Detection of manganese superoxide dismutase mRNA in the theca interna cells of rat ovary during the ovulatory process by in situ hybridization

To determine the possible involvement of reactive oxygen species in ovulation, dynamic aspects of superoxide dismutase (SOD) isozyme were studied in the ovaries of rats by in situ hybridization histochemistry. Previously, mRNA levels of ovarian manganese superoxide dismutase (Mn-SOD) were reported markedly to increase whilst enzymic activity of Mn-SOD decreased during the ovulatory process after treating immature rats with 10 and 5 Units, respectively, of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Levels of Cu/Zn-SOD activity and Cu/Zn-SOD mRNA were reported to remain unchanged throughout ovulation. This increase in the Mn-SOD mRNA level was shown in the present study by in situ hybridization to be localized to the theca interna cells throughout the PMSG/HCG-induced ovulatory process. The observations suggest that the turnover rate of Mn-SOD but not Cu/Zn-SOD increases specifically in the mitochondria of these cells. SOD has been postulated to play important roles in steroidogenesis. The relationship is discussed between mitochondrial functions in steroid-secreting cells and superoxide radicals and related metabolite(s).     

Diethyldithiocarbamate inhibits in vivo Cu,Zn-superoxide dismutase and perturbs free radical processes in the yeast Saccharomyces cerevisiae cells

Copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in some model experiments in vitro demonstrated antioxidant as well as pro-oxidant properties. In the present study, yeast Saccharomyces cerevisiae lacking Mn-SOD were studied using Cu,Zn-SOD inhibitor N-N'-diethyldithiocarbamate (DDC) as a model system to study the physiological role of the yeast Cu,Zn-SOD. Yeast treatment by DDC caused dose-dependent inhibition of SOD in vivo, with 75% inhibition at 10mM DDC. The inhibition of SOD by DDC resulted in modification of carbonylprotein levels, indicated by a bell-shaped curve. The activity of glutathione reductase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase (enzymes associated with antioxidant) increased, demonstrating a compensatory effect in response to SOD inhibition by different concentrations of DDC. A strong positive correlation (R2=0.97) was found between SOD and catalase activities that may be explained by the protective role of SOD for catalase. All observed effects were absent in the isogenic SOD-deficient strain that excluded direct DDC influence. The results are discussed from the point of view that in vivo Cu,Zn-SOD of S. cerevisiae can demonstrate both anti- and pro-oxidant properties.     

低氧-复氧胁迫对鲢抗氧化酶活性及Cu/Zn-SOD和Mn-SOD基因表达的影响

为探究低氧-复氧胁迫对鲢(Hypophthalmichthys molitrix)抗氧化酶活性及Cu/Zn-SOD和Mn-SOD基因表达的影响, 对鲢进行急性低氧、持续低氧及复氧实验, 进而分析血清、心脏和肝脏中不同抗氧化酶和SODs基因表达的变化特征。结果表明: 在急性低氧胁迫后, 血清中总抗氧化能力(T-AOC)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)活性随着氧浓度的降低均呈上升趋势, 但超氧化物歧化酶(SOD)活性呈先升后降的趋势。在持续低氧胁迫后, 血清中T-AOC和GSH-PX活性随着低氧胁迫时间的增加显著升高(P<0.05); 心脏中SOD活性显著高于常氧水平(P<0.05), 但Cu/Zn-SOD和Mn-SOD基因表达在低氧胁迫24h时显著低于常氧水平(P<0.05); 肝脏中SOD活性在低氧胁迫24h时显著高于常氧水平(P<0.05), 且Cu/Zn-SOD和Mn-SOD基因表达在低氧胁迫24h时也显著高于常氧水平(P<0.05)。复氧后, 血清、心脏和肝脏中T-AOC、SOD、CAT和GSH-PX活性均能恢复至常氧水平, 且心脏和肝脏中Cu/Zn-SOD和Mn-SOD基因表达的也能恢复至常氧水平, 但肝脏中Mn-SOD基因表达恢复至常氧水平较在心脏中所需时间更少。因而, 鲢可以通过调节抗氧化酶的活性来保护自身免受氧化应激造成的损伤。研究为解析低氧胁迫下鲢抗氧化应激机制提供了基础。     

氯化钠胁迫下嫁接黄瓜叶片SOD和CAT mRNA基因表达及其活性

研究了NaCl胁迫下嫁接和自根黄瓜叶片Cu/Zn-SOD、Mn-SOD和CAT mRNA的表达与其酶活性变化及其MDA含量和电解质渗漏率变化.结果表明：在NaCl胁迫条件下,嫁接黄瓜叶片Cu/Zn-SOD mRNA、Mn-SOD mRNA和CAT mRNA的相对表达量均高于自根黄瓜,SOD、Cu/Zn-SOD、Mn-SOD和CAT活性也均高于自根黄瓜,说明与自根黄瓜相比,嫁接黄瓜叶片较高的Cu/Zn-SOD mRNA、Mn-SOD mRNA和CAT mRNA相对表达量是其维持较高Cu/Zn-SOD、Mn-SOD和CAT活性的重要原因;随着NaCl胁迫时间的延长,嫁接和自根黄瓜叶片Cu/Zn-SOD- mRNA、Mn-SOD mRNA和CAT mRNA的相对表达量均呈上升趋势,但其酶活性变化并不完全一致,说明还有其他因素参与相关酶活性的调控;嫁接黄瓜叶片MDA含量和电解质渗漏率均低于自根黄瓜,说明嫁接黄瓜具有较高的活性氧清除系统,可以减少活性氧物质的危害,提高其耐盐性.     

Assay of superoxide dismutase: cautions relevant to the use of cytochrome c, a sulfonated tetrazolium, and cyanide.

Two commonly used assays for superoxide dismutase (SOD) activity have been compared, one using cytochrome c and the other using XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) as the indicating scavenger of superoxide. The use of cyanide to selectively suppress Cu,Zn-SOD and thus to allow assay of both Cu,Zn-SOD and Mn-SOD in mixtures of the two was also explored, as was the influence of pH. The XTT assay became more sensitive at elevated pH, because the rate of the superoxide/XTT reaction declines with increasing pH. This was clearly seen with the Cu,Zn-SOD but barely with Mn-SOD because the former retains full activity from pH 5 to 10 while the latter does not. Cyanide reacted with cytochrome c, but not XTT, in a concentration- and time-dependent manner and thus diminished its reducibility by superoxide. Cytochromes endogenous to tissue fractions were reduced by the xanthine oxidase reaction and this caused a decrease in absorbance 470 nm which interfered with the XTT assay. The alkalinizing effect of cyanide salts and the problems encountered in neutralizing cyanide stock solutions are discussed.     

Occurrence of Cu,Zn-Superoxide Dismutase in the Intrathylakoid Space of Spinach Chloroplasts

Thylakoids obtained from intact spinach chloroplasts showedno superoxide dismutase (SOD) activity, but Cu,Zn- and Mn-SODactivities were detected in the presence of Triton X-100. Thylakoidmembranes and the lumen fraction were separated by centrifugationafter treatment of the thylakoids with a Yeda pressure cell.Cu,Zn-SOD was found in the lumen fraction. Mn-SOD was detectedin the thylakoid fraction only after addition of 1% Triton X-100.Antibody against spinach Cu,Zn-SOD did not interact with thelatent Cu,Zn-SOD in the thylakoids unless Triton was added.These results indicate that Cu,Zn-SOD occurs in the lumen inaddition to the stroma of spinach chloroplasts, and Mn-SOD bindsto the thylakoid membranes. (Received February 29, 1984; Accepted May 28, 1984)     

低温胁迫下嫁接对黄瓜叶片SOD和CAT基因表达与活性变化的影响

研究了低温胁迫下嫁接和自根黄瓜叶片Mn-SOD、Cu/Zn-SOD和CAT mRNA基因表达和酶活性变化及其与抗冷性的关系.结果表明:低温胁迫下,嫁接与自根黄瓜叶片Cu/Zn-SOD、Mn-SOD mRNA基因相对表达量变化分别与其Cu/Zn-SOD、Mn-SOD活性变化相吻合,而CATmRNA相对表达量变化与其CAT活性变化并不一致;嫁接黄瓜叶片Cu/Zn-SOD和Mn-SOD mRNA相对表达量及SOD、Cu/Zn-SOD和Mn-SOD活性均高于自根黄瓜,MDA含量和电解质渗漏率均低于自根黄瓜,嫁接黄瓜较高的SOD基因表达量调控的较高SOD活性是其抗冷性强于自根黄瓜的主要因素;嫁接黄瓜的功能叶CAT mRNA相对表达量略高于自根黄瓜,而幼叶CAT mRNA相对表达量低于后者,但两者CAT活性差异不大,说明低温胁迫对嫁接黄瓜叶片CAT mRNA相对表达量及CAT活性的影响不大.     

Exposure to anoxia of the clam, Chamelea gallina: II: Modulation of superoxide dismutase activity and expression in haemocytes

In order to investigate the influence of anoxic stress on haemocyte immune response, specimens of Chamelea gallina were exposed to 24 and 48 h anoxia. To evaluate recovery capacity, clams were maintained, at the end of the anoxic phase, for 24 h in reoxygenated seawater. In this paper, activity and expression of the antioxidant enzyme superoxide dismutase (SOD) were studied on haemocyte lysate and haemolymph. Reported results have shown that the anoxic stress changed strongly the response of C. gallina blood cells. Indeed, at the end of the anoxic phase in both experiments (24 and 48 h of anoxia exposure), SOD activity in haemocyte lysate decreased significantly with respect to the control, likely because of a decreasing superoxide anion generation in anoxia. Expression analyses were coherent with activity values.In the first experiment (24 h anoxia), reoxygenation determined an increase in activity of both Cu/Zn-SOD and Mn-SOD, but with values that remained significantly lower than those of the controls. It seems that after the applied anoxic stress, 24 h of recovery is not sufficient to restore pre-anoxic conditions. In the second experiment (48 h anoxia), SOD isoforms showed a different response during the recovery of animals. Cu/Zn-SOD activity dropped below the values showed by haemocytes of anoxic bivalves, while Mn-SOD activity values exceeded significantly those of controls. The different haemocyte response could be probably due to a further stress suffered by the clams because of a massive spawning during the reoxygenation phase. Therefore, the high values of activity shown by Mn-SOD during the recovery are likely to be due to the high inducibility of this isoform.In Cu/Zn-SOD expression analyses, two immunoreactive bands were highlighted in both experiments. The former (apparent molecular weight of 16 kDa) corresponds to the expression of SOD1 and the latter (apparent molecular weight of 28-30 kDa) could be attributed to EC-SOD (SOD3), a Cu/Zn-SOD isoform located in extracellular ambient and identified both in vertebrates and invertebrates. The strong SOD3 expression during anoxia exposure and the further spawning stress (second experiment) testified its inducibility in C. gallina haemocytes and haemolymph in response to stressful conditions.     

Effect of sample preparation on analysis of superoxide dismutase activity and isoenzymes

The effect of superoxide dismutase (SOD) activity and isoenzyme pattern of detergents, incubation time, and sonication in the preparation of rat liver samples was investigated. The activity of the manganese form of the enzyme (Mn-SOD) was found to decrease significantly after 4 hr of incubation at room temperature, and activity of the copper, zinc form of the enzyme (Cu, Zn-SOD) was not changed significantly even after 24 hr, although levels were somewhat decreased. Sonication of the sample did not affect Cu, Zn-SOD activity, but total Mn-SOD activity was increased. Addition of detergents did not increase Mn-SOD activity when homogenates were sonicated, indicating that Mn-SOD is not membrane bound. Detergents also had no effect on Cu, Zn-SOD activity. None of the treatments investigated altered the isoenzyme patterns, providing evidence that these isoenzymes are not degradation products.     

Superoxide dismutase in vesicular arbuscular-mycorrhizal red clover plants

The isoenzymatic pattern of Superoxide dismutase (SOD; EC 1.15.1.1) was studied in the symbiosis of Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe-Trifolium prarense L A Cu.Zn-SOD (M, 40500) was found in spores of G. mosseae . while one Mn-SOD (Mn-SOD I) and two Cu.Zn-SODs (Cu.Zn-SOD 1 and Cu.Zn-SOD II) were present in both roots and leaves of T. pratense . Molecular masses for Cu.Zn-SOD I and Cu.Zn-SOD II were 31000 and 34300. respectively. However, when T. prateme and G. mosseae were associated, mycorrhizal roots showed two new iso-zymes, Mn-SOD II and mycCu.Zn-SOD, which have relative molecular masses of 37 800 and 33 300, respectively. The mycCu.Zn-SOD was found to be specific for this association, whereas Mn-SOD II was also present in nodules of Rhizobium-T. pra-tense . Results suggest that both enzymes are induced in the T. praiense roots in response to invasion by mycorrhizal fungi, perhaps as a result of an increase in the generation of O-₂ radicals in plant roots.     

Changes in Superoxide Dismutase Activity in Liver and Lung of Old Rats

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.     

Changes in Superoxide Dismutase Activity in Liver and Lung of Old Rats

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.     

Subcellular distribution of superoxide dismutase in leaves of ureide-producing leguminous plants

The subcellular localization of superoxide dismutase (SOD; EC. 1.15.1.1) was studied in leaves of two ureide-producing leguminous plants ( Phaseolus vulgaris L. cv. Contender and Vigna unguiculata [L.] Walp). In leaves of Vigna and Phaseolus , three superoxide dismutases were found, an Mn-SOD and two Cu, Zn-containing SODs (I and II). Chloroplasts, mitochondria, and peroxisomes were purified by differential and density-gradient centrifugation using either Percoll or sucrose gradients. The yields obtained in intact chloroplasts and peroxisomes from Vigna were considerably higher than those achieved for Phaseolus . Purified chloroplasts only contained the Cu, Zn-SOD II isozyme, but in mitochondria both Mn-SOD and Cu, Zn-SOD I isozymes were present. In purified peroxisomes no SOD activity was detected. The absence of SOD activity in leaf peroxisomes from Vigna contrasts with results reported for the amide-metabolizing legume Pisum sativum L. where the occurrence of Mn-SOD was demonstrated in leaf peroxisomes (del Río et al. 1983. Planta 158: 216–224; Sandalio et al. 1987. Plant Sci. 51: 1–8). This suggests that in leaf peroxisomes from Vigna plants the generation of O₂^- radicals under normal conditions probably does not take place.     

Species and Organ Diversity in the Effects of Hydrogen Peroxide on Superoxide Dismutase Activity In Vitro

Superoxlde dlsmutase （SOD） is ubiquitous in aerobic organisms and constitutes the first link In the enzyme scavenging system of reactive oxygen species. In the present study, species and organ diversity of SOD activity In a solution and In an in-gel assay system, as well as the effects of hydrogen peroxide （H202） on SOD activity, were Investigated. In a solution assay system, SOD activity of jackfruIt root, shoot, leaves, axes, and cotyledons, of maize embryos and endosperms, of mung bean leaves and seeds, of sacred lotus axes and cotyledons, and of rice and wheat leaves was Increased by 1-15 mmol/L H2O2. However, SOD activity In rice root and seeds, maize roots and leaves, mung bean roots and shoots, and wheat seeds was decreased by 1-15 mmol/L H2O2. The SOD activity of wheat root and soybean roots, leaves, axes, and cotyledons was Increased by 1-4 mmol/L H2O2, but was decreased by concentrations of H2O2 〉4 mmol/L. The SOD activity of soybean shoots was not affected by 1-15 mmol/L H2O2. The SOD activity In crude mltochondrla of jackfruIt, maize, and upas seeds, as well as In purified mitochondria of jackfruIt, was also Increased by 1-15 mmol/L H2O2. In the In-gel assay system, the SOD In jackfruIt cotyledons was comprised of Mn-SOD, Cu/Zn-SOD, and Fe-SOD, the crude mltochondria of jackfruit seeds and maizes embryo was comprised of Mn-SOD and Cu/ Zn-SOD, and the crude mltochondria of maize seeds was comprised of Mn-SOD only. In the present study, H2O2 markedly Inhibited Cu/Zn-SOD and Fe-SOD activity.     

Rescue of the corpus luteum and an increase in luteal superoxide dismutase expression induced by placental luteotropins in the rat: action of testosterone without conversion to estrogen

The superoxide radical and its scavenger, superoxide dismutase (SOD), play important roles in the regulation of corpus luteum function. The present study was undertaken to investigate whether SOD is related to pregnancy-induced maintenance of corpus luteum function. Placentae obtained from rats on Day 12 of pregnancy were incubated for 24 h, and the supernatant was used as placental luteotropins. Pseudopregnant rats were given the placental incubation medium from Day 9 to Day 12 of pseudopregnancy. The treatment significantly increased serum progesterone concentrations on Day 12 of pseudopregnancy. Both activities and mRNA levels of copper-zinc SOD (Cu,Zn-SOD) and manganese SOD (Mn-SOD) in the corpus luteum were also increased on Day 12 of pseudopregnancy. Treating the placental incubation medium with charcoal significantly eliminated the stimulatory effects of placental incubation medium on serum progesterone concentrations and luteal Mn-SOD expression, but not on Cu,Zn-SOD expression. The inhibitory effect of the charcoal treatment on luteal Mn-SOD expression was reversed by supplementation with testosterone or dihydrotestosterone (DHT), but serum progesterone concentrations were recovered only by DHT. Testosterone or DHT alone had no effect on serum progesterone concentrations and luteal SOD expression. In conclusion, placental luteotropins increased SOD expression in the corpus luteum and stimulated progesterone production, suggesting that SOD is involved in the maintenance of the corpus luteum function by placental luteotropins. In addition, androgen, with other placental luteotropins, acted to stimulate progesterone production and Mn-SOD expression in pseudopregnant rats.     

Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress

Superoxide dismutases (SODs) are important antioxidant enzymes responsible for the elimination of superoxide radical (O(2)(-)). The manganese-containing SOD (Mn-SOD) has been suggested to have tumor suppressor function and is located in the mitochondria where the majority of O(2)(-) is generated during respiration. Although increased reactive oxygen species (ROS) in cancer cells has long been recognized, the expression of Mn-SOD in cancer and its role in cancer development remain elusive. The present study used a human tissue microarray to analyze Mn-SOD expression in primary ovarian cancer tissues, benign ovarian lesions, and normal ovary epithelium. Significantly higher levels of Mn-SOD protein expression were detected in the malignant tissues compared with normal tissues (p < 0.05). In experimental systems, suppression of Mn-SOD expression by small interfering RNA caused a 70% increase of superoxide in ovarian cancer cells, leading to stimulation of cell proliferation in vitro and more aggressive tumor growth in vivo. Furthermore, stimulation of mitochondrial O(2)(-) production induced an increase of Mn-SOD expression. Our findings suggest that the increase in Mn-SOD expression in ovarian cancer is a cellular response to intrinsic ROS stress and that scavenging of superoxide by SOD may alleviate the ROS stress and thus reduce the simulating effect of ROS on cell growth.     

Catalase activity and isozyme pattern of the metalloenzyme system, superoxide dismutase, as a function of leaf development during growth of Pisum sativum L. plants

The catalase activity and the isozyme pattern of the metalloenzyme system superoxide dismutase (SOD) have been determined in pea ( Pisum sativum L., cv, Lincoln) leaves of different ages (apical, middle and lower), during several stages of plant development. Pea seedlings were grown in full nutrient solution in a phytotron. Catalase activity was determined polarographically, and superoxide dismutase isozymes (Mn-SOD, Cu, Zn-SOD I and Cu, Zn-SOD II) were separated by acrylamide gel electrophoresis and their relative amounts quantified by densitonietry. The results indicate that the relative amounts of SOD isozymes are slightly different in leaves of different ages during plant growth and, interestingly, each molecular form of SOD shows a clearly distinct pattern during plant development. These changes in the relative percentages of SOD isozymes could be due to the induction of the distinct molecular forms of SOD by the metals Mn, Cu and Zn, translocated to the different leaves as a result of plant development. The relative percentage of the Mn-SOD isozyme showed a similar pattern to that of catalase activity, suggesting a possible link between these two metalloenzymes at subcellular level, both cooperating to remove the toxic effects of O^-₂ and H₂O₂.
An additional conclusion is that before a certain metalloenzyme can be used as a marker to assess the plant micronutrient status, it is essential to have a detalled study of its activity pattern in leaves of different age during plant development.     

Oxidative stress induced by intermittent exposure at a simulated altitude of 4000 m decreases mitochondrial superoxide dismutase content in soleus muscle of rats

The effects were examined of 6-month intermittent hypobaric (4000 m) exposure on the antioxidant enzyme systems in soleus and tibialis muscles of rats. At the end of the 6-month experimental exposure, the six rats in both the exposed group and the control group were sacrificed. Immunoreactive mitochondrial superoxide dismutase (Mn-SOD) contents were measured as well as the activities of antioxidant enzymes [Mn-SOD, cytosolic SOD (Cu,Zn-SOD), catalase (CAT), and glutathione peroxidase (GPX)]. Thiobarbituric acid-reactive substances (TBARS) were also determined as an indicator of lipid peroxidation. The high altitude exposure resulted in a marked increase in TBARS content in soleus muscle, suggesting increased levels of oxygen free radicals. Conversely, significant decreases in both Mn-SOD content and activity in solens muscle were oted affer exposure. Such trends were not noticed in tibialis muscle. On the other hand, no significant changes in Cu,Zn-SOD, CAT, or GPX were observed in either muscle. These results suggested that the increases in lipid peroxidation were most probably a result of decreased Mn-SOD function which was more depressed in oxidative than in glycolytic muscle.