Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.     

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。     

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.     

Effect of repeated sampling on haemolymph pH,PO2 and haemocyte activity in the Pacific oyster,Crassostrea gigas (Thunberg)

The effects of repeat bleeding via a novel cannulation technique, on the physiology of the Pacific oyster, Crassostrea gigas (Thunberg), were compared to control animals from which haemolymph was sampled by syringe and needle. The effects of surgery and cannulation were characterized by an initial alkalosis (up to 8 h) in the haemolymph of cannulated animals followed by an acidosis of up to 12 h; which occurred in both cannulated and control oysters. A decline in partial pressure of oxygen of oyster haemolymph, observed in both cannulated and control oysters, was directly related to shell closure following handling. Furthermore, a haemocyte concentration observed at 4 h followed by haemocyte dilution at 12 h post-surgery, suggested a handling-related stress response in both cannulated and control oysters. A heightened phagocytic activity of haemocytes at 4 h also supported the occurrence of handling-related stress response in all animals. The cannulation technique described here provides the investigator with a convenient tool for chronic, and repeated sampling of haemolymph with a transient disturbance to the animal.     

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.     

Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation

A battery of enzymes from the eukaryotic antioxidant defense system was measured in salivary gland and in whole body extract of fourth instar larvae of Chironomus ramosus with an objective of finding any clue for the dipteran insect's capacity to tolerate heavy doses of ionizing radiation. Levels of activity of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSH-Px) were quantified in 30 days old larvae exposed to LD₂₀ dose of gamma radiation. Compared to controls, activity of Cu,Zn-SOD increased 3 to 4 fold and catalase 2 fold in response to ionizing radiation stress, while activities of GR and GSH-Px enzymes were decreased. Among the other SOD isoenzymes, our results showed comparable levels of Mn-SOD and Cu,Zn-SOD activity in control and irradiated groups of larvae. The increase in levels of the Cu,Zn-SOD isoenzyme was also confirmed by Western blot and zymography supported by densitometric quantification. No evidence of Fe-SOD was found in C. ramosus larvae. These findings could help to explain the persistence of natural populations of Chironomus in radioactively contaminated regions.     

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.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

Superoxide Dismutase as an Anaerobic Polypeptide : A Key Factor in Recovery from Oxygen Deprivation in Iris pseudacorus?

The perennating organ, the rhizome, was chosen for examination of response to anoxia in the species Iris pseudacorus L., Iris germanica L. var Quechei, and Glyceria maxima (Hartm.) Holmberg. These monocots are known to differ in their tolerance of anoxia. Intact rhizomes were subjected to periods of prolonged anoxia of up to 28 days and superoxide dismutase (SOD) activity was determined in a 48 hour postanoxic recovery phase. Tests were performed to ensure the accuracy of the measured enzyme activities. In the most anoxia tolerant species, I. pseudacorus, SOD activity rose continuously during the period of imposed anoxia, and levels were maintained in the postanoxic recovery phases: 28 days brought about a 13-fold increase to 1576 U SOD per milligram protein. Small increases were found in the less anoxia tolerant I. germanica during anoxic/postanoxic phases, while a drop in activity was recorded in the least anoxia tolerant G. maxima. However, initial levels in G. maxima were more than twice as high as in the other two species. Experiments applying cycloheximide to anoxic rhizome slices of I. pseudacorus inhibited the increase in SOD activity. This indicates that SOD is, paradoxically, induced under anoxia and we suggest that in this species SOD is one of the enzymes identified as anaerobic polypeptides. The significance of the induction of an `oxygen-protecting' enzyme during complete oxygen deprivation is discussed with regard to a possible critical role during recovery from anoxic stress.     

海洋污染物对菲律宾蛤仔的免疫毒性

环境污染能够影响养殖贝类的免疫能力,是导致贝类大规模死亡的重要原因之一.探讨了大连周边4个海区污染物对采集的菲律宾蛤仔(Ruditapes philippinarum)免疫毒性影响.结果发现:污染物浓度和种类对蛤仔的免疫和生理指标具有重要影响,在重金属和石油污染物浓度较低的皮口海区,血细胞总数、亮氨酸氨基肽酶活性和血淋巴溶菌酶活性均显著高于其它3个海区(P＜0.05),而蛤仔的脂质氧化水平则较低;在重金属和石油污染物浓度较高的黑石礁海区,蛤仔血淋巴谷胱甘肽含量显著高于其它3个海区(P＜0.05);在重金属浓度较高的庄河海区,蛤仔表现出较高的超氧化物歧化酶活性(P＜0.05).     

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot^?1, 0.40 ± 0.06 U mgprot^?1, P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot^?1, 93.0 ± 6.3 nmol mgprot^?1, P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.     

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 as modulator of immune function in American white shrimp (Litopenaeus vannamei)

The immunomodulatory action of superoxide dismutase (SOD) and its possible use as an indicator of immune responses in American white shrimp (Litopenaeus vannamei) were studied. Juvenile shrimp were immersed in aerated beta-glucan and sulfated polysaccharide solutions for 6 h. SOD activity in haemocytes and muscle was quantified to evaluate whether beta-glucan and sulfated polysaccharide induce immunostimulatory activity. Haemocytes and muscle showed similar increased levels of SOD activity (1.5- and 1.4-fold that of control, respectively). Total haemocyte count decreased within the first 24 h after challenge with immunostimulants, but total haemocyte count and total soluble haemocyte protein increased over normal values after 48-120 h. Single immunostimulation with beta-glucan and sulfated polysaccharide is sufficient to generate an increase in the antioxidant activity of L. vannamei SOD.     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

Superoxide dismutase of plant cell vacuoles

Metal-dependent superoxide dismutases (SOD; EC 1.15.1.1) are present in many cell compartments (mitochondria, plastids, nuclei, peroxisomes, endoplasmic reticulum, cell wall and cytosol). We have established that SOD is also localized in the central vacuole. Cyanide-sensitive Cu, Zn-SOD was found in the fraction of isolated vacuoles of red beet roots (Beta vulgaris L.). The enzyme was represented by three isoforms. Comparison of isoenzyme composition and the level of SOD activity in vacuoles, nuclei, plastids and mitochondria isolated from root cells has shown that Cu, Zn-SOD is present in vacuoles and nuclei, two SOD forms (Cu, Zn- and Fe-SOD) are present in plastids, and two SOD forms (Cu, Zn- and Mn-SOD) are present in mitochondria. Cu, Zn-SOD of organelles, unlike vacuolar Cu, Zn-SOD, had only one isoform. The level of enzyme activity from the vacuolar fraction was twice higher than the level of SOD activity from the fractions of isolated organelles. Previously it has been suggested that Cu, Zn-SOD may be localized on the vacuolar membrane or in the near-membrane space from the side of cytoplasm. Our tests have revealed the Cu, Zn-SOD activity in water-soluble extracts of isolated vacuole fractions in the absence of detergent, which may confirm localization of the enzyme inside the organelles.     

Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to beta-glucan and sulphated polysaccharide

Juvenile American white shrimp (Litopenaeus vannamei) were immersed in aerated beta-glucan and sulphated polysaccharide solutions for 1, 3 and 6 h. Superoxide anion and SOD activity in haemocytes and muscle were investigated to evaluate whether beta-glucan and sulphated polysaccharide induce any immunostimulatory activity. Haemocytes and muscle showed different levels of superoxide anion generation and SOD activity (2.0 and 14 times that of control, respectively) when shrimp were immersed for 6 h in aerated sea water containing beta-glucan and sulphated polysaccharide. Total haemocyte count (THC) decreased within the first 24 h after challenge with immunostimulants, but THC and total soluble haemocyte protein increased over normal values after 48-120 h. Single immunostimulation with beta-glucan and sulphated polysaccharide is capable of generating an increase in the respiratory burst of L. vannamei haemocytes.     

水分胁迫及复水过程中小麦抗氧化酶的变化

对两个抗旱性不同的小麦品种进行水分胁迫和复水处理,研究其抗氧化酶活性的响应。在水分胁迫下,陇春-20的相对含水量高于优鉴-24,复水24h后,优鉴-24的相对含水量恢复较快且高于陇春-20。水分胁迫下,优鉴-24中H2O2含量增加迅速,而且各阶段含量均高于陇春-20,复水后两个品种的H2O2含量都下降,这表明优鉴-24在水分胁迫时受到更严重的氧化胁迫。采用温和胶电泳结合抑制剂实验发现小麦有3条Mn—SOD,一条Fe—SOD和Cu／Zn-SOD同工酶带,CAT同工酶有3条谱带。在水分胁迫和复水期间,优鉴-24的SOD和CAT活性高于陇春-20,随着水分胁迫程度的增加,两个品种的SOD和CAT活性都增强,复水后,优鉴-24的SOD活性继续增强,而陇春-20的Mn—SOD—3活性略微降低,Fe—SOD和Cu／Zn—SOD活性略微升高,陇春-20的CAT活性降低。水分胁迫诱导了Mn—SOD—1在优鉴-24及Mn—SOD-2和Fe—SOD在陇春-20中的表达。