Efeito de indutores bióticos e abióticos na atividade de quitinase e peroxidase e no controle da ferrugem causada por Puccinia psidii em eucalipto

Verificou-se o efeito de indutores de resistência bióticos e abióticos nas atividades de quitinase e peroxidase e na redução da severidade da ferrugem do eucalipto causada por Puccinia psidii. Para isso, mudas de dois clones de eucalipto (Eucalyptus grandis x E. urophylla) denominados VR e C0, com sessenta dias de idade, mantidas em casa de vegetação, receberam tratamentos com Bion® (Acibenzolar-S-metil-ASM), Agro-Mos®, Dipel®, Ecolife40®, Crop-set® e uma preparação obtida a partir de Saccharomyces cerevisiae, 5 dias antes da inoculação com o patógeno. Uma suspensão de uredósporos de P. psidii, coletados a partir de plantas naturalmente infectadas, foi calibrada para 5 x 10(4) uredósporos/ mL. A inoculação foi realizada na face abaxial das folhas e a avaliação se deu 15 dias após, estimando-se a severidade da doença por meio de escala de notas. Os tratamentos ASM, preparado de S. cerevisiae e Ecolife® apresentaram os melhores resultados de controle da doença e os demais tratamentos não se mostraram eficazes para o controle. O aumento de atividade das enzimas quitinase e peroxidase foi observado em ambos os clones, previamente tratados com os indutores(ASM e S. cerevisiae), 48 horas após a inoculação com o fungo.

Effect of phytoplasma infection on the activity of peroxidase, β-1,3 glucanase and chitinase in corn plants

In the present work we studied the effect of inoculating corn plants with the maize bushy stunt phytoplasma on the activity of the enzymes peroxidase, β-1,3 glucanase and chitinase. The experiments were carried out inside a greenhouse. Plants of a resistant and a susceptible corn hybrid were inoculated by using infective Dalbulus maidis leafhoppers 10 days after sowing. When symptoms started to appear, leaf samples were collected at different periods to quantify enzyme activity. The results showed an increase in the activity of the three enzymes in inoculated plants of both hybrids. In general, the values observed for the level of the different enzymes were higher in the susceptible hybrid when compared to the resistant one. Thus, the increases in peroxidase, β-1,3 glucanase and chitinase levels in inoculated plants are evidence of changes in the host metabolism caused by the phytoplasma. On the other hand, since the increases could not be correlated with plant resistance further studies are needed to explain such changes.

Expression of P-glycoprotein, multidrug resistance-associated protein, glutathione-S-transferase pi and p53 in canine transmissible venereal tumor

The overexpression of proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1), mutant p53, and the enzyme glutathione-S-transferase (GSTpi) are related to resistance to chemotherapy in neoplasms. This study evaluated the expression of these markers by immunohistochemistry in two groups of canine TVT, without history of prior chemotherapy (TVT1, n=9) and in TVTs presented unsatisfactory clinical response to vincristine sulfate (TVT2, n=5). The percentage of specimens positively stained for P-gp, MRP1, GSTpi and p53 were, respectively 88.8%, 0%, 44.5% and 22.2% in TVT1 and 80%, 0%, 80% and 0% in TVT2. In TVT1, one specimen presented positive expression for three markers and four specimens for two markers. In TVT2, three specimens expressed P-gp and GSTpi. In conclusion, the canine TVTs studied expressed the four markers evaluated, but just P-gp and GSTpi were significantly expressed, mainly at cytoplasm and cytoplasm and nuclei, respectively, either before chemotherapy as after vincristine sulfate exposure. Future studies are needed to demonstrate the function of these two markers in conferring multidrug resistance (MDR) or predict the response to chemotherapy in canine TVT.

Efeito do cobre na atividade da enzima pirogalol peroxidase em plantas de Myriophyllum aquaticum cultivadas em solução nutritiva

Myriophyllum aquaticum é uma planta perene, herbácea, que pode se desenvolver totalmente submersa ou com a porção terminal dos ramos acima da superfície da água. É também considerada uma planta daninha que possui elevado potencial de colonização, o qual, dependendo da densidade populacional, pode causar aumento no teor de matéria orgânica e redução de oxigênio na água, comprometendo a qualidade da água e seus usos múltiplos. O objetivo do presente trabalho foi verificar a influência do cobre na atividade da pirogalol peroxidase de plantas de M. aquaticum submetidas à solução nutritiva contendo concentrações de cobre de 1,2; 11,2; 21,2; 31,2; e 41,2 µg L-1. O experimento foi conduzido em um delineamento experimental inteiramente casualizado, com quatro repetições e cinco tratamentos, aos quais as plantas foram submetidas durante 21 dias. Aos 81 dias após a instalação das mudas em solução nutritiva contendo os diferentes níveis de cobre, as folhas foram colhidas a partir do ápice da planta até o final do ramo, que não estavam em contato com a solução. Esse material fresco foi envolvido por plástico transparente e papel-alumínio e, a seguir, congelado em nitrogênio líquido, sendo armazenado em freezer a -20 ºC até o momento da determinação da atividade da enzima pirogalol peroxidase. A atividade da enzima foi progressiva com o aumento das doses de cobre. As plantas cultivadas com 40 µg L-1 de Cu2+ após três semanas, com base em avaliação visual, apresentaram redução no desenvolvimento.

Detection of adrenocortical autoantibodies in Addison's disease with a peroxidase-labelled protein A technique

Adrenocortical autoantibodies (ACA), present in 60-80% of patients with idiopathic Addison's disease, are conventionally detected by indirect immunofluorescence (IIF) on frozen sections of adrenal glands. The large-scale use of IIF is limited in part by the need for a fluorescence microscope and the fact that histological sections cannot be stored for long periods of time. To circumvent these restrictions we developed a novel peroxidase-labelled protein A (PLPA) technique for the detection of ACA in patients with Addison's disease and compared the results with those obtained with the classical IIF assay. We studied serum samples from 90 healthy control subjects and 22 patients with Addison's disease, who had been clinically classified into two groups: idiopathic (N = 13) and granulomatous (N = 9). ACA-PLPA were detected in 10/22 (45%) patients: 9/13 (69%) with the idiopathic form and 1/9 (11%) with the granulomatous form, whereas ACA-IIF were detected in 11/22 patients (50%): 10/13 (77%) with the idiopathic form and 1/9 (11%) with the granulomatous form. Twelve of the 13 idiopathic addisonians (92%) were positive for either ACA-PLPA or ACA-IIF, but only 7 were positive by both methods. In contrast, none of 90 healthy subjects was found to be positive for ACA. Thus, our study shows that the PLPA-based technique is useful, has technical advantages over the IIF method (by not requiring the use of a fluorescence microscope and by permitting section storage for long periods of time). However, since it is only 60% concordant with the ACA-IIF method, it should be considered complementary instead of an alternative method to IIF for the detection of ACA in human sera.

DL-methionine supplementation of rice-and-bean diets affects gamma-glutamyltranspeptidase activity and glutathione content in livers of growing rats

Gamma-glutamyltranspeptidase (GGT-EC 2.3.2.2) activity and glutathione (GSH) content were measured in livers of female weanling Wistar rats (N = 5-18), submitted to rice-and-bean diets (13 and 6% w/w protein), both supplemented or not with DL-methionine (0.5 and 0.23 g/100 g dry diet, respectively). After 28 days, the rats on the rice-and-bean diets showed significantly higher levels (four times higher) of liver GGT activity and a concomitant 50% lower concentration of liver GSH in comparison with control groups feeding on casein. The addition of DL-methionine to rice-and-bean diets significantly increased the liver GSH content, which reached levels 50% higher than those found in animals on casein diets. The increase in GSH was accompanied by a decrease in liver GGT activity, which did not reach levels as low as those observed in the control groups. No significant correlation could be established between GGT and GSH changes under the present experimental conditions. Linear correlation analysis only revealed that in animals submitted to unsupplemented rice-and-bean diets GSH concentration was positively associated (P<0.05) with weight gain, food intake and food efficiency. GGT, however, was negatively correlated (P<0.05) with food intake only, and exclusively for supplemented rice-and-bean diets. The high levels of GGT activity observed in the present study for rats receiving a rice-and-bean mixture could be a result of the poor quality of these diets associated with their deficiency in sulfur amino acids. The results also suggest that diet supplementation with methionine could be important in the reduction of the deleterious effects of GSH depletion by restoring the intracellular concentration of this tripeptide.

Thyroid peroxidase activity is inhibited by amino acids

Normal in vitro thyroid peroxidase (TPO) iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml) or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml). A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml) and some amino acids (cysteine, tryptophan and methionine, 50 µM each) also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml), and tyrosine, phenylalanine and histidine (50 µM each) inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml) or any other amino acid (50 µM) tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine) or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine). Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2) concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin

Changes in glutathione levels were determined in tissues of 11- to 12-week-old Swiss albino mice at different stages of Dalton's lymphoma tumor growth and following cisplatin (8 mg/kg body weight, ip) treatment for 24-96 h, keeping 4-5 animals in each experimental group. Glutathione levels increased in spleen of tumor-bearing compared to normal mice (9.95 ± 0.14 vs 7.86 ± 1.64 µmol/g wet weight, P<=0.05) but decreased in blood (0.64 ± 0.10 vs 0.85 ± 0.09 mg/ml) and testes (9.28 ± 0.15 vs 10.16 ± 0.28 µmol/g wet weight, P<=0.05). Dalton's lymphoma cells showed an increase in glutathione concentration (4.43 ± 0.26 µmol/g wet weight) as compared to splenocytes, their normal counterpart (3.62 ± 0.41 µmol/g wet weight). With the progression of tumor in mice, glutathione levels decreased significantly in testes (~10%) and bone marrow cells (~13%) while they increased in Dalton's lymphoma cells (28-46%) and spleen (15-27%). Glutathione levels in kidney, Dalton's lymphoma cells and bone marrow cells (8.50 ± 1.22, 4.43 ± 0.26 and 3.28 ± 0.17 µmol/g wet weight, respectively) decreased significantly (6.04 ± 0.42, 3.51 ± 0.32 and 2.17 ± 0.14 µmol/g wet weight, P<=0.05) after in vivo cisplatin treatment for 24 h. Along with a decrease in glutathione level, the glutathione-S-transferase (GST) activity also decreased by 60% in tumor cells after cisplatin treatment. The elevated drug uptake by the tumor cells under the conditions of reduced glutathione concentration and GST activity after treatment could be an important contributory factor to cisplatin's anticancer activity leading to tumor regression. Furthermore, lower doses of cisplatin in combination with buthionine sulfoximine (an inhibitor of glutathione synthesis) may be useful in cancer chemotherapy with decreased toxicity in the host.

Ethnicity and glutathione S-transferase (GSTM1/GSTT1) polymorphisms in a Brazilian population

The distribution of polymorphisms related to glutathione S-transferases (GST) has been described in different populations, mainly for white individuals. We evaluated the distribution of GST mu (GSTM1) and theta (GSTT1) genotypes in 594 individuals, by multiplex PCR-based methods, using amplification of the exon 7 of CYP1A1 gene as an internal control. In São Paulo, 233 whites, 87 mulattos, and 137 blacks, all healthy blood-donor volunteers, were tested. In Bahia, where black and mulatto populations are more numerous, 137 subjects were evaluated. The frequency of the GSTM1 null genotype was significantly higher among whites (55.4%) than among mulattos (41.4%; P = 0.03) and blacks (32.8%; P < 0.0001) from São Paulo, or Bahian subjects in general (35.7%; P = 0.0003). There was no statistically different distribution among any non-white groups. The distribution of GSTT1 null genotype among groups did not differ significantly. The agreement between self-reported and interviewer classification of skin color in the Bahian group was low. The interviewer classification indicated a gradient of distribution of the GSTM1 null genotype from whites (55.6%) to light mulattos (40.4%), dark mulattos (32.0%) and blacks (28.6%). However, any information about race or ethnicity should be considered with caution regarding the bias introduced by different data collection techniques, specially in countries where racial admixture is intense, and ethnic definition boundaries are loose. Because homozygous deletions of GST gene might be associated with cancer risk, a better understanding of chemical metabolizing gene distribution can contribute to risk assessment of humans exposed to environmental carcinogens.

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (~40%) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.

Association between dental-oral health in young adults and salivary glutathione, lipid peroxidation and sialic acid levels and carbonic anhydrase activity

The aim of the present study was to evaluate the relationship between salivary oxidative stress and dental-oral health. Healthy young adults, matched for gender and age, with (N = 21, 10 men, mean age: 20.3 ± 1 years) and without (N = 16, 8 men, mean age: 21.2 ± 1.8 years) caries were included in this study. The World Health Organization (WHO) caries diagnostic criteria were used for determining the decayed, missing, filled teeth (DMFT) index. The oral hygiene and gingival status were assessed using the simplified oral hygiene index and gingival index, respectively. Unstimulated salivary total protein, glutathione (GSH), lipid peroxidation and total sialic acid levels, carbonic anhydrase activity, and salivary buffering capacity were determined by standard methods. Furthermore, salivary pH was measured with pH paper and salivary flow rate was calculated. Simplified oral hygiene index and gingival index were not significantly different between groups but DMFT scores were significant (P < 0.01). Only, GSH values were significantly different (P < 0.05) between groups (2.2 and 1.6 mg/g protein in young adults without caries and with caries, respectively). There was a significant negative correlation between DMFT and GSH (r = -0.391; P < 0.05; Pearson's correlation coefficient). Our results suggest that there is an association between caries history and salivary GSH levels.

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8% inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

Preventive effect of reduced glutathione on contrast-induced nephropathy in elderly patients undergoing coronary angiography or intervention: a randomized, controlled trial

Coronary angiography can be a high-risk condition for the incidence of contrast-induced nephropathy (CIN) in elderly patients. Reduced glutathione, under a variety of mechanisms, may prevent CIN in this procedure. We prospectively examined whether hydration with reduced glutathione is superior to hydration alone for prevention of CIN in an elderly Han Chinese population. A total of 505 patients (271 males and 234 females) aged 75 years or older who underwent non-emergency coronary angiography or an intervention were randomly divided into two groups. The treatment group received hydration with reduced glutathione (n=262) and the control group received hydration alone (n=243). Serum creatinine and blood urea nitrogen levels were measured prior to coronary angiography and 48 h after this procedure. The primary endpoint was occurrence of CIN, which was defined as 25% or 44.2 µmol/L above baseline serum creatinine levels 48 h after the procedure. The overall incidence of CIN was 6.49% in the treatment group and 7.41% in the control group, with no significant difference between the groups (P=0.68). In subgroup analysis by percutaneous coronary intervention, no significant differences were found between the two groups. In summary, reduced glutathione added to optimal hydration does not further decrease the risk of CIN in elderly patients undergoing coronary angiography or an intervention.

EFEITO DO TRATAMENTO TÉRMICO SOBRE A ATIVIDADE DE PEROXIDASE (POD) E POLIFENOLOXIDASE (PPO) EM MAÇÃ (Mallus comunis)

O extrato enzimático foi preparado a partir da polpa e casca da maçã de cultivares Fuji e Gala utilizando tampão fosfato de sódio 100mM, pH 5,0 como solução extratora. Dentre as análises determinou-se a concentração de proteína nos extratos enzimáticos concentrados de polpa e casca, sendo que o cultivar Fuji apresentou teores mais elevados em comparação ao cultivar Gala. Os tratamentos térmicos foram realizados nas temperaturas de 60, 65, 70 e 75°C por períodos que variaram de 1 a 10 minutos, sendo observado diminuição da atividade de POD e PPO com o aumento da temperatura e tempo; no entanto a POD não chegou a ser inativada em nenhum dos tratamentos realizados. A PPO foi inativada totalmente após 10 minutos de tratamento a 75°C. A eletroforese mostrou uma composição diferente de isoenzimas aniônicas e catiônicas da peroxidase.

Características da atividade da peroxidase de abacaxis (Ananas comosus (L.) Merrill) da cultivar IAC Gomo-de-mel e do clone IAC-1

No presente trabalho foram estudadas as características bioquímicas das peroxidases de novos abacaxis, cultivar IAC Gomo-demel e clone IAC-1. As peroxidases dos sucos destes abacaxis apresentaram atividade ótima entre 45ºC e 50°C e entre 50°C e 55°C, respectivamente. Estas peroxidases apresentaram atividade ótima em pH 4,5 e mostraram-se estáveis na faixa de pH 4,0 a 9,0, retendo, após 24 horas de incubação a 50ºC, mais de 80% da atividade inicial. Foi observada regeneração parcial da atividade da peroxidase, após tratamento a 75ºC por 10 minutos. As peroxidases dos sucos dos abacaxis estudados foram inativadas, após tratamento a 90ºC por 2 minutos.