Oxidative effects of the human antifungal drug clotrimazole on the eucaryotic model organism Saccharomyces cerevisiae.

Clotrimazole is a type of antifungal medication developed from azole compounds. It exhibits several biological actions linked to oxidative stress. This study focuses on the oxidative effects of clotrimazole on the eukaryotic model yeast, Saccharomyces cerevisiae. Our results showed that although initial nitric oxide levels were above control in clotrimazole exposed cells, they showed decreasing tendencies from the beginning of incubation and dropped below control at 125 µM from the 60th min. The highest superoxide anion and hydrogen peroxide levels were 1.95- and 2.85-folds of controls at 125 µM after 15 and 60 min, respectively. Hydroxyl radical levels slightly increased throughout the incubation period in all concentrations and reached 1.3-fold of control, similarly at 110 and 125 µM in the 90th min. The highest level of reactive oxygen species was observed at 110 µM, 2.31-fold of control. Although NADH/NADPH oxidase activities showed similar tendencies for all conditions, the highest activities were found as 3.07- and 2.27-folds of control at 125 and 110 µM in the 15th and 30th min, respectively. The highest superoxide dismutase and catalase activities were 1.59- and 1.21-folds of controls at 110 µM clotrimazole in 30 and 90 min, respectively. While the drug generally induced glutathione-related enzyme activities, the ratios of glutathione to oxidized glutathione were above the control only at low concentrations of the drug. The levels of lipid peroxidation in all treated cells were significantly higher than the controls. The findings crucially demonstrate that this medicine can generate serious oxidative stress in organisms.

Chloramine T induced oxidative stress and the response of antioxidant system in Phanerochaete chrysosporium.

In this study, the effect of chloramine T (Chl-T) on the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and glutathione S-transferase (GST); the levels of reduced (GSH) and oxidised glutathione (GSSG) and their ratios; and also membrane lipid peroxidation (LPO) levels in Phanerochaete chrysosporium were investigated in a dose- (0.25-1 mmol/L) and time-dependent (1.5-9 h) manner. The highest SOD activity was observed in 0.5 mmol/L Chl-T at 6th hour as 1.48-fold of its control. The observed highest level in CAT activities was 4.6-fold of control in 0.5 and 0.75 mmol/L at the 6th hour. The GSH levels that were over the control showed decreasing tendency from the beginning of incubation, except 0.25 mmol/L. In contrast with GSH level variations, GSSG levels reached 10.0-fold of its control by showing increasing tendency with the increases in concentration and time. While the GSH/GSSG ratios were over the control at 0.25 mmol/L during all incubation, they fell under the control values at the earlier hours of incubation with the increasing concentrations of Chl-T. Glutathione-related enzymes GSH-Px, GR and GST were also induced with Chl-T treatment, and the highest activities were 3.29-, 7.5- and 6.56-fold of their controls, respectively. On the other hand, the increases in LPO levels with increasing concentration and time up to 5.27-fold of its control showed that the inductions observed in antioxidant system could not prevent the Chl-T-based oxidative stress.

Yeast caspase-dependent apoptosis in Saccharomyces cerevisiae BY4742 induced by antifungal and potential antitumor agent clotrimazole.

Clotrimazole is an antifungal medication commonly used in the treatment of fungal infections. There is also promising research on using clotrimazole against other diseases such as malaria, beriberi, tineapedis and cancer. It was aimed to investigate the apoptotic phenotype in Saccharomyces cerevisiae induced by clotrimazole. The exposure of S. cerevisiae to 10 µM clotrimazole for 3, 6 and 9 h caused to decrease in cell viability by 24.82 ± 0.81, 56.00 ± 1.54 and 77.59 ± 0.53%, respectively. It was shown by Annexin V-PI assay that 110 µM clotrimazole treatment caused to death by 35.5 ± 2.48% apoptotic and only 13.1 ± 0.08% necrotic pathway within 30 min. The occurrence of DNA strand breaks and condensation could be visualised by the TUNEL and DAPI stainings, respectively. Yeast caspase activity was induced 12.34 ± 0.71-fold after 110 µM clotrimazole treatment for 30 min compared to the control. The dependency of clotrimazole-induced apoptosis to caspase was also shown using Δyca1 mutant.

Aqueous two-phase system purification for superoxide dismutase induced by menadione from Phanerochaete chrysosporium.

In the present work, the partitioning behavior of menadione-induced superoxide dismutase (SOD; EC 1.15.1.1), an antioxidant enzyme that has various applications in the medical and cosmetic industries, from the white rot fungus Phanerochaete chrysosporium has been characterized on different types of aqueous two-phase systems (ATPSs) (poly(ethylene glycol)/polypropylene glycol (PEG/PPG)-dextran, PEG-salt and PPG-salt). PEG-salt combinations were found most optimal systems for the purification of SOD. The best partition conditions were found using the PEG-3350 24% and K2HPO4 5% (w/w) with pH 7.0 at 25 °C. The partition coefficient of total SOD activity and total protein concentration observed in this system were 0.17 and 6.65, respectively, with the recovery percentage as 78.90% in the bottom phase and 13.17% in the top phase. The highest purification fold for SOD from P. chrysosporium was found as 6.04 in the bottom phase of PEG 3350%24 - K2HPO4%5 (w/w) system with pH 7.0. SOD purified from P. chrysosporium was determined to be a homodimer in its native state with a molecular weight of 60 ± 4 kDa. Consequently, simple and only one step PEG-salt ATPS system was developed for SOD purification from P. chrysosporium.

The Induction of Growth Inhibition and Apoptosis in HeLa and MCF-7 Cells by Teucrium sandrasicum, Having Effective Antioxidant Properties.

The hidromethanolic (Met/W), ethyl acetate (EA(EA/W)), and water (W(EA/W)) extracts from Teucrium sandrasicum leaves (L) and flowers (F) were investigated for antioxidant properties and antiproliferative effects on HeLa, MCF-7, and L929. The highest DPPH scavenging, metal chelating capacities, and total phenolic and flavonoid contents were observed in Met/WL. The highest hydroxyl scavenging and reducing power capacities were found in EA(EA/W)L. Met/WL, EA(EA/W)L and EA(EA/W)F inhibited cancer cell growths, while they did not show significant cytotoxicity on L929. While the reactive oxygen species (ROS) levels were generally close to controls in HeLa, they were induced in MCF-7 with the treatment of Met/WL, EA(EA/W)L, and EA(EA/W)F and acted as antioxidant for L929. The highest apoptosis inductions were observed in Met/WL-treated HeLa and EA(EA/W)L-treated MCF-7, which were supported with the changes in mitochondrial membrane potentials. The highest caspase-9 activities were found in Met/WL-treated HeLa and EA(EA/W)F-treated MCF-7. Caspase-3 activity was only induced in EA(EA/W)F-treated HeLa.

In vitro and in vivo inhibitory effects of some fungicides on catalase produced and purified from white-rot fungus Phanerochaete chrysosporium.

In this study, in vitro and in vivo effects of some commonly used fungicides, antibiotics, and various chemicals on isolated and purified catalase from Phanerochaete chrysosporium were investigated. The catalase was purified 129.10-fold by using 60% ammonium sulfate and 60% ethanol precipitations, DEAE-cellulose anion exchange and Sephacryl-S-200 gel filtration chromatographies from P. chrysosporium growth in carbon- and nitrogen-limited medium for 12 days. The molecular weight of native purified catalase from P. chrysosporium was found to be 290 ± 10 kDa, and sodium dodecyl sulfate (SDS)-PAGE results indicated that enzyme consisted of four apparently identical subunits, with a molecular weight of 72.5 ± 2.5 kDa. Kinetic characterization studies showed that optimum pH and temperature, Km and Vmax values of the purified catalase which were stable in basic region and at comparatively high temperatures were 7.5, 30°C, 289.86 mM, and 250,000 U/mg, respectively. The activity of purified catalase from P. chrysosporium was significantly inhibited by dithiothreitol (DTT), 2-mercaptoethanol, iodoacetamide, EDTA, and sodium dodecyl sulfate (SDS). It was found that while antibiotics had no inhibitory effects, 45 ppm benomyl, 144 ppm captan, and 47.5 ppm chlorothalonil caused 14.52, 10.82, and 38.86% inhibition of purified catalase, respectively. The inhibition types of these three fungicides were found to be non-competitive inhibition with the Ki values of 1.158, 0.638, and 0.145 mM and IC50 values of 0.573, 0.158, 0.010 mM, respectively. The results of in vivo experiments also showed that benomyl, captan and chlorothalonil caused 15.25, 1.96, and 36.70% activity decreases after 24-h treatments compared to that of the control.

The glutathione-related detoxication responses to juvenile and ecdysone hormones in Galleria mellonella.

The effect of 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the glutathione pathway of the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) was determined by investigating glutathione peroxidase (GSH-Px), glutathione S-transferases (GST), and glutathione reductase (GR) activities as well as reduced and oxidized glutathione (GSH and GSSG) content with respect to developmental stage. The continuous decreases of GSH-Px and GST activities dependent on the growth period of G. mellonella occurred in JH and 20E groups over and under their controls, respectively. While the GR activities of G. mellonella showed increases in young pupa (YP) for both control and in old larvae (OL) for the 20E groups after the minimum at these periods, they also increased after old pupa (OP) for the JH group with a maximum in OL period. Although GR activity levels in the JH group were significantly higher compared with controls and 20E groups up to OP period, the activity levels for the control and 20E groups were higher than those of the JH group at adult (AD) and old pupa (OP) periods, respectively. In spite of increases in the GR activity of 20E and control groups of G. mellonella, decreased GSH and increased GSSG levels were observed at aging period. GSH levels in the JH group reached a maximum at prepupa (PP) and then decreased with non-significant changes from OL to AD period. According to the results, GSH and GSSG levels, as well as GSH/GSSG ratios, were below and over control levels in 20E and JH groups, respectively, during all of the investigated developmental stages. On the contrary, the LPO levels were higher than the control for 20E and lower for the JH groups during the developmental period. These results show that while ecdysone hormone has a negative effect on the glutathione-related detoxication capacity of G. mellonella, the juvenile hormone has a positive effect on this process.

The variation of antioxidant defense system of Streptomyces sp. M4018 with respect to carbon sources.

The effect of glycerol, glucose, and starch as carbon sources on the antioxidant defense system such as superoxide dismutase (SOD) and catalase (CAT) activities, pyruvate levels, and membrane lipid peroxidation (LPO) levels of Streptomyces sp. M4018, after isolation from the rhizosphere samples of Colutea arborescens and identification as a strain of S. hiroshimensis based on phenotypic and genotypic characteristics, were investigated. As an antioxidant defense enzyme, SOD activities increased up to 20 g/L of glycerol and 15 g/L of starch, while they showed negative correlation with glucose concentration. CAT activity variations of glycerol- and glucose-supplemented mediums showed significant positive correlations with the trend of SOD activities. However, CAT activity, in contrast to SOD, in Streptomyces sp. M4018 tended to decrease as the starch concentration increased. The production of pyruvate increased with respect to glycerol and starch up to 15 g/L, while it was positively correlated with glucose concentration. The highest pyruvate production was seen at 20 g/L glucose. Membrane LPO levels were negatively correlated with the activities of SOD and CAT enzymes, and the minimum LPO level was determined at 5 g/L of glucose, where SOD and CAT activities reached their maximum levels. Nevertheless, the higher SOD and CAT activities in a wider range of incubation period compared to the beginning by resulting in insignificant increases in membrane LPO levels showed the unusual antioxidant response capacities of the in Streptomyces sp. M4018 against the potentially deleterious effects of reactive oxygen species (ROS) for glycerol, glucose, and starch as carbon sources.

Carbohydrate metabolite pathways and antibiotic production variations of a novel Streptomyces sp. M3004 depending on the concentrations of carbon sources.

To determine the variations of growth, some key enzyme activities such as glucose kinase (GK), glucose-6-phosphate dehydrogenase (G6PDH), α-ketoglutarate dehydrogenase (KGDH), and isocitrate lyase (ICL) besides metabolite levels of pyruvate and antibiotic production of newly isolated Streptomyces sp. M3004 were grown in culture media which contain 10-20 g/l concentration with either glucose or glycerol as carbon source. Biomass and intracellular glucose and glycerol levels of Streptomyces sp. M3004 showed positive correlation with the concentration of these carbon sources, and these levels were higher in glucose compared with the glycerol-supplemented mediums. GK, G6PDH, and KGDH activities showed marked correlation with the concentration of both glucose and glycerol, and the activity levels were 4.14-, 1.47-, and 1.27-fold higher in glucose than glycerol. A key enzyme of the glyoxalate cycle, ICL activities decreased with increasing glucose concentrations from 10 to 20 g/l, but increased up to 15 g/l of glycerol. The positive correlations were also determined between intracellular glucose and glycerol levels besides pyruvate and protein variations with respect to concentrations of the carbon sources. Antibacterial activities of Streptomyces sp. M3004 reached maximum on the stationary phase, while it did not change significantly with respect to glucose and glycerol.

Immobilization of phenylalanine dehydrogenase and its application in flow-injection analysis system for determination of plasma phenylalanine.

Phenylalanine dehydrogenase (L-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized L-PheDH was shifted from pH 10.4 to 11.0. The immobilized L-PheDH showed activity variations close to the maximum value in a wider temperature range of 45-55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized L-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K (m) values of the native and the immobilized L-PheDH were determined as K(m Phe) = 0.118, 0.063 mM and K(m NAD)(+) = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5-600 µM Phe at 9 mM NAD(+) with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.

The correlations between TCA-glyoxalate metabolite and antibiotic production of Streptomyces sp. M4018 grown in glycerol, glucose, and starch mediums.

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5-20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.

Balance between aerobic and anaerobic metabolites production of Amycolatopsis orientalis depending on initial glucose concentration.

The effect of glucose concentration as a carbon source in the range of 5-20 g/L on the fermentative productions of intra-and extra-cellular ethanol, acetate, formate, oxalate, lactate, and pyruvate, as well as pyruvate decarboxylase in A. orientalis were investigated, depending on the incubation period. Intra-and extra-cellular pyruvate levels increased with rising glucose concentrations up to 15 and 20 g/L of glucose, respectively. In addition, intra-cellular pyruvate levels reached their maximum on the 48th hour in the range of 12.5-20 g/L of glucose, except for 5 and 10 g/L while extra-cellular pyruvate were at the 48th and 60th hours. As a fermentative end product, intra-and extra-cellular ethanol levels increased with increasing glucose concentrations of the growth medium and with incubation period. Activity of pyruvate decarboxylase, one of the key enzymes of the alcoholic fermentation, increased significantly with increasing glucose concentrations up to the 48th hour. Intra-and extra-cellular acetate levels increased significantly with increasing glucose concentrations of the growth medium and reached their maximums on the 48th hour, as was the case also for pyruvate. Intra-cellular formate levels increased up to 15 g/L, while extra-cellular levels increased with increasing glucose concentration. The maximum intra-and extra-cellular lactate levels were determined at 12.5 g/L and 20 g/L of glucose on the 48th hour, respectively. The results suggest that elevated ethanol production suppressed lactate and formate production, supported via possibly formed CO(2). In addition, pyruvate, as well as acetate, were used as carbon sources due to the depletion of glucose contents in the growth medium.

In vitro antioxidant properties of Cucurbita pepo L. male and female flowers extracts.

Total antioxidant capacities, 2,2-diphenyl-1-picrylhydrazyl (DPPH.), hydroxyl (HO.), scavenging activities, and total phenolic values were determined in extracts of Cucurbita pepo L. female and male flowers. Powdered C. pepo L. samples were extracted in aqueous ethyl acetate (EA: W1, 17:3), ethanol (E), and water (W) by agitating in magnetic stirrer for 80 degrees C, 15 min and also by in aqueous ethyl acetate (EA: W2, 17:3) at 25 degrees C, 15 min. DPPH., HO. scavenging capacities and total phenolic values of C. pepo L. female and male were higher in EA:W2 than in other extracts. In addition, all determined antioxidant capacities of female were significantly higher than male.

Functions of antioxidant enzyme activities on the membrane bound total sialic acid and lipid peroxidation level in F. equiseti and F. acuminatum.

The variations of membrane bound total sialic acid (TSA) and lipid peroxidation level dependent on the antioxidant enzyme activities such as Superoxide Dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GSH-Px) have been studied in yeast extract supplemented medium. The maximum SOD and CAT activities of F. equiseti tended to increase with raises of yeast extract concentration up to 25 g/L where they were determined to be 78.6 +/- 0.96 and 312.7 +/- 5.6 IU/mg. On the other hand, SOD and CAT activities in F. acuminatum significantly increased with the rise of yeast extract concentration up to 10 g/L (p < 0.01) and maximum activities were observed at this concentration as 36.3 +/- 0.54 and 115.3 +/- 2.19 IU/mg on the 12th day incubation. Other H2O2 scavenger enzyme, GSH-Px activities of F. equiseti and F. acuminatum were reached the maximum at 5 and 25g/L yeast extract and determined as 5.06 +/- 0.04 and 4.74 +/- 0.09 IU/mg, respectively. TSA level showed positive correlation with SOD and CAT activities while LPO levels variations negatively correlated. The results may indicate that these antioxidant enzymes also appeared to be involved in protecting membrane bound sialic acids as well as membrane lipid of the fungus from exogenous reactive oxygen species.

A comparative study of the metal ion uptake and antioxidant enzyme activities of Fusarium equiseti and Fusarium acuminatum as a function of external magnesium concentration.

The increase of Mg2+, from 1.3 to 3 microM, in growth medium of F. equiseti and F. acuminatum increased intracellular magnesium levels from 0.83 and 0.81 microM to 1.75 and 1.42 microM on the 12th day, respectively. Intracellular magnesium levels also elevated depending upon the number of incubation days. The maximum manganese levels of F. equiseti and F. acuminatum obtained in 1.6 microM Mg2+ culture medium were 0.67 and 1.23 microM, while maximum iron levels were determined to be 1.3 microM Mg2+ as 0.51 and 0.29 microM, respectively. The maximum intracellular iron and manganese levels were decreased significantly with increasing Mg2+ concentration in the culture medium and were increased depending upon the incubation period. However, intracellular zinc levels of these strains didn't change with Mg2+ concentration and incubation period. The maximum superoxide dismutase (MnSOD) activities of F. equiseti and F. acuminatum, related to increased intracellular manganese levels up to 1.6 microM Mg2+ in growth medium, were determined to be 78 and 110 IU/mg, respectively. CAT activity variations showed agreement with SOD activity and reached a maximum at 320 and 225 IU/mg under the same conditions. The minimum LPO levels of the Fusarium strains with the maximum MnSOD and CAT activities were determined as 1.2 and 0.9 nmol MDA/g., wet weight. The higher LPO level of F. equiseti grown at the same condition, in spite of 1.42-fold higher CAT activity due to the 1.41-fold lower SOD activity, as well as a 2.0-fold higher iron level, indicated increases in the generation of reactive oxygen species via the Fenton reaction.

Role of pyruvate and ascorbate production in regulation of antioxidant enzymes and membrane LPO levels in Fusarium acuminatum.

The role of pyruvate and ascorbate in the regulation of superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase enzymes; and, therefore, membrane lipid peroxidation (LPO) levels in Fusarium acuminatum was investigated in media containing either glycerin or glucose as a carbon source, depending on the incubation period, in the range of 5-25 g/L. Increasing SOD activity between d 9 and 16 of the incubation period showed a positive correlation with a significant increase in pyruvate production up to 15 g/L of glycerin and glucose. In addition, maximum ascorbate production was observed at 15 g/L of glycerin as 82.5+/-2.1 and 20 g/L of glucose as 54+/-1.51, whereas CAT activity decreased with an increased concentration of both carbon sources. When compared with the LPO levels determined in media supplemented with glycerin and glucose, the minimum LPO level was 1.88+/-0.028 nmol of malondialdehyde/g wet wt at 15 g/L of glycerin on d 16, at which it was also observed to have a maximum pyruvate and ascorbate production and SOD, CAT, and GSH-Px activities of 75+/-1.42 microg/mL, 82.5+/-2.1 microg/mL, 32.5+/-0.634 microg/mL, 86.8+/-2.58 IU/mg, and 1.867 IU/mg, respectively. These results indicate that the biosynthesis of pyruvate and ascorbate may be involved in the regulation of antioxidant enzymes, depending on the glycerin and glucose concentrations, and also this defense network was effective in preventing membrane damage from oxidative stress.

Effects of vitamin-mineral supplementation on cardiac marker and radical scavenging enzymes, and MDA levels in young swimmers.

The relationship among the enzyme activities of cardiac markers, the antioxidant defense system, and erythrocyte membrane malonyldialdehyde (MDA) levels related to vitamin-mineral supplementation in swim exercise was investigated. Swimmers aged 11 - 13 years were divided into 2 separate groups as control and vitamin-mineral supplemented. Swimmers participated in a monthly swimming program (4 times/wk) and swam approximately 2- 2.5 km/d. Cardiac markers such as creatine kinase (CK), creatine kinase-MB (CK - MB), glutamic oxaloacetic transaminase [GOT (AST)], lactate dehydrogenase (LDH), and anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities in post-training samples were found to be significantly (p<.05) higher than in pre-training samples. Except for GOT (AST), the activity increases in CK, CK-MB, and LDH in female and male supplemented groups were significantly (p<.05) lower than those of control groups during the 1-month period of swim training. Antioxidant enzyme activity increases in the male vitamin-mineral group were significantly (p <.05) higher when compared with the other groups. Post-training MDA levels were significantly (p <.001) higher than pre-training MDA levels in the control groups, whereas no significant (p<.05) differences were found between the vitamin-mineral supplemented groups. Vitamin-mineral supplementation was found to attenuate cardiac and muscle damage markers while also enhancing antioxidant levels and reducing membrane LPO levels in response to 1 month of swim training.

Dismutation properties of purified and GDA modified CuZnSOD from chicken heart.

Superoxide dismutase (SOD, 1.15.1.1) from chicken heart has been purified 139-fold with specific activity of 2130 IU/mg. Purified SOD has a molecular weight 31.0 +/- 1.0 kDa and is composed of two equally sized subunits each having 1.1 +/- 0.03 and 0.97 +/- 0.02 atoms of Cu and Zn elements, respectively. Purified CuZnSOD modified by covalent attachment of the glutaraldehyde (GDA) in presence and absence of bovine serum albumin (BSA). The optimum conditions were obtained with a series of modification reactions as 0.25 mg/mL CuZnSOD in 50 mM phosphate buffer, pH 7.5 containing 3% GDA in presence and absence of 0.25 mg/mL BSA. The highest recovery activity of modified SODs was determined as 23.4 and 18.5% for the designated SOD-I and SOD-II derivatives, respectively. The recovery activity of SOD-I reached 28.6% while SOD-II didn't change significantly and determined as 19% after the reaction with 1% ethylendiamine. The activity variations of native and modified CuZnSODs were investigated depending on the pH and temperature. Optimum pH values for native and modified SOD-I, -II were determined as 8.8, 8.3, and 8.2, respectively. The native and modified SODs have the same optimum temperatures approximately as 35 degrees C. The pH- and thermal-stability properties of modified SODs were found to be better than native SOD, in the pH range of 6.5-8.5 at 25 degrees C after 6 h, and up to 40 degrees C at pH 7.4 after 3 h incubation period. Inhibitory effects of ditiothreitol (DTT), beta-mercaptoethanol, and iodoacetamide were not observed on the native and modified SODs activities after 5 h incubation period. Phenylmethylsulfonylfloride (PMSF), H20O2, and EDTA were caused by slight inhibition on the enzyme activities.

The relationship between total sialic acid levels and antioxidant status in the tadpoles of Bufo viridis and Rana ridibunda ridibunda.

Total sialic acid levels (TSA), antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) levels were investigated during the developmental period in tadpoles of the predominantly terrestrial amphibian B. viridis and the predominantly aquatic amphibian R. r. ridibunda. Maximum TSA levels were observed in B. viridis and R. r. ridibunda at the fifth and third week of their development, respectively. SOD and CAT activity variations during development in B. viridis were higher than in R. r. ridibunda. Although SOD activity in B. viridis was higher than R. r. ridibunda at the eighth week, SOD activity increased 19.2-fold in R. r. ridibunda and 10.4-fold in B. viridis between the first and eighth week. CAT activity in R. r. ridibunda did not significantly change (p>0.001) until the fifth week then increased, whereas in B. viridis CAT increased after the third week. In contrast to the rise in the antioxidant enzyme activities, LPO levels tended to decrease during the developmental period. Levels of LPO showed a similar trend until the third week for both species. The minimum LPO levels in B. viridis and R. r. ridibunda were 23+/-1.2 and 146+/-7.3 nmol MDA g(-1) tissue, at the eighth week, respectively. While decreasing LPO levels correlated with increasing antioxidant enzyme activities, TSA tended to decrease after reaching a maximum point.

Glutathione redox system, GSH-Px activity and lipid peroxidation (LPO) levels in tadpoles of R.r.ridibunda and B.viridis.

Total glutathione (t-GSH), reduced glutathione (GSH), glutathione disulphide (GSSG) levels, t-GSH/GSSG ratio, glutathione peroxidase (GSH-Px) activity and lipid peroxidation (LPO) levels were investigated during the development period of a predominantly aquatic amphibian R.r.ridibunda and a predominantly terrestrial amphibian B. viridis. While t-GSH and GSH showed a similar trend, GSSG concentration increased significantly (p<0.05) during the larval stages in R.r.ridibunda larvae. In contrast to R.r.ridibunda larvae, there was no significant (p>0.05) change between 1 and 5 weeks in the t-GSH and GSH concentrations of B. viridis. t-GSH and GSH concentrations of B. viridis larvae became sharply elevated after the fifth week, GSSG levels increased 3.25-fold during the metamorphosis. The t-GSH/GSSG ratio fluctuated and the lowest t-GSH/GSSG ratios were observed at the third week for both species. GSH-Px activities for both species increased significantly (p<0.05) during the growing period. The highest GSH-Px activities in R.r.ridibunda and B.viridis were observed at the eighth week and they were 3.45 +/- 0.17 and 4.1 +/- 0.21 IU mg(-1), respectively. The membrane LPO levels in the R.r.ridibunda and B. viridis tadpoles significantly (p<0.001) decreased from 206 +/- 10.3 to 146 +/- 7.3 and from 198 +/- 9.9 to 23 +/- 1.15 nmol MDA g(-1) w.w., respectively.