Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 μg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.     

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特 的关键作用,因而也称为微囊藻毒素去毒酶. 从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列. 序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920 bp,其中5′-UTR长74 bp,3′-UTR长174 bp,编码区长672 bp,编码223个氨基酸. 应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878 bp序列. 与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

鲢鱼、鳙鱼、草鱼谷胱甘肽过氧化物酶cDNA的克隆及肝组织表达

利用RT-PCR技术分别从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)、鳙鱼(Aristichthys nobilis)及草食性鱼类草鱼(Ctenopharyngodon idella)肝组织扩增出谷胱甘肽过氧化物酶(GPX)cDNA核心序列。序列分析表明鲢鱼、鳙鱼、草鱼GPX的cDNA序列均为263bp,编码87个氨基酸。鲢鱼、鳙鱼、草鱼与斑马鱼(Danio rerio)(同属鲤科)、条石鲷(Oplegnathus fasciatus)、虹鳟(Oncorhynchus mykiss)(鲈形目)等鱼类GPX氨基酸序列同源性很高,为75%～93%;与人(Homo sapiens)、小鼠(Mus musculus)、大鼠(Rattus norvegicus)、猪(Sus scrofa)、牛(Bos taurus)等哺乳动物GPX氨基酸序列的同源性也较高,为76%～79%。以β-肌动蛋白为内参照,比较鲢鱼、鳙鱼、草鱼肝组织GPX cDNA的表达水平,鲢鱼、鳙鱼肝组织GPX的表达量远高于草鱼肝的GPX表达量,这与鲢鱼、鳙鱼大量摄入微囊藻毒素在鱼体内引发产生过量的脂过氧化物相适应。     

Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus)

Glutathione peroxidase (GPX) and glutathione S-transferase (GST) are key enzymes of cellular detoxification systems that defend cells against reactive oxygen species (ROS). In this study, we isolated the GPX and GST full-length cDNA and investigated the expression of these mRNAs from livers of olive flounder during salinity changes (35, 17.5, 8.75, 4 and 0 psu) by quantitative PCR (QPCR). GPX cDNA consists of 429 base pairs (bp) and encodes a protein of 142 amino acids. GST cDNA consists of 663 bp and encodes a protein of 220 amino acids. Both of GPX and GST mRNA expressions were the highest in 4 psu and then decreased in 0 psu. Also, the levels of Na(+) and Cl(-) decreased, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased during the experimental period. These findings provide molecular characterization of GPX and GST in olive flounder and suggest that GPX and GST play important roles in detoxification of ROS, thereby these maybe indicators of oxidative stress responses by salinity changes in olive flounder.     

斑马鱼Abcc2介导微囊藻毒素解毒功能探究

研究克隆了斑马鱼（Danio rerio）abcc2基因序列,探讨了其在微囊藻毒素（MC-LR）解毒中的潜在功能。结果表明:斑马鱼abcc2具有同哺乳动物ABCC2相似的介导荧光底物MCB外排的转运活性,MC-LR处理可显著诱导其在斑马鱼幼体中的转录表达;过表达Abcc2蛋白能显著增强ZF4细胞和斑马鱼胚胎对MC-LR的耐受性;Abcc2作为MC-LR的主要耐受因子,在组织防御和有毒物质的排泄中起重要作用,但其解毒功能还不清楚。研究结果为进一步揭示鱼类抗MC-LR积累的分子机理及培育低MC-LR残留的养殖新品种提供理论基础。     

Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major

Four overlapping cDNA fragments encoding a partial sequence for uncoupling protein 2 (UCP2) were amplified by PCR using degenerate primers from the liver of a marine teleost fish, red sea bream (Pagrus major). The partial sequence was 674 bp long, encoding 224 amino acids. The deduced amino acid sequence from the cDNA partial sequence contained the signature motifs for mitochondrial transporter protein and revealed positional identity higher than 72.8% with UCP2 from mammals. The fish UCP2 gene was highly expressed in the liver but almost undetectable in the visceral mesenteric adipose tissue. Using beta-actin as control, the UCP2 mRNA level was determined to be at least 20-fold higher in the liver than in the visceral mesenteric adipose tissues. Neither 48 h starvation nor high lipid diet had any significant effect on liver UCP2 gene expression, indicating that the abundant UCP2 gene expression was stable and might have some basic function in a fish liver that always contains high lipid content. The striking contrast of UCP2 gene expression in the two fish fat-depot organs is consistent with their large differences in oxidative capacity. We suggest that the fish liver may adapt to a constantly high fat deposit by maintaining high UCP2 expression to constrain reactive oxygen species (ROS) production and protect hepatocytes from apoptosis.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 microgram/ml) and tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-alpha exerted a transient effect on the GPX activity. The treatment with SNAP (200 microM) or sodium nitroprusside (200 microM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-alpha accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

alpha-Tocopherol transfer protein expression in rat liver exposed to hyperoxia

-Tochopherol transfer protein ( TTP), a 32 kDa protein exclusively expressed in liver cytosol, has a high binding affinity for -tochopherol. The factors that regulate the expression of hepatic TTP are not clearly understood. In this study, we investigated whether or not exposure to hyperoxia (95% O 2 for 48 h) could alter the expression of hepatic TTP. We also examined the association between the expression of antioxidant enzymes (hepatic glutathione peroxidase (GPX) and Mn-superoxide dismutase (Mn-SOD)) and the expression of hepatic TTP. The levels of thiobarbituric acid-reactive substances (TBARS) in both plasma and liver were significantly higher after rats were exposed to hyperoxia for 48 h when compared with the levels in control rats. Northern blotting showed a decrease in the expression of TTP messenger RNA (mRNA) after hyperoxia, although the TTP protein level remained constant. Expression of Mn-SOD mRNA and protein, as well as the expression of GPX mRNA, were stable after hyperoxia. These findings indicate that mRNA for hepatic TTP, rather than Mn-SOD or GPX, may be highly responsive to oxidative stress.     

Bioaccumulation, oxidative stress and HSP70 expression in Cyprinus carpio L. exposed to microcystin-LR under laboratory conditions

Microcystin-LR (MC-LR) produced by cyanobacteria are potent specific hepatotoxins. So far the pathogenesis of environmental MC-LR toxicity to aquatic organisms has not been fully elucidated. In the present study the accumulation of MC-LR was investigated in various organs/tissues of Cyprinus carpio L. (C. carpio) following exposure to MC-LR for 14 d at environmentally relevant concentrations (0.1 to 10 μg L(-1)). Results showed that the presence of MC-LR enhanced toxin accumulation in all investigated organs and the highest accumulation was found in the liver of fish exposed to 5.0 μg L(-1) of MC-LR. An EPR analysis indicated ·OH intensity in liver was significantly induced at 0.1 μg L(-1) of MC-LR and then restored when the MC-LR concentration was greater than 0.1 μg L(-1). After 14-day exposure, MC-LR (1.0-10.0 μg L(-1) of MC-LR) caused a pronounced promotion of glutathione S-transferase (GST) activity and a depletion of reduced glutathione (GSH) content in fish liver, which indicated that GSH was involved in detoxification of MC-LR and the conjugation reaction of MC-LR and GSH occurred. A mild oxidative damage was evidenced by the accumulation of malondialdehyde (MDA) level at 5.0 μg L(-1) of MC-LR exposure, but which was restored when the MC-LR concentration was increased to 10.0 μg L(-1). The responses of antioxidant enzymes and the induction of HSP70 expression might contribute to MC-LR tolerance of C. carpio. However, the protein phosphatase (PP) activities were strikingly inhibited in all treated groups. Thus, the overall toxicity of environmental MC-LR on C. carpio seems to be initiated in the liver via both the ROS pathway and the PP inhibition pathway, and the latter might be more important when ambient MC-LR concentration is greater than 0.1 μg L(-1). More importantly, these results can help to support the evaluation on the potential effects of MC-LR under common environmental concentrations.     

Thermal sensitivity of uncoupling protein expression in polar and temperate fish

Uncoupling proteins (UCP), capable of increasing proton leakage across the inner mitochondrial membrane, may play a role in the temperature-dependent setting of energy turnover in animals (and their mitochondria). Therefore, the genes and expression of fish UCP were investigated in the Antarctic eelpout Pachycara brachycephalum and a temperate confamilial species, the common eelpout Zoarces viviparus. UCP full-length cDNA was amplified from liver and muscle using RT–PCR and rapid amplification of cDNA ends (RACE). The fish UCP mRNA consists of 1906 bp in P. brachycephalum and of 1876 bp in Z. viviparus. Both zoarcid sequences contain open reading frames of 939 bp, encoding 313 amino acids, with 98% and 99% identity, respectively. Protein sequences of zoarcid UCP are closest related to fish and mammalian UCP2. For analysis of temperature-dependent expression common eelpouts were cold-acclimated from 10 °C to 2 °C and Antarctic eelpouts were warm-acclimated from 0 °C to 5 °C. Identical cDNA probes for both species were developed to investigate fish UCP mRNA expression, and protein expression levels were detected by Western Blot in the enriched membrane fraction. During cold-acclimation in Z. viviparus, mRNA levels increased by a factor up to 2.0, protein levels increased up to 1.5, in line with mitochondrial proliferation during cold-acclimation. Despite decreased mitochondrial protein content, in Antarctic eelpout UCP levels rose upon warm acclimation by a factor up to 2.0 (mRNA) and 1.6 (protein), respectively. Besides the ongoing discussion of UCP function in vertebrates, the data are indicative of a significant role of fish UCP in thermal adaptation of fish mitochondria.     

Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation

Uncoupling protein 2 (UCP2) belongs to the mitochondrial anion carrier family and partially uncouples respiration from ATP synthesis when expressed in recombinant yeast mitochondria. We generated a highly sensitive polyclonal antibody against human UCP2. Its reactivity toward mitochondrial proteins was compared between wild type and ucp2(-/-) mice, leading to non-ambiguous identification of UCP2. We detected UCP2 in spleen, lung, stomach, and white adipose tissue. No UCP2 was detected in heart, skeletal muscle, liver, and brown adipose tissue. The level of UCP2 in spleen mitochondria is less than 1% of the level of UCP1 in brown adipose tissue mitochondria. Starvation and LPS treatments increase UCP2 level up to 12 times in lung and stomach, which supports the hypothesis that UCP2 responds to oxidative stress situations. Stimulation of the UCP2 expression occurs without any change in UCP2 mRNA levels. This is explained by translational regulation of the UCP2 mRNA. We have shown that an upstream open reading frame located in exon two of the ucp2 gene strongly inhibits the expression of the protein. This further level of regulation of the ucp2 gene provides a mechanism by which expression can be strongly and rapidly induced under stress conditions.     

Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-alpha

Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-alpha in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-alpha has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-alpha.     

Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction

Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.     

Fenofibrate activates the biochemical pathways and the de novo expression of genes related to lipid handling and uncoupling protein-3 functions in liver of normal rats

Fibrates (anti-hyperlipidemic agents) enhance the mRNA expression of uncoupling protein 2 (UCP2) in the liver and that of uncoupling protein 3 (UCP3) in skeletal muscle in standard-diet-fed rats and induce a de novo expression of UCP3 (mRNA and protein) in the liver of high-fat-fed rats. Here, we report that in the liver of normal rats, fenofibrate induces a de novo expression of UCP3 and a 6-fold increase in UCP2 mRNA, whereas UCP2 protein was not detectable. Indeed, we evidenced an ORF in UCP2 exon 2 potentially able to inhibit the expression of the protein. Fenofibrate increases the expression and activity of hepatic enzymes and cofactors involved in lipid handling and UCP3 activity and, as is the case for UCP3, induces other muscle-specific genes (e.g., Carnitine palmitoyl transferase 1b and Ubiquinone biosynthesis protein COQ7 homolog). In addition, we demonstrated that in mitochondria from fenofibrate-treated rats a palmitoyl-carnitine-induced GDP-sensitive uncoupling takes place, involving UCP3 rather than other uncouplers (i.e., UCP2 and Adenine Nucleotide Translocase). Thus, the liver of fenofibrate-treated standard-diet- fed rat is a useful model for investigations of the biochemical functions of UCP3 and allowed us to demonstrate that fenofibrate programs a gene-expression pattern able to modulate lipid handling and UCP3 activation.     

The acute phase response alters cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus)

The effect of an acute phase response (APR) on cationic amino acid transporter (CAT1-3) mRNA expression in liver, muscle, bursa and thymus was determined in broiler strain chickens. The APR was initiated by injecting Salmonella typhimurium lipopolysaccharide subcutaneously (LPS; 1 mg/kg bw). In Experiment 1, CAT1-3 mRNA expression was determined at multiple time points following LPS administration. LPS increased bursa and liver total and high affinity CAT mRNA expression (P<0.05) and transiently increased pectoralis total CAT mRNA expression (P<0.05). Total CAT mRNA expression in the thymus decreased 7.7-fold from 0 to 8 h after LPS injection (P<0.05). In Experiment 2, fasted chicks were uninjected or LPS-injected. LPS increased total and high affinity CAT mRNA 2-fold in both the bursa and liver (P<0.05) and did not change thymus total and high affinity CAT mRNA expression (P>0.05). LPS increased liver weight only (P<0.05) and did not alter the plasma lysine and arginine concentration (P>0.05). In Experiments 3 and 4, thymocyte proliferation and total protein content were dependent upon the media lysine concentration (P<0.001). The inability of the thymus to compete for lysine and arginine during the APR may limit the ability of thymocytes to develop during infections.