Antioxidant enzyme activities and trace element concentrations in ischemia-reperfusion

In this study, we investigated the impact of ischemia-reperfusion on antioxidant enzyme activities and trace element concentrations. For this purpose, ischemia was initiated by clamping superior mesenteric artery of Wistar (albino) rats for 30 min, followed by reperfusion for 20 min. Immediately after reperfusion, blood samples were taken and examined for red cell copper-zinc superoxide dismutase (Cu-Zn-SOD), catalase (CAT), and glutathione peroxidase (GPx) activities spectrophotometrically and plasma zinc, copper, and magnesium concentrations by atomic absorption spectrophotometer. In the ischemiareperfusion group, red cell Cu-Zn-SOD activity and plasma zinc and copper concentrations were increased significantly (p<0.001) when compared to the control group; however, the increases in GPx activity and plasma magnesium concentration were not significant (p>0.05). We also found a significant (p<0.01) decrease in catalase activity. Free radicals released as a consequence of ischemia-reperfusion caused significant alterations in antioxidant enzymes and in the concentrations of trace elements. Presented at III International Congress of Pathophysiology 1998, Lahti, Finland.     

Antioxidant enzyme activities and lipid peroxidation induced by eicosapentaenoic acid (EPA) in PC12 cells

Eicosapentaenoic acid (EPA) is one of the major dietary polyunsaturated fatty acids and induces apoptosis in several cancer cells. In this study, the EPA induced lipid peroxidation and response of antioxidative enzymes have been investigated in rat pheochromocytoma PC12 cells to elucidate the mechanisms of apoptosis induced by the polyunsaturated fatty acid EPA. We have analyzed superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and glutathione (GSH) contents in PC12 cells after exposure to different concentrations of EPA. Lipid peroxidation was shown to increase in the presence of EPA as an indication of the oxidative damage. Lipid peroxidation was enhanced by EPA in a dose-dependent manner, and the loss of cell viability was partially reversed by vitamin E. In the case of antioxidant enzyme activities, SOD and GPX activities and GSH contents increased significantly at 50 μmol/L EPA and were respectively 2.41-fold (p < 0.01), 3.49-fold (p < 0.05), and 1.43-fold (p < 0.05) higher than controls. The CAT activity at 10 μmol/L had the highest value and was increased by 25.83% (p < 0.05) compared to control. The results suggest that in PC12 cells the mechanism of apoptosis induced by EPA may be partly due to lipid peroxidation.     

Vibrio splendidus biotype 1 as a cause of mortalities in hatchery-reared larval turbot, Scophthalmus maximus (L.)

AIMS: To characterize bacteria associated with turbot larvae feeding on Artemia and identify pathogens causing mortalities in larvae. METHODS AND RESULTS: To identify bacteria associated with mortalities in larval turbot rearing, bacteria were isolated from homogenates of Artemia or from several batches of well-performing or poorly performing turbot larvae. Samples were plated onto marine agar and were characterized using biochemical tests and BIOLOG GN plates. Total culturable aerobic bacteria ranged from 1.9 x 10(5) to 1.8 x 10(6) CFU per larva and >96% of bacteria identified were vibrios. Almost all bacteria were haemolytic and clustered into two phenons represented by Vibrio alginolyticus and Vibrio splendidus. The bacterial flora of Artemia was almost entirely V. alginolyticus, whereas V. splendidus biotype 1 dominated the larval turbot gut flora (69/115 isolates in seven experiments) and formed four different groups based on BIOLOG GN reactions. Of 16 isolates tested for virulence towards turbot larvae, four of the 11 V. splendidus biotype 1 isolates were lethal and all belonged to the same group of V. splendidus biotype 1 isolates. CONCLUSIONS: In a commercial turbot hatchery, the microbial flora of the larval gut was dominated by V. splendidus biotype 1. Four of the 11 V. splendidus biotype 1 isolates caused mortalities in larval turbot and all belonged to one group of the biotype 1 strains identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of four isolates of V. splendidus that are pathogenic for turbot larvae from three separate batches of larval turbot will allow these to be compared with avirulent isolates to define how V. splendidus causes mortalities in larval turbot.     

Lipid peroxidation and antioxidant enzyme activities in erythrocytes of type I and II alcoholics

Reduced and oxidized glutathione (GSH and GSSG), protein-bound glutathione, lipid peroxidation and antioxidant enzyme activities were determined in the erythrocyte lysates and membranes of type I and II alcoholics in order to clarify the effect of age-of-onset and the duration of the alcohol consumption on erythrocyte oxidant and antioxidant status. The osmotic fragility and susceptibility of the erythrocytes to haemolysis were also determined. Erythrocyte lipid peroxidation was significantly increased but, GSH and protein-bound GSH, GSH/GSSG ratio and antioxidant enzyme activities were markedly decreased in the erythrocytes of the alcoholic subgroups. Erythrocyte count and haemoglobin content in the blood of alcoholics were found to be decreased in accordance with the finding that erythrocytes were more fragile and less resistant to haemolysis particularly in type II alcoholics. The present study showed that ethanol-induced oxidative stress in erythrocytes can lead to haemolysis and membrane-specific injuries in erythrocytes of the alcoholic subtypes.     

Assessment of digestive enzyme activities during larval development of white bream

Activity of some of the main enzymes involved in protein digestion and absorption (acid and alkaline proteases, leucine-aminopeptidase, acid and alkaline phosphatases) as well as those of amylase and lipase, was assessed during larval development of white sea bream Diplodus sargus . All enzyme activity was detected at the moment of mouth opening. The variations observed in the activity profiles of the digestive enzymes were correlated either to developmental events, such as the functional start of the stomach (22 days after hatching), or to changes in the nature of the diet. The early and noticeable development of digestive enzyme activities was linked to a high survival after weaning.     

Age-related changes in antioxidant enzyme activities and lipid peroxidation in lungs of control and sulfur dioxide exposed rats

Antioxidant defenses within the lung are pivotal in preventing damage from oxidative toxicants. There have also been several reports with conflicting results on the antioxidant system during aging. In this study, we attempted to investigate age-related alterations in both antioxidant enzyme activities and thiobarbituric acid-reactive substances (TBARS), a product of lipid peroxidation, in the whole lung of control and sulfur dioxide (SO₂) exposed rats of different age groups (3-, 12-, and 24-months-old). Swiss-Albino Male rats were exposed to 10 ppm SO₂ 1 hr/day, 7 days/week for 6 weeks. The antioxidant enzymes examined include Cu,Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST). A mixed pattern of age-associated alterations in antioxidant activities was observed. SOD, GSH-Px and GST activities were increased with age, but CAT activity was decreased. Lung SOD, GSH-Px and GST activities were also increased in response to SO₂. The level of TBARS was increased with age. SO₂ exposure stimulated lipid peroxide formation in the lung as indicated by an increase in the level of TBARS. These findings suggest that both aging and SO₂ exposure may impose an oxidative stress to the body. We conclude that the increase in the activities of the antioxidant enzymes of the lung during aging, could be interpreted as a positive feedback mechanism in response to rising lipid peroxidation.     

Interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus)

The present study was to investigate the interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus). The fish were exposed to four total ammonia nitrogen (0, 5, 20, 40 mg/L TAN) and two stocking density. After 96 h of exposure, blood, gill, and liver samples were collected to measure biochemical parameters and mRNA levels of immune-related genes. The results showed that co-exposure to high TAN (20 and 40 mg/L) and high density significantly increased plasma sodium (Na⁺), gill Na⁺/K⁺-ATPase activity and mRNA levels. Following individual and combined exposure to high TAN and high density, the heat shock protein 70 (HSP 70), HSP 90, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) genes expression were obviously higher than their control. Conversely, the lysozyme (LZM) and hepcidin mRNA levels were down-regulated in liver of fish exposed to high TAN alone and combination of high TAN-density. Moreover, glutathione S-transferase (GST) mRNA levels significantly increased in treatments with individual high TAN and high density, but decreased in high TAN-density co-exposed fish. Overall, ion homeostasis and immune status were adversely influenced in high exposed turbot under high density.     

Changes in marine turbot (Scophthalmus maximus) epidermis and skin mucus composition during development from bilateral larvae to juvenile flat fish

Alike other flat fish, marine turbot has the particularity that changes from larvae with bilateral symmetry to adult with asymmetry, in terms of the position of the eyes. As expected, the skin configuration of this species is also affected by the development and transformation suffered by fish during metamorphosis. In this context, changes in the epidermis of marine turbot were studied using conventional staining and histochemical techniques using six lectins (UEA-I, PNA, RCA-I, WGA, Con A and SBA). During development from larvae to juvenile (3–300 days post-hatching), the epidermis increased in both thickness and the number of cell layers. In fact, the simple cuboidal epithelium observed in larvae at day 3 already became stratified at days 10–12, which sequentially increase in thickness with fish development. Turbot epidermis is composed basically of four cell types: epithelial and mucous or secretory cells that are present through the development, and pigmented cells and a type that the authors described as club-like cells that appear during and post-metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method revealed the presence of neutral glycoconjugates in mucous and club-like cells at post-metamorphic stages of fish. Accordingly, lectin analysis showed mucous cells containing glycoproteins rich in fucose (UEA-I labelling) and glycoconjugates rich in the sequence galactose-N-acetyl galactosamine (PNA and RCA-I labelling) when this cell type appears. Interestingly, melanophores were observed in the dorsal epidermis of post-metamorphic juveniles. This type of cell contains a black-to-brown pigment that provides the skin the typical colour of this fish species. Changes in mucous coat composition were observed during fish development, which was attributed to different roles of the glycoconjugates.     

怀头鲇早期发育阶段消化酶及碱性磷酸酶活性变化(英文)

采用动物性饵料和人工饲料培育1～10日龄怀头鲇(Silurus soldatovi)仔稚鱼,分析测定了全鱼酸性、碱性蛋白酶、淀粉酶、脂肪酶以及碱性磷酸酶的活性。结果表明:孵化后3天开口期仔鱼已具有较高的碱性蛋白酶活性,5日龄时碱性蛋白酶比活力达到较高值,8日龄时出现低值,总体变化呈波动上升趋势;酸性蛋白酶活性在1～8日龄处于较低水平,8日龄后开始迅速升高;淀粉酶活性在5日龄左右达到最高值,随后酶活性开始下降至较低水平;脂肪酶活性变化波动较大,表现为双峰型,两个峰值分别出现在3～4日龄和6～8日龄。摄食动物性饵料仔稚鱼消化酶活性和碱性磷酸酶活性均高于摄食人工饲料。在整个早期发育过程中,碱性蛋白酶比酸性蛋白酶活性高,碱性蛋白酶、淀粉酶比活力在约8日龄仔稚鱼转变期明显下降,而酸性蛋白酶活性开始迅速升高,这说明消化酶活性的变化与仔稚鱼发育过程中消化机能转换具有相关性。怀头鲇在10日龄内碱性磷酸酶活性呈上升趋势,表明怀头鲇胃肠道功能的逐步发育完善。     

Population genetic structure of turbot (Scophthalmus maximus L., 1758) in the Black Sea

Turbot, Scophthalmus maximus, is a commercially important demersal flatfish species distributed throughout the Black Sea. Several studies performed locally with a limited number of specimens using both mitochondrial DNA (mtDNA) and microsatellite markers evidenced notable genetic variation among populations. However, comprehensive population genetic studies are required to help management of the species in the Black Sea. In the present study eight microsatellite loci were used to resolve the population structure of 414 turbot samples collected from 12 sites across the Black Sea. Moreover, two mtDNA genes, COI and Cyt-b, were used for taxonomic identification. Microsatellite markers of Smax-04 and B12-I GT14 were excluded from analysis due to scoring issues. Data analysis was performed with the remaining six loci. Loci were highly polymorphic (average of 17.8 alleles per locus), indicating high genetic variability. Locus 3/20CA17, with high null allele frequency (>30%), significantly deviated from HW equilibrium. Pairwise comparison of the F_ST index showed significant differences between most of the surveyed sampling sites (P < 0.01). Cluster analysis evidenced the presence of three genetic groups among sampling sites. Significant genetic differentiation between Northern (Sea of Azov and Crimea) and Southern (Turkish Black Sea Coast) Black Sea sampling sites were detected. The Mantel test supported an isolation by distance model of population structure. These findings are vital for long-term sustainable management of the species and development of conservation programs. Moreover, generated mtDNA sequences would be useful for the establishment of a database for S. maximus.     

Gene diversity analysis in natural populations and cultured stocks of turbot (Scophthalmus maximus L.)

The genetic variability of eight fish-farm and three natural populations of turbot was studied by electrophoretic analysis of 35 enzymatic loci. The results showed low genetic variability in natural populations of turbot ( H _T = 0·029 ± 0·013) in comparison with other flatfish species. Great genetic similarity was revealed among the natural populations studied, which indicates high rates of gene flow in this species. The hatchery stocks showed less genetic variation than the wild populations analysed, which suggests genetic drift phenomena involved in the foundation and management of broodstocks. In addition, the heterozygosity differences detected among the hatchery stocks analysed are correlated with inverted levels of fluctuating asymmetry, which supports the existence of inbreeding depression phenomena in turbot culture.     

大菱鲆白化相关基因(DEN-1、DEN-2)的分离鉴定及特征分析

应用mRNA差异显示技术,对比研究正常与白化大菱鲆有眼侧皮肤组织,克隆差异表达基因,经测序,RT-PCR验证,差异表达基因片段DEN-1(GenBank登录号:DQ886390)与DEN-2(GenBank登录号:DQ886391)均在白化大菱鲆有眼侧皮肤组织中下调表达;通过BLAST检索发现,DEN-1与GenBank中的斑马鱼和牛的类似尿苷二磷酸-葡萄糖:糖蛋白葡糖基转移酶(UGGT)基因、与挪威鼠类似尿苷二磷酸-葡萄糖:神经酰氨葡糖基转移酶1(Ugcgl1)基因有较高同源性;DEN-2与原鸡、斑马鱼、人、挪威鼠、家鼠和狗的眼缺乏同源框4(eya4)基因的同源性均较高。采用相对定量RT-PCR对正常鱼有眼侧皮肤(N1)、白化鱼有眼侧皮肤(A1)、正常鱼无眼侧皮肤(N2)、白化鱼无眼侧皮肤(A2)进行表达谱分析,以内参β-actin基因分别对DEN-1和DEN-2表达量进行标准化的光密度扫描显示:DEN-1和DEN-2在4种皮肤组织中有相似的表达模式,即表达量:N1>N2≈A2>A1。电子延伸将DEN-2延伸为497bp的片段,该延伸片段与多种动物的eya4基因在核酸和蛋白水平均有很高同源性。本研究对DEN-1和DEN-2的不同组织器官和不同发育阶段的电子表达谱分析,将对其进一步研究提供有价值参考。     

Characterization of EST-derived microsatellites for gene mapping and evolutionary genomics in turbot

The detection of microsatellite sequences within expressed sequence tags (ESTs) connects potential markers with specific genes, generating type I markers. We have developed and mapped by linkage analysis a set of EST-derived microsatellites in the turbot, Scophthalmus maximus. One hundred and ninety-one microsatellites were identified from 9256 turbot ESTs. Primer design was possible with 98 microsatellites. After genotyping 25 wild turbot and the parents of two reference families for linkage analysis, 43 EST-derived microsatellites were selected because they met technical and polymorphism criteria. A final set of 31 EST-derived microsatellites could be mapped to 17 linkage groups of the turbot consensus map based on 242 anonymous microsatellites. Twenty-four microsatellite-containing ESTs were functionally annotated, confirming them as type I markers. Nineteen were mapped in the turbot consensus map. These EST-derived microsatellites constitute useful tools for genome scanning of turbot populations, marker-assisted selection programmes and comparative mapping.     

New microsatellite markers in turbot (Scophthalmus maximus) derived from an enriched genomic library and sequence databases

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an H_E ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.     

The interaction of temperature and fish size on growth of juvenile turbot

Growth rate of tagged juvenile turbot was significantly influenced by the interaction of temperature and fish size. The results suggest the optimum temperature for growth of juvenile turbot in the size range 25–75 g is between 16 and 19°C. Optimal temperature for growth decreased rapidly with increasing size, and is between 13 and 16°C for 100 g turbot. Although individual growth rates varied highly at all times within the temperature treatments, significant size rank correlations were maintained during the experimental period. The study confirms that turbot exhibit ontogenetic variation in temperature optimum, which might partly explain different spatial distribution of juvenile and adult turbot in ocean waters.     

Ascorbate peroxidase: A novel antioxidant enzyme in insects

Ascorbate peroxidase (APOX) activity, which catalyzes the oxidation of ascorbic acid with the concurrent reduction of hydrogen peroxide (H₂O₂), was found in larvae of Helicoverpa zea. Since insects apparently lack a Se-dependent glutathione peroxidase and since catalase has a low affinity for H₂O₂, this enzyme may be important in removing H₂O₂ in insects. We partially purified the APOX activity 58x from the whole body homogenates and investigated its activity with model lipid peroxides, electron donors, and known inhibitors of plant APOX. The H. zea APOX has activity with model lipid peroxides. This, along with the APOX activity found in fat body tissues, suggests that ascorbate peroxidase may be important in removing lipid peroxides in insects. The H. zea APOX has broader specificity for electron donors than the plant APOX with activity using cysteine, NADPH, glutathione, and cytochrome C as electron donors (22–93% of activity with ascorbate). The H. zea APOX is also resistant to many of the known inhibitors of plant APOX, suggesting that the enzyme has a different active site and may not be a heme-peroxidase. © 1997 Wiley-Liss, Inc.     

镉胁迫对旱柳细胞膜透性和抗氧化酶活性的影响

以‘Fuyang 3’旱柳为材料,通过水培方法研究了中、高剂量(5、25μmol.L-1)镉胁迫下旱柳器官的镉(Cd2 )积累、叶和根细胞膜渗透及抗氧化酶活性的变化。结果表明:Cd2 主要积累在旱柳的根部(最高达1 297.71μg.g-1),其次是枝条(最高为163.13μg.g-1)。中、高剂量Cd2 胁迫下,旱柳叶相对电导率、根K 渗透以及根和叶丙二醛(MDA)含量均未发生显著变化。中、高剂量Cd2 胁迫使旱柳根系的超氧歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽转移酶(GST)活性,以及叶片的SOD、愈创木酚过氧化酶(POD)、谷胱甘肽过氧化物酶(GPX)和GST活性均比对照显著增强;且高剂量胁迫旱柳根系的SOD和GST活性,及其叶片的POD、GPX和GST活性均显著高于相应中剂量胁迫。研究发现,旱柳在不同浓度镉胁迫条件下,其根是Cd2 主要积累器官,其叶和根细胞质膜能保持相对稳定性,其根和叶各抗氧化酶活性发生不同程度改变,从而使旱柳对Cd2 胁迫表现出一定的忍耐性。     

Differential effects of exposure to parasites and bacteria on stress response in turbot Scophthalmus maximus simultaneously stressed by low water depth

The stress response of turbot Scophthalmus maximus was evaluated in fish maintained 8 days under different water depths, normal (NWD, 30 cm depth, total water volume 40 l) or low (LWD, 5 cm depth, total water volume 10 l), in the additional presence of infection–infestation of two pathogens of this species. This was caused by intraperitoneal injection of sublethal doses of the bacterium Aeromonas salmonicida subsp. salmonicida or the parasite Philasterides dicentrarchi (Ciliophora:Scuticociliatida). The LWD conditions were stressful for fish, causing increased levels of cortisol in plasma, decreased levels of glycogen in liver and nicotinamide adenine dinucleotide phosphate (NADP) and increased activities of G6Pase and GSase. The presence of bacteria or parasites in fish under NWD resulted in increased cortisol levels in plasma whereas in liver, changes were of minor importance including decreased levels of lactate and GSase activity. The simultaneous presence of bacteria and parasites in fish under NWD resulted a sharp increase in the levels of cortisol in plasma and decreased levels of glucose. Decreased levels of glycogen and lactate and activities of GSase and glutathione reductase (GR), as well as increased activities of glucose‐6‐phosphate dehydrogenase (G6PDH), 6‐phosphogluconate dehydrogenase (6PGDH) and levels of nicotinamide adenine dinucleotide phosphate (NADPH) occurred in the same fish in liver. Finally, the presence of pathogens in S. maximus under stressful conditions elicited by LWD resulted in synergistic actions of both type of stressors in cortisol levels. In liver, the presence of bacteria or parasites induced a synergistic action on several variables such as decreased activities of G6Pase and GSase as well as increased levels of NADP and NADPH and increased activities of GPase, G6PDH and 6PGDH.     

Drinking rate, uptake of bacteria and microalgae in turbot larvae

The drinking rate of turbot larvae increased from 14 to 120 nl larva^-1 h^-1 from day 2 to 11 after hatching, which gave a slightly increased specific drinking rate (calculated per biomass) from day 2 to 7 (0·8–1·9 nl μg carbon^-1 h^-1. The clearance rate of both algae and bacteria was 10–100 times higher than the drinking rate, which indicated that the larvae had an active uptake of both algae and bacteria. On day 2 and 4 after hatching highest clearance rate was observed for Tetraselmis sp. On day 6 about the same clearance rate was observed for bacteria, Isochrysis galbana and Tetraselmis sp. Until day 4 the turbot larvae had a higher ingestion rate of Tetraselmis sp. than of I. galbana , whereas on day 6 the rates were similar (28–41 ng carbon larvae^-1 h^-1). The assimilation efficiency was somewhat higher for I. galbana than for Tetraselmis sp., and on day 6 the assimilated algae constituted 1·5 and 0·9% of the larval biomass for I. galbana and Tetraselmis sp., respectively.     

Antioxidant enzyme activity and growth responses of Huangguogan citrus cultivar to nitrogen supplementation

ABSTRACT

This study examined the physiological effects of different amounts of nitrogen (N) supplementation (0 to 2.72 kg/year) on the citrus cultivar Huangguogan (Citrus reticulata × Citrus sinensis). Root activity, chlorophyll content, and fruit quality were measured, and the activities of the antioxidant enzymes superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT), and the contents of malondialdehyde (MDA) and soluble protein in root, leaf, and fruit tissues were examined at different developmental stages. Root activity, chlorophyll content, fruit quality, antioxidant enzyme activity, MDA content, and soluble protein content increased in plants treated with an appropriate amount of N. Both excessive N and N deficiency decreased the content of MDA and the activities of antioxidant enzymes. Application of 1.36–1.81 kg N/year is suggested for citrus fertilization and the lower end of this range is recommended for minimizing environmental impact and production cost.