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Manoel E.A.,Federal University of Rio de Janeiro | dos Santos J.C.S.,ICP CSIC | dos Santos J.C.S.,Federal University of Ceara | Freire D.M.G.,Programa de Pos Graduacao em Bioquimica | And 2 more authors.
Enzyme and Microbial Technology | Year: 2015

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D- pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D- pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1. M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength. The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium. © 2015 Elsevier Inc.

Rodrigues J.A.G.,Programa de Pos graduacao em Biotecnologia | Vanderlei E.S.O.,Programa de Pos Graduacao em Bioquimica | Quindere A.L.G.,Programa de Pos Graduacao em Bioquimica | Fontes B.P.,Programa de Pos Graduacao em Bioquimica | And 2 more authors.
Acta Scientiarum - Biological Sciences | Year: 2011

The reportedly low standard quality of heparin (HEP) for use in cardiac surgeries has led to concern in the Brazilian and international markets. Sulfated polysaccharides (SPs) from seaweeds have been regarded as promising substitutes for HEP. The aim of this study was to sequentially extract total SPs (TSPs) from Caulerpa cupressoides (Chlorophyceae) with papain in 100 mM sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by fractionation by ion-exchange chromatography (DEAE-cellulose), and then evaluate the anticoagulant potential of SP fractions by activated partial thromboplastin time (APTT) using normal human plasma and compare it to standard HEP (193 IU mg-1). The obtained fractions were chemically characterized by chemical composition and agarose gel electrophoresis. The yield was 4.61%, and three fractions of SP (F I, F II and F III) eluted with 0.50, 0.75 and 1.00 M of NaCl, respectively, were observed on chromatography profiles; however, differences in charge densities patterns and degree of resolution among them were revealed by electrophoresis. SPs were capable of modifying APTT only in fractions eluted with 0.75 M of NaCl, whose activities were 23.37 and 25.76 IU mg-1, respectively, and the charge density was prerequisite to activity. Therefore, C. cupressoides is a source of SPs possessing low anticoagulant potential compared to HEP.

Rockenbach F.J.,Federal University of Rio Grande do Sul | Deon M.,Federal University of Rio Grande do Sul | Marchese D.P.,Servico de Genetica Medica | Manfredini V.,Programa de Pos Graduacao em Bioquimica | And 6 more authors.
Molecular Genetics and Metabolism | Year: 2012

Oxidative stress plays an important role in the pathophysiology of neurodegenerative diseases, including X-linked adrenoleukodystrophy (X-ALD). In the present work, we evaluated lipid (malondialdehyde [MDA] content) and protein (sulfhydryl and carbonyl contents) oxidative damage parameters in plasma from X-ALD patients before and after bone marrow transplant (BMT), in order to verify if this treatment is capable to alter the oxidative parameters studied. We also evaluated the plasma concentration of hexacosanoic acid (C26:0) from X-ALD patients and correlated it with the oxidative damage parameters investigated. We observed that MDA content was significantly increased in plasma of X-ALD patients before BMT and after BMT when compared to controls, and that it was significantly reduced in plasma of X-ALD after BMT when compared to the before BMT group. These results indicate that lipid peroxidation is stimulated in X-ALD patients but there is a significant reduction of lipid peroxidation after BMT. Next, we observed a significant reduction of sulfhydryl content in plasma of X-ALD patients before BMT compared to controls indicating protein oxidative damage and that this measurement was increased in these patients after BMT as compared to before BMT. We found no significant differences in plasma carbonyl content in X-ALD patients before and after BMT as compared to controls. However, we observed a significant reduction in this parameter in X-ALD patients after BMT compared to before BMT. Finally, C26:0 plasma concentration was significantly reduced in X-ALD patients after BMT when compared to before BMT. We found no significant correlations between MDA and carbonyl values with C26:0 levels of the patients before BMT and after BMT, but a significant inverse correlation between sulfhydryl content and C26:0 levels was detected. In conclusion, the present study reinforces the hypothesis that lipid peroxidation and protein damage are induced in plasma of X-ALD patients and, in addition, demonstrates that BMT treatment is capable to reduce this pathogenic process. Taken together, the data obtained from plasma of X-ALD patients before and after BMT showing induction and protection, respectively, of oxidative stress, allowed to suggest that BMT, when well succeeded and under the recommendations, is effective to reduce C26:0 plasma levels and the increased lipid and protein oxidative damage in X-ALD. © 2012 Elsevier Inc.

Busanello E.N.B.,Federal University of Rio Grande do Sul | Lobato V.G.A.,Federal University of Rio Grande do Sul | Zanatta A.,Federal University of Rio Grande do Sul | Borges C.G.,Federal University of Rio Grande do Sul | And 8 more authors.
Cerebellum | Year: 2014

Zellweger syndrome (ZS) and some peroxisomal diseases are severe inherited disorders mainly characterized by neurological symptoms and cerebellum abnormalities, whose pathogenesis is poorly understood. Biochemically, these diseases are mainly characterized by accumulation of pristanic acid (Prist) and other fatty acids in the brain and other tissues. In this work, we evaluated the in vitro influence of Prist on redox homeostasis by measuring lipid, protein, and DNA damage, as well as the antioxidant defenses and the activities of aconitase and α-ketoglutarate dehydrogenase in cerebellum of 30-day-old rats. The effect of Prist on DNA damage was also evaluated in blood of these animals. Some parameters were also evaluated in cerebellum from neonatal rats and in cerebellum neuronal cultures. Prist significantly increased malondialdehyde (MDA) levels and carbonyl formation and reduced sulfhydryl content and glutathione (GSH) concentrations in cerebellum of young rats. It also caused DNA strand damage in cerebellum and induced a high micronuclei frequency in blood. On the other hand, this fatty acid significantly reduced α-ketoglutarate dehydrogenase and aconitase activities in rat cerebellum. We also verified that Prist-induced increase of MDA levels was totally prevented by melatonin and attenuated by α-tocopherol but not by the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester, indicating the involvement of reactive oxygen species in this effect. Cerebellum from neonate rats also showed marked alterations of redox homeostasis, including an increase of MDA levels and a decrease of sulfhydryl content and GSH concentrations elicited by Prist. Finally, Prist provoked an increase of dichlorofluorescein (DCFH) oxidation in cerebellum-cultivated neurons. Our present data indicate that Prist compromises redox homeostasis in rat cerebellum and blood and inhibits critical enzymes of the citric acid cycle that are susceptible to free radical attack. The present findings may contribute to clarify the pathogenesis of the cerebellar alterations observed in patients affected by ZS and some peroxisomal disorders in which Prist is accumulated. © 2014, Springer Science+Business Media New York.

Wayhs C.A.Y.,Federal University of Rio Grande do Sul | Mescka C.P.,Servico de Genetica Medica | Mescka C.P.,Federal University of Rio Grande do Sul | Vanzin C.S.,Servico de Genetica Medica | And 8 more authors.
Metabolic Brain Disease | Year: 2013

Diabetes mellitus is characterized by hyperglycemia resulting from defects on insulin secretion, insulin action, or both. It has recently become clear that the central nervous system is not spared from the deleterious effects of diabetes, since diabetic encephalopathy was recognized as a complication of this heterogeneous metabolic disorder. There is a well recognized association between depression and diabetes, once prevalence of depression in diabetic patients is higher than in general population, and clonazepam is being used to treat this complication. Oxidative stress is widely accepted as playing a key mediatory role in the development and progression of diabetes and its complications. In this work we analyzed DNA damage by comet assay and lipid damage in prefrontal cortex, hippocampus and striatum of streptozotocin-induced diabetic rats submitted to the forced swimming test. It was verified that the diabetic group presented DNA and lipid damage in the brain areas evaluated, when compared to the control groups. Additionally, a significant reduction of the DNA and lipid damage in animals treated with insulin and/or clonazepam was observed. These data suggest that the association of these two drugs could protect against DNA and lipid damage in diabetic rats submitted to the forced swimming test, an animal model of depression. © 2013 Springer Science+Business Media New York.

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