Síntese e caracterização do poli(ácido láctico) para potencial uso em sistemas de liberação controlada de fármacos

With the advances in medicine, life expectancy of the world population has grown considerably in recent decades. Studies have been performed in order to maintain the quality of life through the development of new drugs and new surgical procedures. Biomaterials is an example of the researches to improve quality of life, and its use goes from the reconstruction of tissues and organs affected by diseases or other types of failure, to use in drug delivery system able to prolong the drug in the body and increase its bioavailability. Biopolymers are a class of biomaterials widely targeted by researchers since they have ideal properties for biomedical applications, such as high biocompatibility and biodegradability. Poly (lactic acid) (PLA) is a biopolymer used as a biomaterial and its monomer, lactic acid, is eliminated by the Krebs Cycle (citric acid cycle). It is possible to synthesize PLA through various synthesis routes, however, the direct polycondensation is cheaper due the use of few steps of polymerization. In this work we used experimental design (DOE) to produce PLAs with different molecular weight from the direct polycondensation of lactic acid, with characteristics suitable for use in drug delivery system (DDS). Through the experimental design it was noted that the time of esterification, in the direct polycondensation, is the most important stage to obtain a higher molecular weight. The Fourier Transform Infrared (FTIR) spectrograms obtained were equivalent to the PLAs available in the literature. Results of Differential Scanning Calorimetry (DSC) showed that all PLAs produced are semicrystalline with glass transition temperatures (Tgs) ranging between 36 - 48 °C, and melting temperatures (Tm) ranging from 117 to 130 °C. The PLAs molecular weight characterized from Size Exclusion Chromatography (SEC), varied from 1000 to 11,000 g/mol. PLAs obtained showed a fibrous morphology characterized by Scanning Electron Microscopy (SEM)

Calcium gluconate and sodium succinate for therapy of sodium fluoroacetate experimental intoxication in cats: clinical and electrocardiographic evaluation

Sodium fluoroacetate (SFAC) or Compound 1080 is a potent rodenticide, largely used after 1946 for rodent and home pest control. The toxic effects of SFAC are caused by fluorocitrate action, a toxic metabolite, which has a competitive action with aconitase enzyme, leading to citrate accumulation and resulting in interference in energy production by Krebs cycle blockade. In the present study, domestic cats were intoxicated with oral doses of fluoroacetate (0.45 mg/kg). The intoxicated animals presented emesis, diarrhea with abdominal pain posture and an abdominal palpation, tachypnea, bilateral midriasis, hypothermia, hyperexcitability and convulsions. Blood gas analysis indicated decreased pH and bicarbonate levels. Serum ionized calcium was also decreased. ECG showed non-specific changes in ventricular repolarization and ventricular arrhythmias. The survival rate was 75% in the treated group with calcium gluconate and sodium succinate and 37.5% in the non-treated group.

Hematological and biochemical profiles and histopathological evaluation of experimental intoxication by sodium fluoroacetate in cats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aspectos toxicológicos do bissulfito e outros agentes tóxicos sobre a síntese de glicerol-3-fosfato desidrogenase citoplasmá tica de levedura de panificação

The synthesis of intracellular glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) in baker's yeast was investigated in submerged culture supplied with glucose or glycerol as sole carbon sources. Inhibitors of the glycolytic pathway, Krebs cycle and respiratory chain did not stimulate glycerol-3-phosphate dehydrogenase synthesis when added in low concentrations in up 7.5 × 10 -5 mol/L. The repression exercised by glucose on the synthesis of glycerol-3-phosphate dehydrogenase in YP-glucose medium was reduced by the addition of fermentation products and of sodium bisulfite. Synthesis of the enzyme was raised 22-110%. However, in YP-glycerol medium, the addition of 0.06% (w/v) sodium bisulfite reduced (29%) the synthesis of the enzyme, while 0.012% (v/v) acetaldehyde stimulated the synthesis of glycerol-3-phosphate dehydrogenase by 12%.

Variabilidade clínica na determinação da dose tóxica oral em intoxicação experimental por fluoroacetato de sódio em gatos

The sodium monofluoroacetate (FAC) or compound 1080 is a potent rodenticide used for a rodents and vertebrate pest control. It was prohibited in many countries because of its high toxicity, but in Brazil exist evidences of ilegal use causing the intoxication in children and domestic animals. The fluoroacetate metabolite, fluorocitric acid, blocks body energy production by inhibit the Krebs cycle, resulting in neurological and cardiacs signs. In the present study, four group of oral toxic dosis of the FAC were compared in cats. The best oral toxic dose for clinical signs presentation, without cause acute lethality, was 0,45mg/kg. The clinical variability was dosis dependent and its intensity, in crescent order, was: light signs (dose 1: 0,3mg/kg), light to moderate (dose 2: 0,4mg/kg), moderate to severe (dose 3: 0,45mg/ kg) and severe (dose 4: 0,5mg/kg). There was individual clinical variability between animals that received the same oral toxic dose.

Perfil hematológico, bioquímico, histopatológico e toxicológico de gatos induzidos experimentalmente com monofluoroacetato de sódio

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of gamma-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas. Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4 mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. (C) 2012 Elsevier B.V. All rights reserved.

Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins

Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (similar to 2 % CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45 % of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.

Study of photorespiration in marine microalgae through the determination of glycolic acid using hydrophilic interaction liquid chromatography

Determination of organic acids in intracellular extracts and in the cultivation media of marine microalgae aid investigations about metabolic routes related to assimilation of atmospheric carbon by these organisms, which are known by their role in the carbon dioxide sink. The separation of these acids was investigated by hydrophilic interaction liquid chromatography (HILIC) using isocratic elution with a mobile phase composed of 70: 30 v/v acetonitrile/20 mmol/L ammonium acetate buffer (pH 6.8) and detection at 220 nm. HILIC allowed the determinations of glycolic acid, the most important metabolite for the evaluation of the photorespiration process in algae, to be made with better selectivity than that achieved by reversed phase liquid chromatography, but with less detectability. The concentration of glycolic acid was determined in the cultivation media and in intracellular extracts of the algae Tetraselmis gracilis and Phaeodactylum tricornutum submitted to different conditions of aeration: (i) without forced aeration, (ii) aeration with atmospheric air, and (iii) bubbling with N(2). The concentration of glycolic acid had a higher increase as the cultures were aerated with nitrogen, showing higher photorespiratory flux than that occurring in the cultures aerated with atmospheric air.

Planktonic potential CO2 emission calculation: preliminary results by applying an adapted enzymatic methodology to marine ecosystems

[EN]Isocitrate Dehydrogenase (IDH) is a key enzyme in the Krebs cycle, being responsible for the production of one of the three CO2 molecules related to cellular respiration. In order to measure the potential CO2 production linked to the marine planktonic community we have adapted an enzymatic methodology. Preliminary results show that different proportions of autotrophs, heterotrophs and mixotrophs and their metabolic pathways, lead to different relationships between potential CO2 emission and potential O2 consumption during cellular respiration. Although more experiments need to be made, this methodology is leading to a better understanding of cellular respiration in marine samples and their impact on the food chain, vertical Carbon flux and the current sequestering capacity for anthropogenic CO2.

Deficiency in SLC25A1, encoding the mitochondrial citrate carrier, causes combined D-2- and L-2-hydroxyglutaric aciduria

The Krebs cycle is of fundamental importance for the generation of the energetic and molecular needs of both prokaryotic and eukaryotic cells. Both enantiomers of metabolite 2-hydroxyglutarate are directly linked to this pivotal biochemical pathway and are found elevated not only in several cancers, but also in different variants of the neurometabolic disease 2-hydroxyglutaric aciduria. Recently we showed that cancer-associated IDH2 germline mutations cause one variant of 2-hydroxyglutaric aciduria. Complementary to these findings, we now report recessive mutations in SLC25A1, the mitochondrial citrate carrier, in 12 out of 12 individuals with combined D-2- and L-2-hydroxyglutaric aciduria. Impaired mitochondrial citrate efflux, demonstrated by stable isotope labeling experiments and the absence of SLC25A1 in fibroblasts harboring certain mutations, suggest that SLC25A1 deficiency is pathogenic. Our results identify defects in SLC25A1 as a cause of combined D-2- and L-2-hydroxyglutaric aciduria.

[Mitochondrial dysfunction during sepsis, impact and possible regulating role of hypoxia-inducible factor-1alpha]

There is a direct correlation between the development of the multiple organ dysfunction syndrome (MODS) and the elevated mortality associated with sepsis. The mechanisms responsible for MODS development are being studied, however, the main efforts regarding MODS evaluation have focused on oxygen delivery optimization and on the modulation of the characteristic inflammatory cascade of sepsis, all with negative results. Recent studies have shown that there is development of tissue acidosis, even when there are normal oxygen conditions and limited presence of tissue cellular necrosis or apoptosis, which would indicate that cellular energetic dysfunction may be a central element in MODS pathogenesis. Mitochondrias are the main source of cellular energy, central regulators of cell death and the main source for reactive oxygen species. Several mechanisms contribute to mitochondrial dysfunction during sepsis, that is blockage of pyruvate entry into the Krebs cycle, oxidative phosphorylation substrate use in other enzymatic complexes, enzymatic complex inhibition and membrane damage mediated by oxidative stress, and reduction in mitochondrial content. Hypoxia-inducible factor-1alpha (HIF-1alpha) is a nuclear transcription factor with a central role in the regulation of cellular oxygen homeostasis. Its induction under hypoxic conditions is associated to the expression of hundreds of genes that coordinate the optimization of cellular oxygen delivery and the cellular energy metabolism. HIF-1alpha can also be stabilized under normoxic condition during inflammation and this activation seems to be associated with a prominent pro-inflammatory profile, with lymphocytes dysfunction, and to a reduction in cellular oxygen consumption. Further studies should establish a role for HIF-1alpha as a therapeutic target.

Zinc transporters in prostate cancer

Prostate cancer is a major health concern as it has the second highest incidence rate among cancers in men. Despite progress in tumor diagnostics and therapeutic approaches, prognosis for men with advanced disease remains poor. In this review we provide insight into the changes of the intermediary metabolism in normal prostate and prostate cancer. In contrast to normal cells, prostate cancer cells are reprogrammed for optimal energy-efficiency with a functional Krebs cycle and minimal apoptosis rates. A key element in this relationship is the uniquely high zinc level of normal prostate epithelial cells. Zinc is transported by the SLC30 and SLC39 families of zinc transporters. However, in prostate cancer the intracellular zinc content is remarkably reduced and expression levels of certain zinc transporters are altered. Here, we summarize the role of different zinc transporters in the development of prostate cancer.

SLC13 family of Na⁺-coupled di- and tri-carboxylate/sulfate transporters

The SLC13 family comprises five genes (SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC13A5) encoding structurally related multi-spanning transporters (8-13 transmembrane domains) with orthologues found in prokaryotes and eukaryotes. Mammalian SLC13 members mediate the electrogenic Na(+)-coupled anion cotransport at the plasma membrane of epithelial cells (mainly kidney, small intestine, placenta and liver) or cells of the central nervous system. While the two SLC13 cotransporters NaS1 (SLC13A1) and NaS2 (SLC13A4) transport anions such sulfate, selenate and thiosulfate, the three other SLC13 members, NaDC1 (SLC13A2), NaCT (SLC13A5) and NaDC3 (SLC13A3), transport di- and tri-carboxylate Krebs cycle intermediates such as succinate, citrate and α-ketoglutarate. All these transporters play a variety of physiological and pathophysiological roles in the different organs. Thus, the purpose of this review is to summarize the roles of SLC13 members in human physiology and pathophysiology and what the therapeutic perspectives are. We have also described the most recent advances on the structure, expression, function and regulation of SLC13 transporters.

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.