Metabolismo do malondialdeído em peixes: implicações na avaliação da peroxidação lipídica como biomarcador de contaminação aquática

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Metabolismo do malondialdeído em peixes: implicações na avaliação da peroxidação lipídica como biomarcador de contaminação aquática

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Underlying Factors Associated with Anemia in Amazonian Children: A Population-Based, Cross-Sectional Study

Background: Although iron deficiency is considered to be the main cause of anemia in children worldwide, other contributors to childhood anemia remain little studied in developing countries. We estimated the relative contributions of different factors to anemia in a population-based, cross-sectional survey. Methodology: We obtained venous blood samples from 1111 children aged 6 months to 10 years living in the frontier town of Acrelandia, northwest Brazil, to estimate the prevalence of anemia and iron deficiency by measuring hemoglobin, erythrocyte indices, ferritin, soluble transferrin receptor, and C-reactive protein concentrations. Children were simultaneously screened for vitamin A, vitamin B-12, and folate deficiencies; intestinal parasite infections; glucose-6-phosphate dehydrogenase deficiency; and sickle cell trait carriage. Multiple Poisson regression and adjusted prevalence ratios (aPR) were used to describe associations between anemia and the independent variables. Principal Findings: The prevalence of anemia, iron deficiency, and iron-deficiency anemia were 13.6%, 45.4%, and 10.3%, respectively. Children whose families were in the highest income quartile, compared with the lowest, had a lower risk of anemia (aPR, 0.60; 95% CI, 0.37-0.98). Child age (<24 months, 2.90; 2.01-4.20) and maternal parity (>2 pregnancies, 2.01; 1.40-2.87) were positively associated with anemia. Other associated correlates were iron deficiency (2.1; 1.4-3.0), vitamin B-12 (1.4; 1.0-2.2), and folate (2.0; 1.3-3.1) deficiencies, and C-reactive protein concentrations (>5 mg/L, 1.5; 1.1-2.2). Conclusions: Addressing morbidities and multiple nutritional deficiencies in children and mothers and improving the purchasing power of poorer families are potentially important interventions to reduce the burden of anemia.

Assessment of the sublethal toxicity of organochlorine pesticide endosulfan in juvenile common carp (Cyprinus carpio)

This study is aimed at evaluating the sublethal effects of endosulfan (EDS) in juvenile common carp (Cyprinus carpio). For this purpose, fish were exposed for 15 days to the technical EDS (95% pure) diluted in dimethyl sulfoxide (DMSO) 0.1% of the total volume in water solution in a semi-static system at sublethal concentration (1 mu g/L). Subsequently, the liver somatic index (LSI) and factor condition (K) were determined. The total cytocrome P450 (CYP), CYP1A isoform, and the ethoxyresorufin-O-deethylase (EROD) activity were determined from the hepatic microsomal fraction as well as the activity of the oxidative stress enzyme system such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GP(X)), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). Among the parameters assessed, EDS at the sublethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Thus, it is seen that the use of biochemical biomarkers of environmental contamination in this study proved to be an extremely important tool for detecting the adverse effects of xenobiotics in the aquatic environment, even at low concentration.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.

Polymorphisms, linkage and mapping of four enzyme loci in the fish genus Xiphophorus (Poeciliidae).

Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 to MPI region. No significant sex-specific differences in recombination were observed. This group (designated linkage group II) was shown to assort independently from the three loci of linkage group I (adenosine deaminase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) and from glyceraldehyde-3-phosphate dehydrogenase and two isocitrate dehydrogenase loci. Evidence for conservation of the linkage group, at least in part, in other vertebrate species is presented.

Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

Tools for mass screening of G6PD deficiency: validation of the WST8/1-methoxy-PMS enzymatic assay in Uganda.

BACKGROUND The distribution of the enzymopathy glucose-6-phosphate dehydrogenase (G6PD) deficiency is linked to areas of high malaria endemicity due to its association with protection from disease. G6PD deficiency is also identified as the cause of severe haemolysis following administration of the anti-malarial drug primaquine and further use of this drug will likely require identification of G6PD deficiency on a population level. Current conventional methods for G6PD screening have various disadvantages for field use. METHODS The WST8/1-methoxy PMS method, recently adapted for field use, was validated using a gold standard enzymatic assay (R&D Diagnostics Ltd ®) in a study involving 235 children under five years of age, who were recruited by random selection from a cohort study in Tororo, Uganda. Blood spots were collected by finger-prick onto filter paper at routine visits, and G6PD activity was determined by both tests. Performance of the WST8/1-methoxy PMS test under various temperature, light, and storage conditions was evaluated. RESULTS The WST8/1-methoxy PMS assay was found to have 72% sensitivity and 98% specificity when compared to the commercial enzymatic assay and the AUC was 0.904, suggesting good agreement. Misclassifications were at borderline values of G6PD activity between mild and normal levels, or related to outlier haemoglobin values (<8.0 gHb/dl or >14 gHb/dl) associated with ongoing anaemia or recent haemolytic crises. Although severe G6PD deficiency was not found in the area, the test enabled identification of low G6PD activity. The assay was found to be highly robust for field use; showing less light sensitivity, good performance over a wide temperature range, and good capacity for medium-to-long term storage. CONCLUSIONS The WST8/1-methoxy PMS assay was comparable to the currently used standard enzymatic test, and offers advantages in terms of cost, storage, portability and use in resource-limited settings. Such features make this test a potential key tool for deployment in the field for point of care assessment prior to primaquine administration in malaria-endemic areas. As with other G6PD tests, outlier haemoglobin levels may confound G6PD level estimation.

p73 regulates basal and starvation-induced liver metabolism in vivo

As a member of the p53 gene family, p73 regulates cell cycle arrest, apoptosis, neurogenesis, immunity and inflammation. Recently, p73 has been shown to transcriptionally regulate selective metabolic enzymes, such as cytochrome c oxidase subunit IV isoform 1, glucose 6-phosphate dehydrogenase and glutaminase-2, resulting in significant effects on metabolism, including hepatocellular lipid metabolism, glutathione homeostasis and the pentose phosphate pathway. In order to further investigate the metabolic effect of p73, here, we compared the global metabolic profile of livers from p73 knockout and wild-type mice under both control and starvation conditions. Our results show that the depletion of all p73 isoforms cause altered lysine metabolism and glycolysis, distinct patterns for glutathione synthesis and Krebs cycle, as well as an elevated pentose phosphate pathway and abnormal lipid accumulation. These results indicate that p73 regulates basal and starvation-induced fuel metabolism in the liver, a finding that is likely to be highly relevant for metabolism-associated disorders, such as diabetes and cancer.

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na(+)- or H(+)-linked myo-inositol transporters. While Na(+)-coupled myo-inositol transporters are found exclusively in the plasma membrane, H(+)-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H(+)-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.

Metabolic Regulation of mTOR Activation and Endoplasmic Reticulum Stress in the Heart

When subjected to increased workload, the heart responds metabolically by increasing its reliance on glucose and structurally by increasing the size of myocytes. Whether changes in metabolism regulate the structural remodeling process is unknown. A likely candidate for a link between metabolism and growth in the heart is the mammalian target of rapamycin (mTOR), which couples energy and nutrient metabolism to cell growth. Recently, sustained mTOR activation has also been implicated in the development of endoplasmic reticulum (ER) stress. We explored possible mechanisms by which acute metabolic changes in the hemodynamically stressed heart regulate mTOR activation, ER stress and cardiac function in the ex vivo isolated working rat heart. Doubling the heart’s workload acutely increased rates of glucose uptake beyond rates of glucose oxidation. The concomitant increase in glucose 6-phosphate (G6P) was associated with mTOR activation, endoplasmic reticulum (ER) stress and impaired contractile function. Both rapamycin and metformin restored glycolytic homeostasis, relieved ER stress and rescued contractile function. G6P and ER stress were also downregulated with mechanical unloading of failing human hearts. Taken together, the data support the hypothesis that metabolic remodeling precedes, triggers, and sustains structural remodeling of the heart and implicate a critical role for G6P in load-induced contractile dysfunction, mTOR activation and ER stress. In general terms, the intermediary metabolism of energy providing substrates provides signals for the onset and progression of hypertrophy and heart failure.

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation.

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10% of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentosephosphate pathway

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.

Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants1

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH4+ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O2 (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O2-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O2-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.