hepatic glucose levels than the one with 15%. Treatments did not differ for concentrations of liver protein, as well as of plasma glucose, triglycerides, and protein. Treatments with 10 and 15% glycerol showed higher activity of the glucose-6-phosphate-dehydrogenase enzyme than the treatment with 5%; however, there were no significant differences in the hepatic activities of the malic and glycerol kinase enzymes. A linear positive effect of treatments was observed on the activity of the glycerol kinase enzyme in liver. Levels of glycerol inclusion above 10% in the diet of Nile tilapia juveniles characterize it as a lipogenic nutrient.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Yeast vacuoles fragment in an asymmetrical two-phase process with distinct protein requirements.

Yeast vacuoles fragment and fuse in response to environmental conditions, such as changes in osmotic conditions or nutrient availability. Here we analyze osmotically induced vacuole fragmentation by time-lapse microscopy. Small fragmentation products originate directly from the large central vacuole. This happens by asymmetrical scission rather than by consecutive equal divisions. Fragmentation occurs in two distinct phases. Initially, vacuoles shrink and generate deep invaginations that leave behind tubular structures in their vicinity. Already this invagination requires the dynamin-like GTPase Vps1p and the vacuolar proton gradient. Invaginations are stabilized by phosphatidylinositol 3-phosphate (PI(3)P) produced by the phosphoinositide 3-kinase complex II. Subsequently, vesicles pinch off from the tips of the tubular structures in a polarized manner, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol-3,5-bisphosphate and the Fab1 complex. It is accelerated by the PI(3)P- and phosphatidylinositol 3,5-bisphosphate-binding protein Atg18p. Thus vacuoles fragment in two steps with distinct protein and lipid requirements.

Genome sequence of the Drosophila melanogaster male-killing Spiroplasma strain MSRO endosymbiont.

Spiroplasmas are helical and motile members of a cell wall-less eubacterial group called Mollicutes. Although all spiroplasmas are associated with arthropods, they exhibit great diversity with respect to both their modes of transmission and their effects on their hosts; ranging from horizontally transmitted pathogens and commensals to endosymbionts that are transmitted transovarially (i.e., from mother to offspring). Here we provide the first genome sequence, along with proteomic validation, of an endosymbiotic inherited Spiroplasma bacterium, the Spiroplasma poulsonii MSRO strain harbored by Drosophila melanogaster. Comparison of the genome content of S. poulsonii with that of horizontally transmitted spiroplasmas indicates that S. poulsonii has lost many metabolic pathways and transporters, demonstrating a high level of interdependence with its insect host. Consistent with genome analysis, experimental studies showed that S. poulsonii metabolizes glucose but not trehalose. Notably, trehalose is more abundant than glucose in Drosophila hemolymph, and the inability to metabolize trehalose may prevent S. poulsonii from overproliferating. Our study identifies putative virulence genes, notably, those for a chitinase, the H2O2-producing glycerol-3-phosphate oxidase, and enzymes involved in the synthesis of the eukaryote-toxic lipid cardiolipin. S. poulsonii also expresses on the cell membrane one functional adhesion-related protein and two divergent spiralin proteins that have been implicated in insect cell invasion in other spiroplasmas. These lipoproteins may be involved in the colonization of the Drosophila germ line, ensuring S. poulsonii vertical transmission. The S. poulsonii genome is a valuable resource to explore the mechanisms of male killing and symbiont-mediated protection, two cardinal features of many facultative endosymbionts. IMPORTANCE: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. These endosymbionts play key roles in their hosts' fitness, including protecting them against natural enemies and manipulating their reproduction in ways that increase the frequency of symbiont infection. Little is known about the molecular mechanisms that underlie these processes. Here, we provide the first genome draft of a vertically transmitted male-killing Spiroplasma bacterium, the S. poulsonii MSRO strain harbored by D. melanogaster. Analysis of the S. poulsonii genome was complemented by proteomics and ex vivo metabolic experiments. Our results indicate that S. poulsonii has reduced metabolic capabilities and expresses divergent membrane lipoproteins and potential virulence factors that likely participate in Spiroplasma-host interactions. This work fills a gap in our knowledge of insect endosymbionts and provides tools with which to decipher the interaction between Spiroplasma bacteria and their well-characterized host D. melanogaster, which is emerging as a model of endosymbiosis.

Determinação de glicerol livre e total em amostras de biodiesel por método enzimático com detecção colorimétrica

A method based on enzymatic activities was developed using three enzymes (glycerokinase, glycerol-3-phosphate oxidase and peroxidase) and colorimetric detection for the determination of glycerol in biodiesel. The enzymatic conversion of glycerol produces H2O2 that is eliminated by the action of peroxidase, an oxygen acceptor and 4- aminoantipirine, producing water and a colored compound, which was analyzed. This method showed good linear correlation coefficient (r = 0.9937) in the concentration range of 4.95 x 10-5 to 3.96 x 10-4% (w/w) and had experimental limits of detection and quantitation of 7.10 x 10-6 and 2.10 x 10-5% (w/w), respectively.

Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

Glyphosate as a tool to produce shikimic acid in plants

Oseltamivir phosphate is a potent viral inhibitor produced from shikimic acid extracted from seeds of Ilicium verum, the most important natural source. With the site of action 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), glyphosate is the only compound capable of inhibiting its activity with the consequent accumulation of shikimic acid in plants. Corn and soybean plants were sprayed with reduced rates of glyphosate (0.0 to 230.4 g a.i. ha¹) and shikimic acid content in the dry mass was determined by HPLC 3, 7 and 10 days after application. Results showed shikimic acid accumulation in dry mass with increases of up to 969% in corn and 33,000% on soybeans, with peak concentrations 3 days after treatment (DAT). Industrial feasibility for shikimic acid production, combined with favorable climatic conditions for growing corn and soybean in virtually all over Brazil, favor the use of reduced rates of glyphosate in shikimic acid biosynthesis, with potential for use as an inducer in exploration of alternative sources for production of oseltamivir phosphate with low environmental impact.

Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats

Follicle-stimulating hormone (FSH) and insulin regulate glycide metabolism in Sertoli cells, thus stimulating lactate production. These stimulatory effects of FSH and insulin do not require protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The present investigation was performed to characterize the hormonal control of lipid metabolism in Sertoli cells. The data indicate that FSH and insulin have a regulatory effect on lipid metabolism in Sertoli cells. After 8 h of preincubation with insulin (5 µg/ml), the activity of the enzyme ATP-citrate lyase in cultured Sertoli cells was increased from 0.19 to 0.34 nmol NAD+ formed µg protein-1 min-1. FSH (100 ng/ml) had no effect on this enzyme. Glycerol phosphate dehydrogenase activity was not affected by any of the hormones tested. When Sertoli cells from 19-day old rats were incubated with [1,2­14C]acetate for 90 or 360 min, the [14C] label was present predominantly in triglyceride and phospholipid fractions with minor amounts in other lipids. In Sertoli cells pretreated for 16 h with insulin and FSH, an increase in acetate incorporation into lipids was observed. Most of the label was in esterified lipids and this percentage increased with the time of treatment; this increase was remarkable in triglycerides of control cells (18.8% to 30.6%). Since Sertoli cell triglycerides participate in the control of spermatogenesis, the present data suggest that the hormonal control of lipid metabolism in Sertoli cells is important not only for maintaining the energy of the cell itself, but also for the control of the spermatogenesis process.

Changes in the TBARs content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenodemedullated rats

Thiobarbituric acid reactant substances (TBARs) content, and the activities of glucose-6-phosphate dehydrogenase (G6PDh), citrate synthase (CS), Cu/Zn- and Mn-superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) were measured in the lymphoid organs (thymus, spleen, and mesenteric lymph nodes (MLN)) and skeletal muscles (gastrocnemius and soleus) of adrenodemedullated (ADM) rats. The results were compared with those obtained for sham-operated rats. TBARs content was reduced by adrenodemedullation in the lymphoid organs (MLN (28%), thymus (40%) and spleen (42%)) and gastrocnemius muscle (67%). G6PDh activity was enhanced in the MLN (69%) and reduced in the spleen (28%) and soleus muscle (75%). CS activity was reduced in all tissues (MLN (75%), spleen (71%), gastrocnemius (61%) and soleus (43%)), except in the thymus which displayed an increment of 56%. Cu/Zn-SOD activity was increased in the MLN (126%), thymus (223%), spleen (80%) and gastrocnemius muscle (360%) and was reduced in the soleus muscle (31%). Mn-SOD activity was decreased in the MLN (67%) and spleen (26%) and increased in the thymus (142%), whereas catalase activity was reduced in the MLN (76%), thymus (54%) and soleus muscle (47%). It is particularly noteworthy that in ADM rats the activity of glutathione peroxidase was not detectable by the method used. These data are consistent with the possibility that epinephrine might play a role in the oxidative stress of the lymphoid organs. Whether this fact represents an important mechanism for the establishment of impaired immune function during stress remains to be elucidated.

The carotid body of the spontaneous insulin-dependent diabetic rat

The carotid bodies from adult spontaneous insulin-dependent diabetic rats (strain BB/S) were perfusion-fixed at normal arterial blood pressure with 3% phosphate-buffered glutaraldehyde and compared with the organs from control rats (strain BB/Sc) prepared in the same way. Serial 5-µm sections were cut, stained, and using an interactive image analysis system, were analysed to determine the volumes of the carotid body and its vascular and extravascular compartments. There was no evidence of systemic arterial disease in the carotid stem arteries in either group of animals, and the microvasculature of the organs appeared normal by light microscopy. The volume of the carotid body was unchanged 3 months after the onset of diabetes but was increased at 6 months. The total vascular volume of the organ was unchanged, but the volume of the small vessels (5-12 µm) was increased. In the control group the small vessels comprised 5% of the total volume of the carotid body, or about 44% of the vascular compartment. The percentage of small vessels increased at 3 months in the diabetic group, but had returned to normal at 6 months. The extravascular volume followed the same pattern as the total carotid body volume and so did not change appreciably when expressed as a percentage of the total volume of the organ. The increase in size of the carotid body in diabetic rats is due, therefore, to an augmented extravascular volume. In one diabetic specimen the carotid sinus nerve showed signs of diabetic neuropathy, axonal swelling and intramyelinic oedema. The clinical implications of these results are discussed.

Hydration, conformational states and kinetics of yeast hexokinase PII /

The kinetic study of the coupled enzymatic reaction involving monomeric yeast hexokinase PII (HK) and yeast glucose-6-phosphate dehydrogenase (G-6-PDH) yields a Michaelis constant of 0.15 ± 0.01 mM for D-glucose. At pH 8.7 HK is present in monomeric form. The addition of polyethylene glycol (PEG), to the reaction mixture increased the affinity of HK for glucose, independent ofMW of the PEG from 2000 to 10000. The osmotic stress exerted by PEG can be used to measure the change in number of water molecules that accompany enzyme conformational changes (Rand, et al., 1993). Results indicate that the G-6-PDH is not osmotically sensitive and thus, the change in the number of PEG-inaccessible water molecules (ANw) measured in the coupled reaction is only the difference between the glucose-bound and glucosefree conformations of HK. ANw ~ 450 with PEGs of MW > 2000 under conditions for both binding (Reid and Rand, 1997) and kinetic assays. The contribution water may play in the binding of ATP (Km = 0.24 + 0.02 mM) has also been examined. It was found that in this case ANw = (for osmotic pressures < 2.8x10* dynes/cm^), suggesting no additional numbers of waters are displaced when ATP binds to HK. Osmotic pressure experiments were also performed with dimeric HK. It was determined that both the monomeric and dimeric forms of HK give the same ANw under low pressures. If this large ANw is due to conformational flexibility, it would appear that the flexibility is not reduced upon dimerization ofthe enzyme.

Icewine fermentation by Saccharomyces cervisiae : fundamental stress responses and comparative fermentation dynamics

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999.5 B63 P54 2007

Rôle de l'estérification des acides gras dans la régulation de la sécrétion d'insuline et le stress métabolique induits par le glucose

Le diabète est une maladie chronique de l’homéostasie du glucose caractérisée par une hyperglycémie non contrôlée qui est le résultat d’une défaillance de la sécrétion d’insuline en combinaison ou non avec une altération de l’action de l’insuline. La surnutrition et le manque d’activité physique chez des individus qui ont des prédispositions génétiques donnent lieu à la résistance à l’insuline. Pendant cette période dite de compensation où la concentration d’acides gras plasmatiques est élevée, l’hyperinsulinémie compense pleinement pour la résistance à l’insuline des tissus cibles et la glycémie est normale. Le métabolisme du glucose par la cellule pancréatique bêta entraîne la sécrétion d’insuline. Selon le modèle classique de la sécrétion d’insuline induite par le glucose, l’augmentation du ratio ATP/ADP résultant de la glycolyse et de l’oxydation du glucose, induit la fermeture des canaux KATP-dépendant modifiant ainsi le potentiel membranaire suivi d’un influx de Ca2+. Cet influx de Ca2+ permet l’exocytose des granules de sécrétion contenant l’insuline. Plusieurs nutriments comme les acides gras sont capables de potentialiser la sécrétion d’insuline. Cependant, le modèle classique ne permet pas d’expliquer cette potentialisation de la sécrétion d’insuline par les acides gras. Pour expliquer l’effet potentialisateur des acides gras, notre laboratoire a proposé un modèle complémentaire où le malonyl-CoA dérivé du métabolisme anaplérotique du glucose inhibe la carnitine palmitoyltransférase-1, l’enzyme qui constitue l’étape limitante de l’oxydation des acides gras favorisant ainsi leur estérification et donc la formation de dérivés lipidiques signalétiques. Le modèle anaplérotique/lipidique de la sécrétion d'insuline induite par le glucose prédit que le malonyl-CoA dérivé du métabolisme du glucose inhibe la bêta-oxydation des acides gras et augmente la disponibilité des acyl-CoA ou des acides gras non-estérifiés. Les molécules lipidiques agissant comme facteurs de couplage du métabolisme des acides gras à l'exocytose d'insuline sont encore inconnus. Des travaux réalisés par notre laboratoire ont démontré qu’en augmentant la répartition des acides gras vers la bêta-oxydation, la sécrétion d’insuline induite par le glucose était réduite suggérant qu’un des dérivés de l’estérification des acides gras est important pour la potentialisation sur la sécrétion d’insuline. En effet, à des concentrations élevées de glucose, les acides gras peuvent être estérifiés d’abord en acide lysophosphatidique (LPA), en acide phosphatidique (PA) et en diacylglycérol (DAG) et subséquemment en triglycérides (TG). La présente étude a établi l’importance relative du processus d’estérification des acides gras dans la production de facteurs potentialisant la sécrétion d’insuline. Nous avions émis l’hypothèse que des molécules dérivées des processus d’estérification des acides gras (ex : l’acide lysophosphatidique (LPA) et le diacylglycerol (DAG)) agissent comme signaux métaboliques et sont responsables de la modulation de la sécrétion d’insuline en présence d’acides gras. Afin de vérifier celle-ci, nous avons modifié le niveau d’expression des enzymes clés contrôlant le processus d’estérification par des approches de biologie moléculaire afin de changer la répartition des acides gras dans la cellule bêta. L’expression des différents isoformes de la glycérol-3-phosphate acyltransférase (GPAT), qui catalyse la première étape d’estérification des acides gras a été augmenté et inhibé. Les effets de la modulation de l’expression des isoenzymes de GPAT sur les processus d’estérifications, sur la bêta-oxydation et sur la sécrétion d’insuline induite par le glucose ont été étudiés. Les différentes approches que nous avons utilisées ont changé les niveaux de DAG et de TG sans toutefois altérer la sécrétion d’insuline induite par le glucose. Ainsi, les résultats de cette étude n’ont pas associé de rôle pour l’estérification de novo des acides gras dans leur potentialisation de la sécrétion d’insuline. Cependant, l’estérification des acides gras fait partie intégrante d’un cycle de TG/acides gras avec sa contrepartie lipolytique. D’ailleurs, des études parallèles à la mienne menées par des collègues du laboratoire ont démontré un rôle pour la lipolyse et un cycle TG/acides gras dans la potentialisation de la sécrétion d’insuline par les acides gras. Parallèlement à nos études des mécanismes de la sécrétion d’insuline impliquant les acides gras, notre laboratoire s’intéresse aussi aux effets négatifs des acides gras sur la cellule bêta. La glucolipotoxicité, résultant d’une exposition chronique aux acides gras saturés en présence d’une concentration élevée de glucose, est d’un intérêt particulier vu la prépondérance de l’obésité. L’isoforme microsomal de GPAT a aussi utilisé comme outil moléculaire dans le contexte de la glucolipotoxicité afin d’étudier le rôle de la synthèse de novo de lipides complexes dans le contexte de décompensation où la fonction des cellules bêta diminue. La surexpression de l’isoforme microsomal de la GPAT, menant à l’augmentation de l’estérification des acides gras et à une diminution de la bêta-oxydation, nous permet de conclure que cette modification métabolique est instrumentale dans la glucolipotoxicité.