GSTA is a major glutathione S-transferase gene responsible for 4-hydroxynonenal conjugation in largemouth bass liver

We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic α,β-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein. Liquid chromatography mass spectrometry (LC–MS) and electrospray ionization analysis of the major bass GST subunit yielded a molecular weight of 26,396 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST peak demonstrated that this protein was encoded by bass GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that contained GSTA, and was similar to a GST gene cluster characterized by Leaver et al., in plaice. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. This GST is part of a gene cluster that may be conserved in certain freshwater and marine fish.     

Comparison of xenobiotic metabolizing enzymes of Tilapia with those of other fish species and interspecies relationships between gene families

Baseline data for hepatic xenobiotic metabolizing biomarker enzyme activities were obtained for artificially reared tilapia Oreochromis niloticus, and were compared with those of the plaice (Pleuronectes platessa) and rainbow trout (Onchorynchus mykiss). Basal activities exhibited species variations with notably higher CYP1A and phenol UGT activities and lower GST activity in plaice than the freshwater species. Interspecies relationships between gene families determined by immunoblotting and substrate-activity profiles demonstrated the presence of homologous CYP1A and CYP3A enzymes in all three species, alpha class GSTs in plaice and trout, mu and pi class GSTs in trout and theta class GSTs in plaice and tilapia. CYP1A of tilapia was induced by 3-MC or PBO treatment, whilst CYP3A was induced by PCN treatment.     

Regulation of hepatic glutathione S-transferase expression in flounder

The major glutathione S-transferase isoform of flounder liver, an antigenically related structural homologue of plaice GST-A, also displays mRNA homology. A 901bp cRNA probe for plaice GST-A cross-hybridised to a 1100bp flounder mRNA on northern blot analysis. The plaice antibody and cRNA probes were used to study effects of inducer treatment on GST-A expression in flounder liver. Six days after PAH treatment (3-methylcholanthrene) total hepatic GST activity was halved, levels of GST-A were 80% and GST-A mRNA levels were 25% of controls. A commercial PCB mixture (Aroclor 1254^TM) had little effect on total GST or GST-A levels despite halving GST-A mRNA levels. An epoxide, trans-stilbene oxide induced total GST activity 1·4 fold and GST-A protein levels 1·8-fold and its mRNA levels 3-fold. This reduced expression of the major flounder hepatic GST by agents which induce cytochrome P4501A1 may modulate cytoxicity of environmental pollutants in this species.     

GSTA is a major glutathione S-transferase gene responsible for 4-hydroxynonenal conjugation in largemouth bass liver

We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic alpha,beta-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein. Liquid chromatography mass spectrometry (LC-MS) and electrospray ionization analysis of the major bass GST subunit yielded a molecular weight of 26,396 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST peak demonstrated that this protein was encoded by bass GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that contained GSTA, and was similar to a GST gene cluster characterized by Leaver et al., in plaice. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. This GST is part of a gene cluster that may be conserved in certain freshwater and marine fish.     

Structural Studies of a UDP-glucuronosyltransferase gene from the plaice (Pleuronectes platessa)

A partial length cDNA coding for the putative PAH-inducible phenol-conjugating UDP-glucururonosyltransferases (UGT) isoform of plaice was used to isolate overlapping clones from a plaice genomic library. Sequence analysis revealed the presence of a complete gene spanning 4.1 kb for a plaice UGT which showed a strong conservation in exon structure, amino acid character and amino acid sequence with mammalian UGT1 family genes, although additional alternative upstream exon 1s were not identified in the present study. Southern blot analysis revealed a low copy number for the gene and some degree of structural polymorphism in gene structure between individuals. This was also reflected in the finding that there were significant variations between the nucleotide sequences of the plaice gene and the cDNA previously isolated from a different individual fish. Future studies will investigate the possibility that there may be phenotypic variations which could lead to alterations in susceptibility to pollutant toxicity.     

A piscine glutathione S-transferase which efficiently conjugates the end-products of lipid peroxidation

A cDNA clone for glutathione S-transferaseA (GSTA) from plaice (Pleuronectes platessa) was expressed in Eschericia coli (E. coli) and purified to homogeneity by S-hexylglutathione affinity chromatography. When compared to literature values for a variety of purified mammalian GSTs, the heterologously expressed purified plaice enzyme had moderate activity towards the model substrate 1,2-chloro-2,4-dinitrobenzene (CDNB) and exhibited a Km of 2.5 ± 2 mM and Vmax of 30.9 ± 2.3 μmol min⁻¹ mg⁻¹. It had little or no activity towards several other model GST substrates including 1,2-dinitrochloro-4-benzene (DCNB), ethacrynic acid (EA), and p-nitrobenzylchloride (NBC). However plaice GSTA was a relatively efficient catalyst for the conjugation of a series of alk-2-enals and alk-2,4-dienals and also 4-hydroxynonenal. The highest activity observed with this series of substrates was with trans-non-2-enal with a Km of 17.9 ± 2.2 μM and a Vmax of 3.01 ± 0.57 μmol min⁻¹ mg⁻¹. These unsaturated alkenals have been identified in cells and cell extracts as highly toxic products arising from peroxidation of unsaturated fatty acids particularly during periods of oxidative stress. Fish are relatively rich in polyunsaturated fatty acids and thus GSTA mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products.     

The cytochrome P450 1A gene (CYP1A) from European flounder (Platichthys flesus), analysis of regulatory regions and development of a dual luciferase reporter gene system.

Concensus primers designed to CYP1A-conserved regions were used to amplify a 1.3 kb probe from flounder genomic DNA via polymerase chain reaction (PCR). A 14-kb clone was isolated from a flounder genomic library constructed in lambda FIXII. Of this clone, 8 kb was sequenced, including 3 kb of upstream sequence. The predicted amino acid sequence showed closest similarity to plaice CYP1A1 (98%). Gene structure conformed to the seven exons and six introns common to previous CYP1A sequences, but intron lengths were not conserved. Concensus sequences corresponding to xenobiotic and other response elements as well as TATA, CAAT and GC boxes were identified. Upstream sequence (3.5 kb) including the first exon and intron up to the putative start codon were amplified via PCR and inserted upstream of the luciferase gene in a pGL3 reporter gene construct. The HepG2 mammalian hepatoma cell line was transiently co-transfected with the flounder CYP1A reporter gene construct and the pRL-CMV internal control construct. The maximal induction upon exposure to 100 nM 3-MC was 4.4-fold in comparison with carrier-treated cells. Use of deletion constructs resulted in loss of inducibility.     

The effect of salinity on growth and weight loss of juvenile plaice (Pleuronectes platessa, L): An experimental test

Previous population estimates of the 0+ plaice (Pleuronectes platessa L.) in the Firth of Forth, east central Scotland, did not take account of the Forth estuary west of the Forth bridges. Previous work found plaice in the estuary grew as fast as, or faster than, the outer firth plaice. It was hypothesised that salinity may affect growth rates of early 0+ plaice. This hypothesis was tested in a laboratory experiment, by exposing juvenile plaice to three different, but naturally — experienced by the juveniles, salinities; 25, 30 and 35. Plaice fed a minimum ration did not grow in length. Mean weight decreased at all three salinities, however, the lowest weight loss was found at the lowest salinity (25) and the highest weight loss was found at the highest salinity (35). The minimum feeding ration was halted and plaice were then fed ad libitum. Consumption rates were not significantly different during the ad libitum feeding, while significant differences in mean weight change were found between the highest and lowest salinities.     

大叶藻微粒体GST基因对温度胁迫响应的分子机制

为了分析鳗草（Zostera marina）中微粒体谷胱甘肽S-转移酶在不同温度下的基因表达规律以及响应机制，本文通过在大肠杆菌中表达ZmGST，纯化重组蛋白以及热稳定性分析，以此为进一步阐述Z. marina的种群退化机制并提供理论依据。总之，鳗草中的微粒体GST的热稳定性和其对温度变化的响应决定了其对温度的耐受范围，并进而影响其恢复力。     

Continental shelf nurseries and recruitment variability in American plaice and yellowtail flounder on the Grand Bank: insights into stock resiliency

In 1994, a directed fishing moratorium was declared for Grand Bank American plaice (Hippoglossoides platessoides) and yellowtail flounder (Limanda ferruginea) stocks because both stocks showed severe declines in abundance from heavy exploitation during the mid 1980s and early 1990s. Four years later, the fishery for yellowtail re-opened while the plaice stock has shown little recovery and the moratorium is still in effect. To assess the possible causes of the differences in recovery between species, we examined the spatial structure and environmental characteristics of the continental shelf nursery habitats of plaice and yellowtail, and their relationship to recruitment variability and overall population size. Depth plays a major influential role determining the spatial pattern and the abundance of juveniles of both species and in the case of plaice the spatial structure of the adult population also determines the amount of nursery area utilised by juveniles. Recruitment variability was higher in plaice than in yellowtail. We found year class synchrony in both species indicating that common environmental conditions and/or biological processes are affecting recruitment in a similar manner. Density-dependent regulation appears to be more severe in yellowtail and this should contribute to a more stable population when compared to plaice. These results are discussed in terms of resiliency of both stocks to over-exploitation.     

Characterization and molecular analysis of hepatic microsomal UDP-glucuronosyltransferases in the marine fish, Pleuronectes platessa

Here we report the results of the first molecular enzymological study of fish UDP-glucuronosyltransferase (UDPGT). UDPGT activities of plaice liver microsomes were greatest for planar phenols and there was low but measurable conjugation of bilirubin, testosterone and androsterone. A highly purified preparation was isolated which possessed activities towards 1-naphthol, bilirubin and testosterone, containing several molecular weight species of M_r 52–56 kDa. On immunoblot analysis these proteins cross-reacted with a polyspecific anti-rat UDPGT antibody, suggesting that a number of plaice UDPGT isoforms with epitopes in common with the corresponding rat enzymes had been purified.     

Changes in the spatial distribution of North Sea plaice (Pleuronectes platessa) and implications for fisheries management

To protect the main nursery area of plaice, an area called the ‘Plaice Box’ was closed to trawl fisheries with large vessels in 1989, with the expectation that recruitment, yield and spawning stock biomass would increase. However, since then the plaice population has declined and the rate of discarding outside the Plaice Box has increased, suggesting an offshore shift in spatial distribution of juvenile plaice. Using research vessel survey data collected since 1970, the change in distribution of juvenile age groups was analysed in relation to the distance to the coast. Further, a comparison of the distribution of different length classes of plaice between three historic periods was made (1902–1909; 1983–1987; 1999–2003). A shift towards deeper water of larger-sized plaice (20–39 cm) is apparent already before the 1980s and may be related to the decrease in the number of competitors or predators. An offshore shift in the distribution of young plaice occurred in the 1990s most likely in response to higher water temperatures that may have exceeded the maximum tolerance range or increased the food requirements above the available food resources. A decrease in competition with larger plaice offshore, possibly in combination with increased inshore predation by cormorants and seals, may also have played a role. The offshore shift in distribution has reduced the effectiveness of the Plaice Box as a technical measure to protect the under-sized plaice from discarding, since an increased proportion of the population of undersized plaice is moving to the more heavily exploited offshore areas.     

杜氏盐藻叶绿体ATP合酶α亚单位基因启动子的克隆及初步功能分析

采用DraⅠ,EcoRⅤ,PvuⅡ,ScaⅠ,SmaⅠ和StuⅠ六种内切酶消化杜氏盐藻（Dunaliella salina）叶绿体基因组DNA,与相应DNA接头连接,构建成无载体连接的盐藻叶绿体Genome Walker DNA文库。利用长距离PCR（long distance-polymerase chain rea...     

A comparative analysis of recruitment variability in North Atlantic flatfishes — testing the species range hypothesis

The hypothesis that recruitment variation in flatfishes should be most variable at the northern edge of the species range, least near the centre of the range, and intermediate near the southern limit was tested using stock and recruitment data generated from sequential population analysis for several different flatfish stocks involving four species (plaice Pleuronectes platessa, sole Solea vulgaris from the eastern Atlantic, American plaice Hippoglossoides platessoides, and yellowtail flounder Limanda ferruginae from the western Atlantic). Several groundfish species have been found to conform to this so-called species range hypothesis with the suggestion that density-independent processes predominate at the edges of the distributional range and density-dependent processes dominate in the centre of the range. Our results were generally inconsistent with the hypothesis: the coefficient of variation (CV) of recruitment for plaice in the eastern Atlantic was independent of latitude, the CV of recruitment for sole exhibited a dome-shaped relationship with latitude with the highest CVs occurring at the mid-point of the range, and the CV of recruitment for the western Atlantic stocks exhibited a monotonic decrease with latitude. We extended our latitudinal analyses by assessing both the degree of dependency of recruitment on spawning stock biomass and the spatial and temporal scales of variability in recruitment and pre-recruit survival for the eastern Atlantic stocks. In general our analysis revealed no evidence of a strong stock and recruitment relationship for any of the stocks examined, and previously published analyses revealed no such patterns with latitude. Analysis of both de-trended recruitment and pre-recruit survival time series over the species ranges of sole and plaice revealed strong positive correlations among adjacent stocks and inverse correlations among stocks at the extremes of the range. Recruitment variation in the flatfish stocks examined appears to be dominated by density-independent factors, operating at a local scale, on the egg and larval stages.     

Marine invertebrate glutathione-S-transferases: purification, characterization and induction

High glutathione-S-transferase (GST) activity was found in hepatopancreas and gill cytosol of the blue crab (Callinectes sapidus) and the digestive gland cytosol of two marine gastropods (Nassarius obsoletus and Cerithium floridanum).Purification of GST from crab hepatopancreas by Sephadex G-200, DEAE-Sephacel and chromofocusing resulted in the isolation of two isoenzymes with isoelectric points of 5·9 and 5·7 (GST 5·9 and GST 5·7). Antibodies were prepared to these two isoenzymes and the two forms cross-reacted immunologically. The two transferases had similar molecular weights, amino acid compositions, substrate specifities and kinetic parameters.Crab gill cytosol showed one isoenzyme which reacted with antibodies to GST 5·9 and GST 5·7. The major isoenzyme of N. obsoletus was a basic form while C. floridanum showed a homodimer acidic form. The gastropod GST forms did not react with antibodies to crab GST. The presence of the phenolic antioxidant, butylated hydroxytoluene, in the diet of blue crab or shrimp (Penaeus aztecus) resulted in high hepatic GST activity.     

Carbon stable isotopes in estuarine sediments and their utility as migration markers for nursery studies in the Firth of Forth and Forth Estuary, Scotland

The stable carbon isotope ratios (δ¹³C) of the organic fraction of intertidal sediments in the Forth Estuary and the Firth of Forth, Scotland, were measured to determine if terrestrially derived carbon was present in the estuarine sediments. It was hypothesised that differences in the inputs from marine vs. terrestrial sources to the organic carbon of estuarine and marine sediments, as well as differences in ambient seawater stable oxygen isotope (δ¹⁸O) ratios between the estuary and the Outer Firth, would allow the use of these two stable isotopes as habitat markers for juvenile plaice (Pleuronectes platessa), to allow determination of nursery habitats. Muddy and sandy sediments from the estuary and sandy sediments from the Outer Firth were sampled and δ¹³C measured. Juvenile plaice were caught at two estuarine sites and at two Outer Firth sites and otoliths were removed for δ¹³C and δ¹⁸O analysis. The sandy sediments in the estuary showed a strong gradient of δ¹³C enrichment with distance down the estuary, while the muddy sediments showed a much shallower gradient. δ¹³C and δ¹⁸O measured in the carbonate of juvenile plaice otoliths showed no clear difference between otoliths of fish caught at one of the estuarine sites and at the two Outer Firth sites. However, the isotope ratios of both carbon and oxygen in plaice otoliths from the other estuarine site showed the expected trend of depletion in the heavier isotopes. While the measurements recorded here did not conclusively distinguish between otoliths from juveniles caught in the estuarine site and those caught in the other three sites, they show that stable isotopes have potential to distinguish between estuarine habitats with terrestrial carbon inputs, and coastal marine habitats with predominantly marine carbon inputs.     

The impact of intrapopulation variability in reproductive traits on population reproductive potential of Grand Bank American plaice (Hippoglossoides platessoides) and yellowtail flounder (Limanda ferruginea)

In geographically extensive fish populations the potential exists for reproductive traits to vary over the population’s range but, the impact that such intrapopulation variability has on overall population reproductive potential has not been formally assessed. Here intrapopulation spatial variability in size at maturity and fecundity are demonstrated for Grand Bank American plaice (Hippoglossoides platessoides) and yellowtail flounder (Limanda ferruginea). Recognition of intrapopulation variability in these reproductive traits coupled with spatial variability in abundance resulted in an increase in estimated population total egg production (TEP) by as much as 10¹⁴ eggs for American plaice and 10¹⁵ eggs for yellowtail flounder as compared to assessment of TEP for the population as a whole. Results highlight the need to explore variability in life history traits not only between but, also within populations and emphasizes the need for sufficient spatial coverage during sampling in order to assess the reproductive potential of fish populations.