Phenotypic expression of homozygous hemoglobin S in relation to β-globin haplotypes, glutathione S-transferase polymorphisms and detoxification enzymes

Sickle cell anemia (SCA) shows a pathophysiology that involves multiple changes in sickle cell erythrocytes, vaso-occlusive episodes, hemolysis, activation of inflammatory mediators, endothelial cell dysfunction, and oxidative stress. These events complicate treatment and culminate in the development of manifestations such as anemia, pain crises and multiorgan dysfunction. The aim of this study was to evaluate, in SCA patients, oxidative stress and antioxidant capacity markers, correlating them to treatment with hydroxyurea (HU), β-globin haplotypes and glutathione S-transferase polymorphisms (GSTT1, GSTM1 and GSTP1), in comparison to a control group (CG). The study groups were composed of 48 individuals without hemoglobinopathies (CG), SCA patients treated with HU [AF (+HU), N = 13] and untreated SCA patients [AF (-HU), N = 15], after informed consent. The groups were analyzed using cytological, electrophoretic, chromatographic and molecular methods and information from medical records. The GSTM1 and GSTT1 polymorphisms were determined by multiplex PCR, while the GSTP1 polymorphism by PCR-RFLP. Biochemical parameters were measured using spectrophotometric methods [TBARS, TEAC and catalase (CAT) and GST activities] and a chromatographic method [glutathione (GSH)]. The fetal Hb (Hb F) levels observed in the SCA (+HU) group (10.9%) confirmed the already well-described pharmacological effect of HU, but the SCA (-HU) group also had high Hb F levels (6.1%), which may have been influenced by genetic factors not targeted in this study. We found a higher frequency of the Bantu haplotype (48.2%), followed by the Benin (32.1%) and also Cameroon haplotypes, rare in our population, and 19.7% of atypical haplotypes. The presence of Bantu haplotype was related to higher lipid peroxidation levels in patients, but also, it conferred a differential response to HU treatment, raising Hb F levels in 52.6% (P = 0.03). The protective effect of Hb F was confirmed, because the increase in their levels resulted in a 41.3% decrease in lipid peroxidation levels (r = -0.74, P = 0.0156). The genotypic frequency of the GST polymorphisms observed was similar to that of other studies in the Brazilian population, and its association with biochemical markers revealed a significant difference only for the GSTP1 polymorphism, where patients with genotype V/V showed higher GSH and TEAC levels (P = 0.04 and P = 0.03, respectively) compared to patients with genotype I/I. The TBARS levels were about five to eight times higher in the SCA (+HU) and SCA (-HU) groups, respectively, compared to controls, and HU produced a 35.2% decrease in lipid peroxidation levels in the SCA (+HU) group (P < 0.0001). Moreover, the SCA (+HU) group showed higher TEAC levels when compared to CG (P = 0.002). We did not find any significant difference in GST activity between the groups studied (P = 0.76), but CAT activity was about 17 and 30% lower in SCA (+HU) and SCA (-HU) groups, respectively (P < 0.00001). Plasma GSH levels were ~2 times higher in SCA patients than in the control group (P = 0.0005) and showed a positive correlation with TBARS levels, confirming its antioxidant function. HU treatment contributed to higher CAT activity and TEAC levels and lower lipid peroxidation, and its pharmacological effect showed a “haplotype-dependent” response. These findings may contribute to elucidating the potential of HU in ameliorating oxidative stress in SCA subjects.

Bromofhenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1 carbodiimide and 1 mol.L-1 ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1 and specific peroxidase activity was 86.06 ± 1.52 µmol min-1 . mg-1, using 1 mmol.L-1 ABTS as substrate. Ten mmol.L-1 and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1 aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1 bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1 and 50 mmol.L-1 derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability.

Tropical soils with high aluminum concentrations cause oxidative stress in two tomato genotypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as delta lesions were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. © 1991.

Cadmium stress antioxidant responses and root-to-shoot communication in grafted tomato plants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The triad indole-3-acetic acid ethyl ester/esterase/horseradish peroxidase as a new cytotoxic prodrug/enzyme combination

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peroxidase and polyphenol oxidase activity on the yield of grafted and ungrafted cucumber plants

The first experiment was carried out under green house and involved nine treatments: ‘Tsuyoi’ cucumber, ‘Shelper’ squash and ‘Green-stripped cushaw squash’ ungrafted plants and ‘Tsuyoi’ cucumber plants grafted onto ‘Shelper’ squash and ‘Green-stripped cushaw squash (lower, mid and upper region of the recommended and non-recommended rootstock, respectively). After grafting, plant tissue samples were collected 1, 4, 7, 10 and 13 days after grafting for analysis of peroxidase (EC 1.11.1.7) and polyphenol oxidase (EC 1.10.3.1) activity. In the second experiment, yield and number of marketable fruits were evaluated. The differences in peroxidase activity at the rootstock region and in polyphenol activity at the region between the scion and the rootstock seem to be determining factors for a successful grafting process, increasing the yield and the number of marketable fruits.

Changes in polyamine, total protein and total carbohydrate content and peroxidase activity during the lifetime of chrysanthemum faroe

The aim of the present work was to evaluate the changes in polyamine (PA) content, peroxidase (POX) activity and levels of total protein and total soluble carbohydrates throughout the lifetime of leaves and inflorescences of chrysantemum 'Faroe' treated with gibberellic acidd (GA3) (used in production practices) and kept at room temperature and cold storage. The treatments were composed of four doses of GA3 (0, 15, 30 and 45 mg L-1) applied at the beggining of flower bud formation (28 days after transplanting of seedlings). After harvesting, the stems (95% of the expanded ligule) were stored at 10ºC and 95% relative humidity for 48 hrs, or kept at room temperature. For biochemical analysis samples of leaves and inflorescences were collected at the 4th, 8th, 12th and 16th day after harvest. The application of GA3 in the field and cold storage increased the content of PAs. There was an increase in POX activity in leaves and inflorescences during postharvest and this increase was related to oxidation of the PAs studied. The amount of proteins and carbohydrates in chrysantemum 'Faroe' decreased during the storage at 25ºC and under cold conditions.

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Background: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 mu M) and PPADS (50 mu M) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 mu M), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 mu M) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 mu M), TNP-ATP (50 mu M) or the purinergic blockers KN-62 (10 mu M) and Ip5I (10 mu M). Incubating P. berghei infected cells with KN-62 (200 mu M) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 mu M) led to an increase in rings forms (82% +/- 4, n = 11) and a decrease in trophozoite forms (18% +/- 4, n = 11). Conclusions: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Evaluation of the Effects of a Preoperative 2-Hour Fast With Maltodextrine and Glutamine on Insulin Resistance, Acute-Phase Response, Nitrogen Balance, and Serum Glutathione After Laparoscopic Cholecystectomy: A Controlled Randomized Trial

Background: Prolonged preoperative fasting increases insulin resistance (IR). The authors investigated whether an abbreviated preoperative fast with glutamine (GLN) plus a carbohydrate (CHO)-based beverage would improve the organic response after surgery. Methods: Forty-eight female patients (19-62 years) were randomized to either standard fasting (control group) or to fasting with 1 of 3 different beverages before video-cholecystectomy. Beverages were consumed 8 hours (400 mL; placebo group: water; GLN group: water with 50 g maltodextrine plus 40 g GLN; and CHO group: water with 50 g maltodextrine) and 2 hours (200 mL; placebo: water; GLN: water with 25 g maltodextrine plus 10 g GLN; and CHO: water with 25 g maltodextrine) before anesthesia. Blood samples were collected pre- and postoperatively. Results: The mean (SEM) postoperative homeostasis model assessment-insulin resistance was greater (P < .05) in control patients (4.3 [1.3]) than in the other groups (placebo, 1.6 [0.3]; CHO, 2.3 [0.4]; and GLN, 1.5 [0.1]). Glutathione was significantly higher (P < .01) in the GLN group than in both CHO and control groups. Interleukin-6 increased in all groups except the GLN group. The C-reactive protein/albumin ratio was higher (P < .05) in controls than in CHO and GLN groups. The nitrogen balance was less negative in GLN (-2.5 [0.8] gN) than in both placebo (-9.0 [2] gN; P = .001) and control (-6.6 [0.4] gN; P = .04) groups. Conclusions Preoperative intake of a GLN-enriched CHO beverage appears to improve IR and antioxidant defenses and decreases the inflammatory response after video-cholecystectomy. (JPEN J Parenter Enteral Nutr. 2012; 36: 43-52)

Experimental hyperprolinemia induces mild oxidative stress, metabolic changes, and tissue adaptation in rat liver

The present study investigated the effects of chronic hyperprolinemia on oxidative and metabolic status in liver and serum of rats. Wistar rats received daily subcutaneous injections of proline from their 6th to 28th day of life. Twelve hours after the last injection the rats were sacrificed and liver and serum were collected. Results showed that hyperprolinemia induced a significant reduction in total antioxidant potential and thiobarbituric acid-reactive substances. The activities of the antioxidant enzymes catalase and superoxide dismutase were significantly increased after chronic proline administration, while glutathione (GSH) peroxidase activity, dichlorofluorescin oxidation, GSH, sulfhydryl, and carbonyl content remained unaltered. Histological analyses of the liver revealed that proline treatment induced changes of the hepatic microarchitecture and increased the number of inflammatory cells and the glycogen content. Biochemical determination also demonstrated an increase in glycogen concentration, as well as a higher synthesis of glycogen in liver of hyperprolinemic rats. Regarding to hepatic metabolism, it was observed an increase on glucose oxidation and a decrease on lipid synthesis from glucose. However, hepatic lipid content and serum glucose levels were not changed. Proline administration did not alter the aminotransferases activities and serum markers of hepatic injury. Our findings suggest that hyperprolinemia alters the liver homeostasis possibly by induction of a mild degree of oxidative stress and metabolic changes. The hepatic alterations caused by proline probably do not implicate in substantial hepatic tissue damage, but rather demonstrate a process of adaptation of this tissue to oxidative stress. However, the biological significance of these findings requires additional investigation. J. Cell. Biochem. 113: 174183, 2012. (C) 2011 Wiley Periodicals, Inc.

USE OF ADDITIVES IN THE WOOD BIODEGRADATION BY THE FUNGUS Ceriporiopsis subvermispora: EFFECT IN THE MANGANESE PEROXIDASE-DEPENDENT LIPID PEROXIDATION

USE OF ADDITIVES IN THE WOOD BIODEGRADATION BY THE FUNGUS Ceriporiopsis subvermispora: EFFECT IN THE MANGANESE PEROXIDASE-DEPENDENT LIPID PEROXIDATION. Ceriporiopsis subvermispora is a selective fungus in the wood delignification and the most promising in biopulping. Through the lipid peroxidation initiated by manganese peroxidase (MnP), free radicals can be generated, which can act in the degradation of lignin nonphenolic structures. This work evaluated the prooxidant activity (based in lipid peroxidation) of enzymatic extracts from wood biodegradation by this fungus in cultures containing exogenous calcium, oxalic acid or soybean oil. It was observed that MnP significant activity is required to promote lipid peroxidation and wood delignification. Positive correlation between prooxidant activity x MnP was observed up to 300 IU kg(-1) of wood.

Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1 mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5 mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

EVALUATION OF GLUTATHIONE S-TRANSFERASE GSTM1 AND GSTT1 POLYMORPHISMS AND METHYLMERCURY METABOLISM IN AN EXPOSED AMAZON POPULATION

Over the last decades, the presence of methylmercury (MeHg) in the Amazon region of Brazil and its adverse human health effects have given rise to much concern. The biotransformation of MeHg occurs mainly through glutathione (GSH) in the bile mediated by conjugation with glutathione S-transferases (GST). Epidemiological evidence has shown that genetic polymorphisms may affect the metabolism of MeHg. The aim of this study was to evaluate the association between GST polymorphisms, GSH, and Hg levels in blood (B-Hg) and in hair (H-Hg) of an Amazon population chronically exposed to the metal through fish consumption. Blood and hair samples were collected from 144 volunteers (71 men, 73 women). B-Hg and H-Hg levels were determined by inductively coupled plasma-mass spectrometry, and GSH levels were evaluated by a spectrophotometric method. GSTM1 and T1 genotyping evaluation were carried out by multiplex polymerase chain reaction (PCR). Mean levels of B-Hg and H-Hg were 37.7 +/- 24.5 mu g/L and 10.4 +/- 7.4 mu g/g, respectively; GSH concentrations ranged from 0.52 to 2.89 mu M/ml of total blood. Distributions for GSTM1/T1, GSTM1/GSTT1*0, GSTM1*0/T1, and GSTM1*0/GSTT1*0 genotypes were 35.4, 22.2, 25.0, and 17.4%, respectively. GSTT1 genotype carriers presented lower levels of B-Hg and H-Hg when compared to other genotypes carriers. In addition, GSTM1*0/GSTT1*0 individuals presented higher Hg levels in blood and hair than subjects presenting any other genotypes. There appeared to be no evidence of an effect of polymorphisms on GSH levels. Therefore, our data suggest that GST polymorphisms may be associated with MeHg detoxification.

Analysis of the Organic Hydroperoxide Response of Chromobacterium violaceum Reveals That OhrR Is a Cys-Based Redox Sensor Regulated by Thioredoxin

Organic hydroperoxides are oxidants generated during bacterial-host interactions. Here, we demonstrate that the peroxidase OhrA and its negative regulator OhrR comprise a major pathway for sensing and detoxifying organic hydroperoxides in the opportunistic pathogen Chromobacterium violaceum. Initially, we found that an ohrA mutant was hypersensitive to organic hydroperoxides and that it displayed a low efficiency for decomposing these molecules. Expression of ohrA and ohrR was specifically induced by organic hydroperoxides. These genes were expressed as monocistronic transcripts and also as a bicistronic ohrR-ohrA mRNA, generating the abundantly detected ohrA mRNA and the barely detected ohrR transcript. The bicistronic transcript appears to be processed. OhrR repressed both the ohrA and ohrR genes by binding directly to inverted repeat sequences within their promoters in a redox-dependent manner. Site-directed mutagenesis of each of the four OhrR cysteine residues indicated that the conserved Cys21 is critical to organic hydroperoxide sensing, whereas Cys126 is required for disulfide bond formation. Taken together, these phenotypic, genetic and biochemical data indicate that the response of C. violaceum to organic hydroperoxides is mediated by OhrA and OhrR. Finally, we demonstrated that oxidized OhrR, inactivated by intermolecular disulfide bond formation, is specifically regenerated via thiol-disulfide exchange by thioredoxin (but not other thiol reducing agents such as glutaredoxin, glutathione and lipoamide), providing a physiological reducing system for this thiol-based redox switch.