Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog,Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (K_m = 0.098 ± 0.003 and K_m = 0.092 ± 0.008) than PK from control and anoxic conditions (K_m = 0.065 ± 0.003 and K_m = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.     

Creatine kinase regulation by reversible phosphorylation in frog muscle

Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent K_m creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with ³²P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of ³²P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.     

Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog

Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K _m and V _max) of G6PDH showed a significant increase in K _m G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K _m NADP⁺ (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K _m G6P of 94.1 ± 1.1 μM and K _m NADP⁺ of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K _m G6P was 172 ± 4.3 μM, K _m NADP⁺ was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.     

Glycolysis and the regulation of cryoprotectant synthesis in liver of the freeze tolerant wood frog

Summary Wood frogs,Rana sylvatica, were sampled after freezing at –4°C (a short time course from 2 to 70 min after the appearance of the freezing exotherm) and thawing (20 h at 3°C after 70 min of freezing) and the regulation of liver glycolysis with respect to cryoprotectant glucose synthesis was examined. Within 5 min of the initiation of freezing, cryoprotectant concentrations in blood and liver had begun to increase. This was correlated with a rapid rise in the levels of hexose monophosphates in liver, including a 2.5 fold increase in glucose-6-P and 10 fold rise in fructose-6-P contents within the first 5 min post-exotherm. Contents of fructose-1,6-P₂, fructose-2,6-P₂, triose phosphates, P-enolpyruvate, and pyruvate did not significantly change over the course of freezing. Thawing sharply reduced the levels of hexose monophosphates in liver but raised P-enolpyruvate content by 2.3 fold. Changes in the contents of glycolytic intermediates over the freeze/thaw course are consistent with an inhibitory block of glycolysis at phosphofructokinase during freezing in order to facilitate a rapid glycogenolysis and production of cryoprotectant; during thawing, however, glycolysis appears to be inhibited at the level of pyruvate kinase.Possible regulatory control of cryoprotectant synthesis by covalent modification of liver glycolytic enzymes was examined. Glycogenolysis during freezing was facilitated by an increase in the percentage of glycogen phosphorylase in the activea (phosphorylated) form and also by an increase in the total amount (a+b) of enzyme expressed. For phosphofructokinase, kinetic changes as a result of freezing included a 40% reduction inK _m for fructose-6-P, a 60% decrease inK _a for fructose-2,6-P₂, and a 2 fold increase in I₅₀ for ATP. These changes imply a freezing-induced covalent modification of the enzyme but are not, apparently, the factors responsible for inhibition of glycolytic flux at the phosphofructokinase locus during glucose synthesis. Kinetic parameters of pyruvate kinase were not altered over the freeze/thaw course.     

Post-translational Regulation of Hexokinase Function and Protein Stability in the Aestivating Frog <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Xenopus laevis endure substantial dehydration which can impose hypoxic stress due to impaired blood flow. Tissues may increase reliance on anaerobic glycolysis for energy production making the regulation of hexokinase (HK) important. We investigated the enzymatic properties and phosphorylation state of purified HK from the muscle of control and dehydrated (30 % total body water lost) frogs. Bioinformatic tools were also applied to analyze the structural implication of HK phosphorylation in silico. HK from the muscle of dehydrated frogs showed a significantly higher V_max (3.4-fold) and K_m for glucose (2.4-fold) compared with control HK but the K_m for ATP was unaltered. HK from dehydrated frogs also showed greater phosphoserine content (20 % increase) and lower phosphothreonine (22 % decrease) content compared to control HK. Control HK had a higher melting temperature (T_m = 61.9 °C) than from dehydrated (T_m = 54.2 °C) frogs when thermostability was tested using differential scanning fluorimetry. In silico phosphorylation of a Xenopus HK caused alterations in active site binding, corroborating phosphorylation as the probable mechanism for kinetic regulation. Physiological consequences of dehydration-induced HK phosphorylation appear to facilitate glycolytic metabolism in hypoxic situations. Augmented HK function increases the ability of Xenopus to overcome compromised oxidative phosphorylation associated with ischemia during dehydration.     

Synthesis and kinetic evaluation of a trifunctional enzyme mimic with a dimanganese active centre

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) play crucial roles in the metabolism and homeostasis of reactive oxygen species (ROS) in living organisms. From examination of the steady state and pre-steady state kinetic behavior of natural GPX it was found that, in contrast to accepted theories, the affinity of the enzyme for H₂O₂ rather than reduced glutathione (GSH) most significantly affects its kinetic behavior. Consequently, an enzyme mimic was produced with a similar affinity for the substrate H₂O₂. A salicylaldehyde Schiff base containing a dimanganese centre was selected as a precursor, because it has high H₂O₂-binding affinity for such a relatively small molecule and similar catalytic activity to that of SOD and CAT. Selenium was also incorporated into the catalytic center to provide activity similar to that of GPX, and thus trifunctional enzymatic activity. The K_mH2O2 of the mimic (7.32 × 10^-2 mM) was found quite close to that of natural enzyme (1.0 × 10^-2 mM), indicating that the affinity of the mimic to H₂O₂ was successfully increased to approach natural GPX. The steady state kinetic performance of the enzyme mimic showed that the ratio between k_cat/K_mGSH and k_cat/ K_mH2O2 was quite similar to that of native GPX, indicating that the Mn(III)₂(L-Se-SO₃Na) had the same selectivity for both substrates GSH and H₂O₂ as native GPX, which put it among the best existing GPX mimics. Moreover, the new mimic was confirmed to strongly inhibit lipid peroxidation and mitochondrial swelling, probably due to the synergism between the three antioxidant enzymatic activities.     

Responses of marine macroalgae to hydrogen-peroxide stress

In this study, we determined the antioxidative potential of 15 marine macroalgae by measuring the photosynthetic efficiency under artificial oxidative stress after a 30-min exposure to a series of ascending H₂O₂ concentrations. Species exhibiting high maximum quantum yields (F_v/F_m values) were regarded as not susceptible towards H₂O₂ stress. In addition to the short-term stress experiments, the antioxidative defense systems (enzymatic and non-enzymatic) of selected algal species under longer exposure times to H₂O₂ were investigated.Species with striking photosynthetic activity under H₂O₂ stress were Chaetomorpha melagonium (Chlorophyta), showing 40% reduced F_v/F_m as compared to the control after 8 days of exposure to 20 mM H₂O₂. In Fucus distichus (Phaeophyta) F_v/F_m decreased to 50% of the control under the same exposure conditions. Polysiphonia arctica (Rhodophyta) exhibited highest F_v/F_m values with a reduction of only 25%, therefore possessing the highest antioxidative potential of the investigated species.In P. arctica the activities of the antioxidative enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), as well as the pool size of the antioxidant ascorbic acid were investigated. When exposed to different H₂O₂ concentrations (0-2 mM) over 6 days, the intrinsic activities of SOD and GR were stimulated. In a kinetic study over 8 days, the activity of antioxidative enzymes APX and CAT as well as ascorbic acid content were recorded. APX activity was much higher in H₂O₂-treated thalli at the end of the experiment than in the control, also CAT activity increased significantly with increasing H₂O₂ stress. In parallel, ascorbic acid content was reduced under high H₂O₂ concentrations. Furthermore, by using GC-MS techniques in P. arctica bromophenolic compounds with antioxidative properties were identified.This study shows that the measurement of the in vivo fluorescence of photosystem II is a suitable tool to determine the effect of oxidative stress on macroalgae. From these studies it is obvious that different algal species have varying strategies against oxidative stress which correlate with zonation on the shore.     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Oxyleghemoglobin was used to supply low concentrations of O₂ to H₂-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H₂ oxidation system of these bacteroids was capable of effectively utilizing O₂ at the low concentrations of O₂ expected to be found in soybean nodules. Apparent K_m values of approximately 10 nanomolar O₂ have been calculated for the oxyhydrogen reaction. These values include the K_m values for both H₂ oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H₂ additions stimulated C₂H₂ reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H₂ oxidation system is capable of recycling all of the H₂ evolved during the N₂ fixation process.     

Development of novel flexible sugar ester vesicles as carrier systems for the antioxidant enzyme catalase for wound healing applications

The antioxidant enzyme catalase (CAT) was encapsulated in biocompatible flexible non-ionic sugar esters (SEs) nano-vesicles for potential topical administration. The effects of the SE hydrophilic lipophilic balance (HLB) value and the carbon chain length of the fatty acid ester of different SEs on the encapsulation efficiency (EE) were studied. Morphology of the vesicles was not altered upon CAT encapsulation using freeze fracture electron microscopy. The extrusion measurements indicated that there was an increase in the vesicle's flexibility index upon the inclusion of phospholipids. The mean diameter of the CAT-EV (ester vesicle; HSC and HSC–PL) was 222–275 nm using laser diffraction measurements. The catalytic efficiency (V_max/K_m) of CAT was improved after encapsulation by a factor of 1.7. Both free CAT and CAT-EV showed maximum catalytic activity at pH 7.0, and CAT-EV was more stable than free CAT at acidic pH, which is advantageous for successful topical delivery. Encapsulation of CAT in SE vesicles protected it against trypsin treatment. Encapsulated CAT retained more than 60% residual activity after 12 successive decomposition cycles of H₂O₂. CAT-EV activity was significantly preserved compared to that of free CAT at 4 °C for 180 days. The in vivo study showed a significant effect of the prepared CAT nano-vesicles on wound healing.     

Effects of deuterium oxide on growth, proton extrusion, potassium influx, and in vitro plasma membrane activities in maize root segments

Elongation of subapical segments of maize (Zea mays) roots was greatly inhibited by ²H₂O in the incubation medium. Short-term exposure (30 min) to ²H₂O slightly reduced O₂ uptake and significantly increased ATP levels. ²H₂O inhibited H⁺ extrusion in the presence of both low (0.05 mm) and high (5 mm) external concentrations of K⁺ (about 30 and 53%, respectively at 50% [v/v] ²H₂O). Experiments on plasma membrane vesicles showed that H⁺-pumping and ATPase activities were greatly inhibited by ²H₂O (about 35% at 50% [v/v] ²H₂O); NADH-ferricyanide reductase and 1,3-β-glucan synthase activities were inhibited to a lesser extent (less than 15%). ATPase activities present in both the tonoplast-enriched and submitochondrial particle preparations were not affected by ²H₂O. Therefore, the effect of short incubation time and low concentration of ²H₂O is not due to a general action on overall cell metabolism but involves a specific inhibition of the plasma membrane H⁺ -ATPase. K⁺ uptake was inhibited by ²H₂O only when K⁺ was present at a low (0.05 mm) external concentration where absorption is against its electrochemical potential. The transmembrane electric potential difference (E_m) was slightly hyperpolarized by ²H₂O at low K⁺, but was not affected at the higher K⁺ concentrations. These results suggest a relationship between H⁺ extrusion and K⁺ uptake at low K⁺ external concentration.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

Hydrogen peroxide enhanced Ca(2+)-activated BK currents and promoted cell injury in human dermal fibroblasts

AimsRecent studies have shown that dermal fibroblasts possess multiple types of voltage-dependent K⁺ channels, and the activation of these channels induces apoptosis. In the present study, we aimed to investigate whether hydrogen peroxide (H₂O₂), an oxidative stress inducer, could modulate these channels or induce human dermal fibroblasts injury.Main methodsThe effects of H₂O₂ on K⁺ currents were studied using a whole-cell recording. Intracellular PKC levels were measured with a direct human PKC enzyme immunoassay kit. Cell viability was assessed using PI staining and apoptotic nuclei were detected with TdT-mediated digoxigenin-dUTP nick-end labelling assay (TUNEL) assay.Key findingsTreatment of cells with 100 μM H₂O₂ resulted in a partially reversible increase in non-inactivating outward K⁺ currents and an alteration in the steady-state activation property of the channels. The H₂O₂-induced increase in K⁺ currents was mimicked by a PKC activator, and was blocked by the PKC inhibitor or the large conductance Ca²⁺-activited K⁺ (BK) channel blockers. The intracellular PKC levels were significantly enhanced by H₂O₂ treatment in a concentration-dependent manner. After exposure to H₂O₂, evaluation of fibroblasts survival rate and damaged cell number with TUNEL-positive nuclei revealed an increased cell injury. Blocking the K⁺ channels with blockers significantly decreased the H₂O₂-induced human dermal fibroblasts injury.SignificanceOur results revealed that H₂O₂ could enhance BK currents by PKC pathway. Increased K⁺ currents might be related to H₂O₂-induced human dermal fibroblasts injury. The results reported here contribute to our understanding of the mechanism underlying H₂O₂-induced human dermal fibroblasts injury.     

Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato

The effects of salicylic acid (SA) and hydrogen peroxide (H₂O₂) on freezing tolerance were studied in two potato (Solanum tuberosum) cultivars (Alpha and Atlantic) that differ in cold sensitivity, Alpha being more tolerant to freezing than Atlantic. Lowest freezing survival rates were observed in 4-week-old plants. Freezing treatments consisting of exposure to 6° C for 4 h in the dark were applied 24 h after plants had been transferred from in vitro culture to soil. Catalase activity and H₂O₂ were estimated at the following harvest points: stage (a) 4-week-old in vitro plants treated with either 0.1 mM SA or 5 mM H₂O₂; stage (b) as in (a) but 24 h following transfer to soil prior to freezing treatment; stage (c) as in (b) but measured 15 days after a 4-h freezing treatment. The results show that (1) SA induced freezing tolerance in both cultivars; (2) SA inhibited ascorbate peroxidase activities in both cultivars at all harvest points but inhibited catalase activities in only at stage (a); (3) SA induced H₂O₂ accumulation only in Atlantic at stage (a); (4) H₂O₂ enhanced shoot catalase activities in Atlantic at stages (a) and (b) whereas this treatment had no effect on shoot catalase activities in Alpha; (5) H₂O₂ treatment induced freezing tolerance in Atlantic, even though shoot catalase activities were lower than those of the controls following exposure to freezing temperatures. We conclude that SA does not always lead to H₂O₂ accumulation even though catalase and ascorbate peroxidase activities are decreased as a result of the treatment. Moreover, H₂O₂ accumulation is not always associated with the induction of freezing tolerance, for example at stage (a) where SA-induced tolerance in Alpha was not accompanied by H₂O₂ accumulation. H₂O₂ was able to induce freezing tolerance only in Atlantic, even though H₂O₂ accumulated in both cultivars following this treatment.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Antioxidant response of a novel Streptomyces sp. M3004 isolated from legume rhizosphere to H2O2 and paraquat

This paper aims to investigate the effect of H₂O₂ and paraquat on the activities of superoxide dismutase (SOD) and catalase (CAT), and membrane lipid peroxidation (LPO) levels in newly isolated Streptomyces sp. M3004. SOD activities of Streptomyces sp. M3004, grown in 10 mM and 30 mM H₂O₂, were significantly lower than the control cultures. On the other hand, as an antioxidant enzyme, CAT activity in both H₂O₂ treatment conditions increased significantly compared with the control. These activity values in 10 mM and 30 mM H₂O₂ treatment on the 48th hour of incubation were 3.8- and 6.6-fold higher than the control, respectively. SOD activity decreased significantly with respect to paraquat concentration, which was added at the start of the incubation. CAT activities increased significantly in 1.0 mM and 3.0 mM paraquat treatments compared to control. As an indicative marker of membrane damage, LPO levels of the novel isolate Streptomyces sp. M3004 treated with H₂O₂, and paraquat stress conditions were significantly higher than the control. Nevertheless, compared with the 30 mM H₂O₂ in both treatment conditions, LPO levels in 10 mM H₂O₂ were significantly higher. The decreases in SOD activities in paraquat and H₂O₂ treatment conditions resulted in the increases in the LPO levels although it increases in CAT activities.     

Melatonin protects rabbit spermatozoa from cryo-damage via decreasing oxidative stress

Mammalian spermatozoa are highly susceptible to reactive oxygen species (ROS) stress. The aim of the present study was to investigate whether and how melatonin protects rabbit spermatozoa against ROS stress during cryopreservation. Semen was diluted with Tris-citrate-glucose extender in presence of different concentrations of melatonin. It was observed that addition of 0.1 mM melatonin significantly improved spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial membrane potential as well as AMP-activated protein kinase (AMPK) phosphorylation. Meanwhile, the lipid peroxidation (LPO), ROS levels and apoptosis of post-thaw spermatozoa were reduced in presence of melatonin. Interestingly, when fresh spermatozoa were incubated with 100 μM H₂O₂, addition of 0.1 mM melatonin significantly decreased the oxidative damage compared to the H₂O₂ treatment, whereas addition of luzindole, an MT1 receptor inhibitor, decrease the effect of melatonin in spermatozoa. It was observed that the glutathione (GSH) content and activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) were significantly increased with addition of melatonin during cryopreservation. In conclusion, addition of melatonin to the freezing extender protects rabbit spermatozoa against ROS attack by enhancing AMPK phosphorylation for increasing the antioxidative defense.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

Arbuscular mycorrhizal symbiosis modulates antioxidant response in salt-stressed Trigonella foenum-graecum plants

An experiment was conducted to evaluate the influence of Glomus intraradices colonization on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (PX), ascorbate peroxidase (APX), and glutathione reductase (GR)] and the accumulation of nonenzymatic antioxidants (ascorbic acid, α-tocopherol, glutathione, and carotenoids) in roots and leaves of fenugreek plants subjected to varying degrees of salinity (0, 50, 100, and 200 mM NaCl) at two time intervals (1 and 14 days after saline treatment, DAT). The antioxidative capacity was correlated with oxidative damage in the same tissue. Under salt stress, lipid peroxidation and H₂O₂ concentration increased with increasing severity and duration of salt stress (DoS). However, the extent of oxidative damage in mycorrhizal plants was less compared to nonmycorrhizal plants. The study reveals that mycorrhiza-mediated attenuation of oxidative stress in fenugreek plants is due to enhanced activity of antioxidant enzymes and higher concentrations of antioxidant molecules. However, the significant effect of G. intraradices colonization on individual antioxidant molecules and enzymes varied with plant tissue, salinity level, and DoS. The significant effect of G. intraradices colonization on antioxidative enzymes was more evident at 1DAT in both leaves and roots, while the concentrations of antioxidant molecules were significantly influenced at 14DAT. It is proposed that AM symbiosis can improve antioxidative defense systems of plants through higher SOD activity in M plants, facilitating rapid dismutation of O₂ ^- to H₂O₂, and subsequent prevention of H₂O₂ build-up by higher activities of CAT, APX, and PX. The potential of G. intraradices to ameliorate oxidative stress generated in fenugreek plants by salinity was more evident at higher intensities of salt stress.