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Paramisgurnus dabryanus (Cypriniformes; Cobitidae), has been an emerging aquaculture species in China since the 1990s. In this study, random amplified polymorphic DNA fingerprinting with 220 primers was used to identify a sex-specific DNA marker in pooled DNA and individual DNA samples from male and female P. dabryanus. One primer, S2115, produced a novel sex-specific DNA fragment found only in tested females. This female-specific fragment was 917 bp with 36% GC content, and was named Pdff1. To further validate the authenticity of this female-specific marker for sexing, two PCR primers (pdff1-F and -R) were designed according to the cloned female-specific sequence. Amplification showed bands specific for females. Dot blot and Southern blot hybridization experiments both displayed female specificity using this marker as the probe. Two other P. dabryanus populations were tested by dot blot hybridization with the Pdff1 probe. The hybridization signals were seen in 33 or 43% of males in addition to all females in the Jinan and Xichuan populations, respectively. We propose to use this sex-specific marker to rapidly and specifically identify the gender of P. dabryanus from the ancient Yellow River Wetland in Yanjin, Henan Province. Our results could assist in cloning sex-specific chromosomal regions.

Cathepsin L is an important protease in the initiation of protein degradation and one of the most powerful endopeptidases. In this study, we cloned mud loach (Misgurnus mizolepis) cathepsin L (MlCtL) cDNA, and the pro-mature enzyme of MlCtL (proMlCtL) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4 T-1 vector. The recombinant proMlCtL was overexpressed in E. coli DH5αMCR as a 62-kDa protein. Its activity was quantified by measuring the cleavage of synthetic fluorogenic peptide substrates, and the protease activity of proMlCtL was also demonstrated by gelatin zymography. Antipain and leupeptin were shown to inhibit the protease activity of proMlCtL. Our results suggest that the structural features and evolutionary relationship of the mud loach cathepsin L gene were similar to that of the other mammalian cathepsin Ls; however, the proMlCtL protein was more stable at neutral and alkaline pH. The optimum temperature for the proMlCtL enzyme was found to be 40 °C. In addition, proMlCtL activity was dependent upon the presence of several metal ions and detergents.

The influence of novel biologically active substance potassium 4-toluenethiosulfonate in concentration of 4 x 10(-5) M on the changes of electrophysiological parameters of embryonic cells in early development of fish (Misgurnus fossilis L.) was investigated including the changes of membrane potential (TMP) and enzyme activity of plasmatic membranes of loach embryos during the period of synchronous division of blastomers in the early period of development. The evaluation of influence of these matters was studied and aperiodic changes of the level of TMP were shown. The diminishing of amplitude in every period by 7/12 mV and diminishing of growth of maximal values of vibrations of TMP by 39 mV in comparison to control was noticed. It was related to inhibition of some biosynthetic processes and results in the decline of activity of membrane enzyme (Na+, K(+)-ATPase) by 75.5 +/- 4.1% and 78.4 +/- 10.4% both at the action of high (4 x 10(-3) M) and low (4 x 10(-9) M) concentrations, accordingly, at first hours of development with subsequent renewal of its activity to the level of control only for the actions of low concentration.

17alpha-ethynylestradiol (EE2), a synthetic estrogen prescribed as a contraceptive, was measured in Shorthead Redhorse Suckers (ShRHSs) (Moxostoma macrolepidotum) collected near a wastewater treatment plant (WWTP) in the St. Clair River (Ontario, Canada). We detected EE2 in 50% of the fish samples caught near the WWTP (Stag Island), which averaged 1.6+/-0.6ng/g (wet weight) in males and 1.43+/-0.96ng/g in females. No EE2 was detected in the samples from the reference site (Port Lambton) which was 26km further downstream of the Stag Island site. Only males from Stag Island had VTG induction, suggesting the Corunna WWTP effluent as a likely source of environmental estrogen. EE2 concentrations were correlated with total body lipid content (R(2)=0.512, p<0.01, n=10). Lipid normalized EE2 concentrations were correlated with delta(15)N (R(2)=0.436, p<0.05, n=10), suggesting higher EE2 exposures in carnivores. Our data support the hypothesis of EE2 bioaccumulation in wild fish., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The shorthead redhorse (sr) Moxostoma macrolepidotum is endemic in North America, while in Canada the copper redhorse (cr) Moxostoma hubbsi is classified as endangered. A commercial heterologous carp vitellogenin (VTG) sandwich ELISA underestimates VTGs in these species. Hybrid ELISAs, using anti-carp VTG antibodies and purified sr- and cr-VTGs as standards, were validated to measure VTGs in plasma and surface mucus of redhorse species. The hybrid ELISAs showed good precision, sensitivity and accuracy. Following induction with beta-estradiol 3-benzoate (E2B), levels of plasma VTG (mean+/-SEM) in male sr increased from 2.7+/-1.5 microg/ml to 16.5+/-2.2mg/ml. In immature cr, plasma VTG increased to 70.1+/-13.8 mg/ml after induction, from an initial value of 37.7+/-51.0 microg/ml. Levels of VTG in mucus from E2B-injected fish were highly correlated (p<0.0001) with levels in plasma, but not in a non-induced mature female. Hybrid assays are a good compromise between homologous and heterologous assays, especially for small-bodied or endangered fish species. The performance of the hybrid assays strongly suggests they could be used to determine exposure of these species to estrogenic contaminants and contribute to restoration efforts of the copper redhorse., (Crown Copyright (c) 2010. Published by Elsevier Inc. All rights reserved.)

Antidepressant pharmaceuticals are widely prescribed in the United States; release of municipal wastewater effluent is a primary route introducing them to aquatic environments, where little is known about their distribution and fate. Water, bed sediment, and brain tissue from native white suckers (Catostomus commersoni) were collected upstream and at points progressively downstream from outfalls discharging to two effluent-impacted streams, Boulder Creek (Colorado) and Fourmile Creek (Iowa). A liquid chromatography/tandem mass spectrometry method was used to quantify antidepressants, including fluoxetine, norfluoxetine (degradate), sertraline, norsertraline (degradate), paroxetine, citalopram, fluvoxamine, duloxetine, venlafaxine, and bupropion in all three sample matrices. Antidepressants were not present above the limit of quantitation in water samples upstream from the effluent outfalls but were present at points downstream at ng/L concentrations, even at the farthest downstream sampling site 8.4 km downstream from the outfall. The antidepressants with the highest measured concentrations in both streams were venlafaxine, bupropion, and citalopram and typically were observed at concentrations of at least an order of magnitude greater than the more commonly investigated antidepressants fluoxetine and sertraline. Concentrations of antidepressants in bed sediment were measured at ng/g levels; venlafaxine and fluoxetine were the predominant chemicals observed. Fluoxetine, sertraline, and their degradates were the principal antidepressants observed in fish brain tissue, typically at low ng/g concentrations. A qualitatively different antidepressant profile was observed in brain tissue compared to streamwater samples. This study documents that wastewater effluent can be a point source of antidepressants to stream ecosystems and that the qualitative composition of antidepressants in brain tissue from exposed fish differs substantially from the compositions observed in streamwater and sediment, suggesting selective uptake.

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na(+)/K(+)-ATPase, H(+)-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC(50) value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na(+)/NH(4)(+)-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H(+)-ATPase and Na(+)/K(+)-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H(+)-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H(+)-ATPase and Rhcg1 to the similar non-Na(+)/K(+)-ATPase immunoreactive cell type would support a role for H(+)-ATPase in ammonia excretion via Rhcg by NH(4)(+) trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H(+)-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H(+)-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH(3) permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.

In this study, male Chinese loaches in a semistatic waterborne exposure system were used to study the effects of tributyltin (TBT) on vitellogenin (Vtg) production induced by 17beta-estradiol (E2), TBT accumulation and distribution in tissues, and the effects of E2 on the distribution of TBT. The results demonstrate that TBT does not induce the synthesis of Vtg in male Chinese loach and that TBT could significantly inhibit Vtg production induced by E2, after exposure to binary mixtures of E2 and TBT for 14 days. TBT was found to accumulate in the liver, kidney, gills, intestines, and muscles of male Chinese loach, wherein the liver, kidney, gills, and intestines were found to have the most TBT accumulation. The existence of E2 did not significantly affect tissue distribution of TBT.

The genomic structure of manganese superoxide dismutase (Mn-SOD) was characterized in Hemibarbus mylodon (Teleostei, Cypriniformes), an endangered freshwater fish species, and changes in Mn-SOD mRNA expression in response to various stimuli, such as challenge with lipopolysaccharide (LPS), bacteria, or heavy metals, were examined. H. mylodon Mn-SOD possesses well-conserved features of vertebrate Mn-SODs, including its homologous quinquepartite gene structure (five exons interrupted by four introns) and high sequence identity with vertebrate orthologs in coding regions, including a typical eukaryotic Mn-SOD signature. The 5'-regulatory upstream region of the H. mylodon Mn-SOD gene lacked a canonical TATA sequence; however, it displayed various transcription factor binding elements that may be involved in immune and stress responses, as well as the modulation of enzymes that mediate the metabolism of metals and other xenobiotics. Bioinformatic analysis of Mn-SOD genes from fish and mammalian genome databases revealed chromosomal synteny of the Mn-SOD locus in the vertebrate lineage. Mn-SOD transcripts were ubiquitously detected in diverse tissues with variable levels of basal expression using an RT-PCR assay. Mn-SOD expression in the liver and kidney were significantly modulated by injection of LPS (1, 5, or 10 microg g(-1) body weight), Edwardsiella tarda challenge (5 x 10(3) or 5 x 10(5) cells/fish), and heavy metal exposure (Cd, Cu, or Zn at 5 microM). Results from this study suggest that Mn-SOD plays an important role in the host defence against oxidative damage caused by infection-mediated inflammation and/or toxicant related stress in this species.

Hexabromocyclododecanes (HBCDs) are now emerging ubiquitous contaminants due to their wide usage, persistence and toxicities. To investigate the bioaccumulative characteristics of HBCDs, sediments, Winkle (Littorina littorea), crucian carp (Carassius carassius) and loach (Misgurnus anguillicaudatus) were collected from two streams near an E-waste dismantling site in China, and HBCD exposure test was then conducted on Chinese rare minnow. The concentration of HBCDs was 14 ng g(-1) dry weight in sediments, 186, 377 and 1791 ng g(-1) lipid weight in winkle, crucian carp and loach, respectively. gamma-HBCD was found to be the dominant diastereoisomer in the sediments (63% of total HBCDs). However, alpha-HBCD was selectively accumulated in the biotic samples and contributed to 77%, 63% and 63% of total HBCDs in winkle, crucian carp and loach, respectively. Moreover, an enrichment of (-)-enantiomers of alpha- and gamma-HBCD were found in the winkle. The reverse results were observed in the crucian carp and loach. Similar observations of diastereoisomeric and enantiomeric composition were obtained in Chinese rare minnow with those found in the crucian carp and loach. These results indicate that the freshwater species from the streams are contaminated by HBCDs. alpha-HBCD can be selectively accumulated in organisms and the accumulative characteristics are enantioselective among species.

The idea of a major developmental transition that includes the activation of the embryonic genome has a long history. In the 1950-1960s this concept was developed to a large extent due to the efforts of Alexander Neyfakh, who described a specific type of deleterious effect resulting from X-ray irradiation of fish eggs. He interpreted the radiation-sensitive target as the nucleus and established the onset of the function of the zygotic genome, naming it the morphogenetic function of nuclei, what we call now the midblastula transition. Most of his studies were performed using the loach (Misgurnus fossilis), a European teleost. Neyfakh's efforts paved the way to understanding the whole phenomenon of the maternal-zygotic transition.

In autumn 2005, the site-specific variability of cytosolic metal and protein concentrations in gills of European chub from the Sava River could be mostly associated with gill mass variability. In spring 2006, the correlations of metals and proteins with gill mass were mainly nonsignificant, and their site-specific variability could be presumably associated with river water pollution. The spring cytosolic concentrations of Zn, Cu, and Mn did not differ significantly between the sites (medians: 8.37-11.34 microg ml(-1), 68.2-86.2 ng ml(-1), and 55.9-68.6 ng ml(-1), respectively). Increased cytosolic Cd concentrations were obtained at Oborovo and Lukavec Posavski (median: 4.01 ng ml(-1))-the sites influenced by pollution sources from two major urban areas-compared with the remaining sampling sites (median: 1.93 ng ml(-1)). Cytosolic Fe concentrations were almost two times higher at Jasenovac (median: 11.98 microg ml(-1)) compared with the concentrations at Sava-Zagreb (median: 6.72 microg ml(-1)). Labile Fe concentrations measured in river water with passive samplers indicated that cytosolic Fe concentrations in the spring possibly reflected water-borne Fe uptake. The spring cytosolic protein concentrations decreased from upstream (Otok Samoborski: 27.2 +/- 5.6 mg ml(-1)) toward the downstream sites (Lukavec Posavski: 14.8 +/- 2.8 mg ml(-1)), possibly because of the influence of organic pollution and water toxicity. The spring period seems to be a more appropriate season for the assessment of the river water pollution if chub gills are used as the target organ.

Two metallothionein genes, MLMT-IA and MLMT-IB, were isolated and characterized from the mud loach Misgurnus mizolepis (Teleostei; Cypriniformes). For these MTs, we determined a tandem "tail-to-head" genomic organizational pattern, identified conserved genomic features, showed high sequence identities in the coding regions, and examined the closest phylogenetic affiliation, suggesting their divergence by a recent gene duplication event. However, the 5'-flanking upstream regions in MLMT-IA and MLMT-IB exposed large differences in the composition and distribution patterns of various transcription factor binding motifs, especially regarding the organization of the metal response element clusters. Real-time RT-PCR assays showed that mRNA levels of both MLMT-IA and MLMT-IB isoforms were variable among tissues and the ratios between them were also variable across tissues, although the MLMT-IA was always predominant in every adult tissue tested. We also found that the MLMT-IA and MLMT-IB mRNA expression levels were regulated dynamically during embryonic and larval development stages, in which the basal expression level of MLMT-IA was also consistently higher than that of MLMT-IB. Upon acute in vivo metal exposure to cadmium, chromium, copper, iron, manganese, nickel, or zinc at 5 microM for 48 h, the transcriptional modulations of MLMT-IA and MLMT-IB were quite different from each other and the type of response was affected significantly by the kind of metals and tissues.

Vitellogenin (VTG), the egg yolk precursor protein, was purified from plasma of estradiol-3-benzoate (E2B)-treated male shorthead redhorse (Moxostoma macrolepidotum) and immature copper redhorse (Moxostoma hubbsi) by a two-step chromatographic procedure without precipitation. Intact VTGs appeared as dimers with apparent molecular masses, determined by gel filtration, of approximately 425 kDa (copper redhorse) and approximately 450 kDa (shorthead redhorse). In native polyacrylamide gel electrophoresis (PAGE), dimeric redhorse VTGs appeared as a 520 kDa band. Both VTGs were reduced to a single monomer of approximately 150 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, indicating that monomers are not linked by disulfide bonds in the dimer form. The purified proteins were characterized as phospholipoglycoproteins. Isoelectric focusing of both VTGs revealed components with isoelectric points ranging from 5.3 to 6.0, suggesting charge heterogeneity. The amino acid composition of both VTGs contains a high proportion of nonpolar amino acids and was similar to those of other teleosts. An antibody developed against carp (Cyprinus carpio) VTG showed cross-reactivity with VTG from both redhorse species. Using this antibody, VTG was detected in plasma and surface mucus of E2B-treated redhorse. This is the most extensive report on purification and characterization of vitellogenin from catostomidid species.

To understand potential ecotoxicological effects of electrical and electronic equipment waste (e-waste) recycling and polybrominated diphenyl ethers (PBDEs) bioaccumulation in loaches, a semi-field experiment using Chinese loach (Misgurnus anguillicaudatus) as experimental fish was performed. Larval loaches were kept in net-cage for three months in an e-wastes recycling site and a reference site in Southeastern China. There was significant difference of the survival rate between the loaches from the e-wastes recycling site (27%, 19/70) and from reference site (70%, 49/70). Histopathological responses were also found in all the livers examined in loaches from the e-wastes recycling site. These results showed that mixed pollutants resulted from e-wastes recycling led to ecotoxicological effects on loaches. The bioaccumulation of polybrominated diphenyl ethers (PBDEs), the main pollutants in e-waste, in loaches was also studied, the mean concentration of total PBDEs in sediment was 6726.17 ng/g wet weight and in water samples was 4.08 ng/L (dissolved phase). BDE 209 was the dominant congener in sediment and with relatively high concentration in water. Relatively low concentration of BDE 209 (less than 0.01% of total PBDEs) and high concentration of BDE47 (up to 39.34% of total PBDEs) were detected in loaches.

Metallothionein gene was characterized in Hemibarbus mylodon, an endangered fish species. H. mylodon MT shared a high homology with other vertebrate MTs, including (1) tripartite exon/intron structure, (2) typical regulatory elements such as MREs and GC boxes in the 5'-flanking region, and (3) high proportion of Cysteines (33.3%) in its amino acid sequence. MT mRNA was ubiquitously detected in various tissues. Basal level of MT mRNA was the highest in ovary while the lowest in heart. Transcription of MT was highly inducible by exposures to waterborne cadmium (0.1-10 microM), copper (2-10 microM) or zinc (2-10 microM), based on real-time RT-PCR. Cadmium was more potent for the stimulation of MT transcripts than copper and zinc. Liver was more responsive to heavy metals than kidney and gill. In overall, the transcriptional activation of MT gene by metal exposures followed a dose- and/or time-dependent fashion.

Transient expression of recombinant plasmids carrying the lacZ gene under the control of either bovine alphaS1-casein gene tissue-specific promoter-enhancer region or highly homologous goat alphaS1-casein gene promoter-enhancer region with supplementary regulatory sequences of the goat gene were studied in Misgurnus fossilis L. loach embryos. It has been shown previously that the expression of the constructs carrying these regulatory elements in transgenic mice occurred primarily in the mammary glands. At early developmental stages, loach embryos and early prelarvae showed nonspecific and mosaic transient expression of lacZ carrying casein regulatory sequences. Transgenic activity was the highest in 1-3-day embryos. At the same time, the efficiency of expression of lacZ gene carrying regulatory sequences of the alphaS1-casein gene of goat was higher than with the promoter-enhancer region of the bovine alphaS1-casein gene. Thus, regulatory sequences of the bovine or goat alphaS1-casein gene appeared capable of providing similar transient expression of reporter gene in the loach embryos. This model can be used for rapid testing of promoter-enhancer activity of transgenes.

The fish lipid was extracted from the body muscle (edible portion) of fresh Lepidocephalus guntea (Ham.) fish and the lipid content of the fish in size-1 (below 8.5 cm), size-2 (8.5-9.5 cm) and size-3 (above 9.5 cm) were found to be 2.515, 3.013 and 3.455%, respectively. The specific gravity and refractive index of the lipid were found to be 0.93 at 30 degrees C and 1.467 at 31.5 degrees C, respectively. The saponification value, saponification equivalent, iodine value, peroxide value and acetyl value of the lipid were found to be 220.325, 254.624, 96.05, 1.993 and 11.32, respectively. The acid value, percentage of free fatty acid as oleic and unsaponifiable matter present in the lipid were found to be 2.005, 1.008 and 0.593, respectively. The fatty acid composition of the lipid was determined qualitatively and quantitatively by TLC and GLC. The analysis revealed that the fatty acid composition of the lipid lies between C14 to C20. The fish lipid of Lepidocephalus guntea (Ham.) was found to contain (average value) myristic acid (3.17%), palmitoleic acid (7.45%), palmitic acid (29.16%), linolenic acid (7.13%), linoleic acid (5.57%), oleic acid (22.93%), stearic acid (17.42%), arachidonic acid (7.17%) and arachidic acid in trace amount.

The modulation influence of Misgurnus anguillicaudatus polysaccharide on the expression of nitric oxide synthase (NOS), B cell lymphoma/leukemia-2 (Bcl-2, hepatocyte apoptosis inhibitor) and Bcl-2 associated X protein (Bax, hepatocyte apoptosis promoter) in mice's liver with immunological hepatic injury was studied. Immunological hepatic injury was induced by lipopolysaccharide (LPS ip, 0.2 mg kg(-1)) in bacillus calmette-guerin (BCG ip, 0.15 g kg(-1), once, before 7 days) primed mice. The mice were treated with M. anguillicaudatus polysaccharides (MAP) at doses of 30 mg kg(-1), 60 mg kg(-1), respectively, ig, once a day, and sacrificed on the 8th day after ip LPS for 4 h. In comparison to the normal mice, the nitric oxide production, serum alanine aminotransferase (sALT) and serum glutathione s-transferase (sGST) levels were increased significantly, iNOS and Bax expression were up-regulated by 16.5 times (P<0.001 vs. normal animal group) and 0.43 times (P<0.05, vs. normal animal group) respectively, cNOS expression was not apparently changed, and no Bcl-2 expression was found in immunological hepatic injury mice. The M. anguillicaudatus polysaccharide (30 mg kg(-1)) could reduce sALT, sGST and nitric oxide production levels (vs. BCG-LPS model control group) by 25.1%, 42.6% and 17.8% respectively, and the expression of iNOS and Bax was decreased (vs. BCG-LPS model control group) by 80.3% and 38.4%, while the expression of cNOS and Bcl-2 increased (vs. BCG-LPS model control group) by 58.7% and 352%, respectively.

Estrogen receptors (ERs) play essential roles in estrogen function. Because they may be targets of environmental chemicals, concern exists that exposure to some anthropogenic agents may result in disruption of endocrine systems in human and wildlife populations. Various kinds of ERs have been reported, and a number of in vitro assays have been established to test hormone-like activity of chemicals. In the present study, cDNAs encoding ERs from fathead minnow (Pimephales promelas) and bluegill (Lepomis macrochirus) fish, which are useful for environmental monitoring, were cloned and sequenced. Four complete, full-length ERalpha and ERbeta cDNA sequences could be derived, with an ATG start site and a TGA termination signal included in each. Fathead minnow ERalpha has 602 amino acid residues, bluegill ERalpha1 has 582, bluegill ERalpha2 has 506, and bluegill ERbeta has 554. When the cDNAs were inserted into the pRc/RSV vector and transfected into HeLa cells (derived from cervical cancer cells) with a reporter plasmid, the encoding proteins were confirmed to be functional through interaction of the receptor-ligand complex with the estrogen-responsive element (ERE). Here, we report the analysis of piscine differences in ER-dependent transactivation with some chemicals using reporter gene assays. In agonist assays, fold-induction of luciferase activity significantly varied among ERs, but responsiveness to each chemical demonstrated certain similarities. These reporter gene assays appear to be very useful for estimating adverse effects of chemicals, including hormone-like or antihormone-like activity, in fish.

The functional confirmation of availability of Ca2+ transport initially-active systems in the embryo cells of loach Misgurnus fossilis L. has been obtained. Using thapsigargin, the specific inhibitor of endoplasmic reticulum of Ca2+, Mg(2+)-ATPase, this enzyme activity was divided into thapsigargin-sensitive (actually endoplasmic reticulum Ca2+, Mg(2+)-ATPase) and thapsigargin-insensitive (plasma membrane Ca2+, Mg(2+)-ATPase) constituents. The Ca(2+)-independent Mg(2+)-dependent ATPase activity makes above 39.7% of the common Ca2+, Mg(2+)-ATPase activity of embryo loach. The periodic changes of Ca2+, Mg(2+)-ATPase activity (except for the changes of plasma membrane Ca2+, Mg(2+)-ATPase activity) were found out, which coincide with periodic [Ca2+]i oscillations during the synchronous divisions of loach blastomers embryos.

The concentrations of six indicator and co-planar polychlorinated biphenyls (PCBs), 17 polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans (PCDD/Fs) were determined in soil and associated biota samples collected from a polluted and wasted farmland in southern China. The sum of six indicator PCBs (Nos 28, 52, 101, 138, 153 and 180) concentrations in biota samples were proportional to the total amount of PCBs that transferred from soil to the corresponding biota samples, and the sum six indicator PCB congeners in samples were about 20% of the total PCBs, respectively. The ratios of the sum six indicator PCBs and the total PCBs in samples collected in the same area were approximately equal. The sums of six indicator PCBs were proportional to the total PCBs that transferred from soil to plant, while the co-planar PCBs were not. Analysis of individual PCB and PCDD/F congener signatures in soil revealed the original pollutant source after transportation and biodegradation for 14 years using principal component analysis (PCA). The pattern of PCBs in soils and plants could reflect the original pollution source after transportation and biodegradation for 14 years, while the pattern of PCBs in the terrestrial animals could not reflect the primary source. The consumption foods in this region such as foraging chicken eggs, foraging duck eggs, and loach (Misgurnus anguillicaudatus) were highly contaminated, the total toxicity equivalent (TEQ) was up to 784 pg WHO-TEQ/g on basis lipid in foraging chicken eggs.

The gills are the major site of acid-base regulation in most fish. Acid-base transfer across fish gills is dominated by carbon dioxide and ammonia excretion, especially the former. Bicarbonate buffering in the blood is less than that found in mammals; regulation of ventilation has little effect on CO(2) levels in the blood and control of ventilation is not used to regulate body pH in fish. Proton ATPase (freshwater fish), Na(+)/H(+) exchangers (marine fish) and anion exchangers (marine and freshwater fish) are located in the gills. These transporters contribute to the regulation of internal pH, but little is known about how this is done in fish. Fish kept in confined water volumes acidify their environment, largely due to CO(2). This acidification augments ammonia excretion and reduces ammonia toxicity. The possible involvement of ammonia recycling in acid excretion is also discussed.

Reference values for background exposures of benzo[a]pyrene (BAP)- and naphthalene (NAPH)-type metabolites were calculated for white sucker, northern hog sucker, and rock bass from the mid-Atlantic region based on the 90th percentile of a probability-based sample. Preliminary findings are presented for common carp. Bile was collected from fish and assayed for polycyclic aromatic hydrocarbon metabolites using the fluorescent method of Lin et al. Exposure reference values for white sucker were 0.3 microg/mg protein for BAP-type metabolites and 40 microg/mg protein for NAPH-type metabolites. Values from the Mid-Atlantic region were similar to those previously reported for the Eastern Corn Belt Plains (ECBP) ecoregion. Exposure reference values of BAP-type metabolites for rock bass were 0.3 microg/mg protein and 0.4 microg/mg protein for northern hog sucker. Exposure reference values of NAPH-type metabolites for rock bass were 30 microg/mg protein for rock bass and 50 microg/mg protein for northern hog sucker. Provisional values for common carp based on 39 observations are 0.4 microg/mg protein for BAP-type metabolites and 120 microg/mg protein for NAPH-type metabolites. Small streams exhibited the greatest range of exposures, 0.015-0.689 microg/mg protein for BAP-type metabolites and from 5.7 to 159 microg/mg protein for NAPH-type metabolites. Larger streams had 0.11-0.859 microg/mg protein for BAP-type metabolites and 10.2-286.5 microg/mg protein for NAPH-type metabolites. Exposure reference values for BAP and NAPH-type metabolites could be used as a basis of comparison of exposure to PAH contamination.

mRNAs of all three types of myosin light chain proteins are expressed in skeletal muscles of both larval and adult stages of loach Misgurnus fossilis (Cobitidae) and these proteins are encoded by different genes (mlc1, mlc2, and mlc3). No difference was revealed between transcripts from larval stage and adult fish for all three mlc proteins. Our approach (RT-PCR with fish-specific mlc1, mlc2, and mlc3 primers) failed to reveal the larval form of myosin light chain protein found previously by protein electrophoresis of loach fry muscle extract. Comparative analysis of the protein structure shows high homology of MLC1 and MLC3 proteins sharing a large EF-hand calcium-binding domain. Phylogenetic analysis of MLC1 from skeletal muscles of fish and other vertebrate species is concordant with the traditional phylogeny of the group. Within the Teleostei, loach MLC1 had the highest homology with other Cyprinidae, and least with Salmonidae fishes.

This study was designed to determine the anti-proliferative, apoptotic properties of a novel polysaccharide from the loach, Misgurnus anguillicaudatus (MAP), using a human promyelocytic leukemia line (HL-60) as a model system. HL-60 cells were cultured in the presence of MAP at various concentrations (50-800 mg/l) for 5 days and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that MAP inhibited the cells viability in time and concentration-dependent characteristics. We found that anti-proliferative effect of MAP was associated with apoptosis on HL-60 cells by determinations of morphological changes and oligonucleosomal DNA fragments. In addition, the content of nitric oxide (NO) and activity of lactate dehydrogenase (LDH) release increased when the cells incubated with MAP at various concentrations and times. These investigations suggest that the polysaccharide from loach has the function of anti-proliferation and induction of apoptosis in tumor cells in vitro.

Recently, eutrophication has induced severe cyanobacterial blooms in the Naktong River, the second largest river of Korea. In the present study, lipid peroxidation and the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, were evaluated in the liver of loach (Misgurnus mizolepis) that were orally exposed to a low dose of Microcystis through dietary supplementation with bloom scum. Loach received 75 mg of dry cells/kg body weight mass (equal to 10 microg microcystin-RR/kg body mass), for 28 days under controlled conditions. Antioxidant enzymatic activity and lipid peroxidation were measured after termination of exposure. The activities of antioxidant enzyme were significantly increased in the livers of toxin-exposed loach after 28 days of exposure, as compared to control fish. However, lipid peroxidation remained stable in both groups. These results suggest that antioxidant enzymes were able to eliminate oxidative stress induced by low concentrations of microcystins and to prevent increased lipid peroxidation in the liver of loach.

This case study examines the ability of the cytochrome P4501A (CYP1A) biomarker to distinguish the pollution status of two small streams, Krähenbach and Körsch, receiving different levels of urban and agricultural impact, with low to moderate contamination by arylhydrocarbon receptor (AhR)-binding PAHs and PCBs. Brown trout, Salmo trutta, exposed in enclosure restrictions, showed significant between-stream differences of hepatic CYP1A levels. EROD activities were the better discriminator than CYP1A protein levels. The CYP1A response was consistent and repeatable over the 5-year observation period from 1995 to 1999. In contrast to brown trout, hepatic CYP1A of stone loach, Barbatula barbatula, did not clearly distinguish the streams. The findings of this long-term study lend support to the use of CYP1A as a biomarker of degraded environmental conditions, provided that sufficiently long observation periods are used to average out confounding factors, that sufficiently sensitive detection methods are used, and that a responsive monitoring species is chosen.

Razorback sucker (Xyrauchen texanus) larvae from adults exposed to selenium at three sites near Grand Junction, Colorado, for 9 months were used in a 30-day waterborne and dietary selenium study. Selenium concentrations in water averaged <1.6 microg/L from 24-Road, 0.9 microg/L from Horsethief, 5.5 microg/L from Adobe Creek, and 10.7 microg/L from the North Pond. Selenium in dietary items averaged 2.7 microg/g in brine shrimp, 5.6 microg/g in zooplankton from Horsethief east wetland, 20 microg/g in zooplankton from Adobe Creek, and 39 microg/g in zooplankton from North Pond. The lowest survival occurred in larvae fed zooplankton rather than brine shrimp. Survival of larvae at Adobe Creek and North Pond was lower in site water than in reference water. Survival of brood stock larvae was higher than Horsethief larvae even though they received the same water and dietary treatments. Arsenic concentrations in brine shrimp may have resulted in an antagonistic interaction with selenium and reduced adverse effects in larvae. Deformities in larvae from North Pond were similar to those reported for selenium-induced teratogenic deformities in other fish species. Selenium concentrations of 4.6 microg/g in food resulted in rapid mortality of larvae from Horsethief, Adobe Creek, and North Pond, and suggested that selenium toxicity in the Colorado River could limit recovery of this endangered fish.

Four groups of larval razorback sucker, an endangered fish, were exposed to selenium-laden zooplankton and survival, growth, and whole-body residues were measured. Studies were conducted with 5, 10, 24, and 28-day-old larvae fed zooplankton collected from six sites adjacent to the Green River, Utah. Water where zooplankton were collected had selenium concentrations ranging from <0.4 to 78 microg/L, and concentrations in zooplankton ranged from 2.3 to 91 microg/g dry weight. Static renewal tests were conducted for 20 to 25 days using reference water with selenium concentrations of <1.1 microg/L. In all studies, 80-100% mortality occurred in 15-20 days. In the 28-day-old larvae, fish weight was significantly reduced 25% in larvae fed zooplankton containing 12 microg/g selenium. Whole-body concentrations of selenium ranged from 3.7 to 14.3 microg/g in fish fed zooplankton from the reference site (Sheppard Bottom pond 1) up to 94 microg/g in fish fed zooplankton from North Roadside Pond. Limited information prior to the studies suggested that the Sheppard pond 1 site was relatively clean and suitable as a reference treatment; however, the nearly complete mortality of larvae and elevated concentrations of selenium in larvae and selenium and other elements in zooplankton indicated that this site was contaminated with selenium and other elements. Selenium concentrations in whole-body larvae and in zooplankton from all sites were close to or greater than toxic thresholds where adverse effects occur in fish. Delayed mortality occurred in larvae fed the two highest selenium concentrations in zooplankton and was thought due to an interaction with other elements.

The effects of agricultural chemicals on cortisol secretion, antioxidants, and lipid peroxidation were investigated in hepatic and adrenal tissue of white sucker (Catostomus commersoni) from a river (Yamaska) that drains an agricultural region in Québec (Canada). Plasma cholinesterase (ChE) activity, used as a biomarker of exposure to pesticides, was elevated in fish from the reference site compared to fish from the contaminated sites. Plasma concentrations of cortisol and thyroid hormones (T3 and T4) were higher in fish from the reference site compared to contaminated sites; reduced glutathione (GSH) levels, catalase (CAT), and glutathione peroxidase (GPx) activities were higher and lipid peroxidation (LPO) was lower. Levels of antioxidants (CAT, Gpx, and GSH) were higher (10-90%) and LPO levels were lower (50%) in the liver than in the adrenal tissue. The present in situ study provided evidence that antioxidants, lipid peroxidation, and plasma hormones were altered in fish sampled in areas impacted by agricultural chemicals. Endocrine-disrupting effects were associated with oxidative stress. The results suggest that antioxidants and lipid peroxidation could be used as markers of contaminant exposure in fish.

This study used an experimental model of a constructed wetland to evaluate the risk of mercury methylation when the soil is amended with sulfate. The model was planted with Schoenoplectus californicus and designed to reduce copper, mercury, and metal-related toxicity in a wastestream. The sediments of the model were varied during construction to provide a control and two levels of sulfate treatment, thus allowing characterization of sulfate's effect on mercury methylation and bioaccumulation in periphyton and two species of fish--eastern mosquitofish (Gambusia holbrooki) and lake chubsucker (Erimyzon sucetta). After one year in the experimental model, mean dry-weight normalized total mercury concentrations in mosquitofish from the non-sulfate treated controls (374+/-77 ng/g) and the reference location (233+/-17 ng/g) were significantly lower than those from the low and high sulfate treatments (520+/-73 and 613+/-80 ng/g, respectively). For lake chubsucker, mean total mercury concentration in fish from the high sulfate treatment (276+/-63 ng/g) was significantly elevated over that observed in the control (109+/-47 ng/g), the low sulfate treatment (122+/-42 ng/g), and the reference population (41+/-2 ng/g). Mercury in periphyton was mostly inorganic as methylmercury ranged from 6.6 ng/g (dry weight) in the control to 9.8 ng/g in the high sulfate treatment, while total mercury concentrations ranged from 1147 ng/g in the control to a high of 1297 ng/g in the low sulfate treatment. Fish methylmercury bioaccumulation factors from sediment ranged from 52 to 390 and from 495 to 3059 for water. These results suggest that sulfate treatments add a factor of risk due to elevated production of methylmercury in sediment and porewater which biomagnified into small fish, and may potentially increase through the food web.

A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCdelta, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCdelta was isolated by screening the cDNA library of mud loach liver and using the 5'-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCdelta gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCdelta isozymes (PH domain, EF-hands, X-Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCdelta shares relatively high sequence identity with mammalian PLCdelta1 (51-52%) and catfish PLCdelta (64%). The recombinant ML-PLCdelta protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni(2+)-NTA affinity chromatography. The recombinant ML-PLCdelta showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP(2)) and its activity was Ca(2+)-dependent, which was similar to mammalian PLCdelta isozymes.

Proteome analysis by two-dimensional gel electrophoresis (2-DE) together with mass spectrometry was applied to screen acute phase response (APR)-related proteins with low molecular weight in loach skin following injury. Furthermore, Western blotting and function tests were applied to confirm the results obtained from the proteomic study. Fifteen APR-related proteins with sixteen spots (PLA with two spots) on a 2-DE map were identified in this study. Furthermore, six were known acute phase proteins including galactose-binding lectin (GBL), lysozyme, C3, CD59, double PLA and 50s ribosomal protein; while ATP kinase, zinc finger protein 183, alpha-neurotoxin homology, angiostatin, serine/threonine kinase, metalloproteinase inhibitor, regulator of G-protein 4, cryptdin-9 and disintegrin trigranin were found by our lab to be APR-related proteins. In addition, our results suggest that proteomes with low molecular weight can be characterized by 2-DE with a Tris-tricine system followed by mass spectrometry.

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.

Previous laboratory studies indicate that coal combustion wastes (a mixture composed of fly ash and other lower volume wastes such as bottom ash; hereafter collectively referred to as ash) adversely affect the health of benthic fish (Erimyzon sucetta; lake chubsucker), but fish in these studies were provided with ample uncontaminated food resources. Because aquatic disposal of ash can also adversely affect food resources for benthic fish, we hypothesized that changes in resources might exacerbate the effects of ash on fish observed in laboratory studies. We exposed juvenile E. sucetta in outdoor microcosms to water, sediment, and benthic resources from an ash-contaminated site or a reference site for 45 days and compared our findings to previous laboratory studies. Benthic invertebrate biomass was nearly three times greater in controls compared to ash microcosms. Total organic content of control sediment (41%) was also greater than in ash sediments (17%), suggesting that additional benthic resources may have also been limited in ash microcosms. Benthic invertebrates isolated from the ash microcosms had trace element concentrations (As, Cd, Co, Cr, Cs, Se, Sr, and V) up to 18 times higher than in weathered ash used in laboratory studies. The concentrations of trace elements accumulated by fish reflected the high dietary concentrations encountered in the ash microcosms and were associated with reduced growth (final mass = 0.07 g) and survival (25%) compared to controls (0.37 g and 67%, respectively). Accumulation of trace elements, as well as reductions in growth and survival, were more pronounced than in previous laboratory studies, suggesting that resource conditions may be important in mediating ash toxicity. Taken together, our studies suggest that ash discharge into aquatic systems is a more serious threat to the health of benthic fish than previously predicted based upon laboratory toxicity tests.

The oriental weatherloach Misgurnus anguillicaudatus is an extremely ammonia-tolerant fish. Many ammonia-protection mechanisms have been reported in this fish. Six strategies used by this fish to deal with the problem of excess ammonia are described. The fish can (1) reduce ammonia production through reduction in protein and/or amino acid catabolism; (2) reduce ammonia production and obtain energy through partial amino acid catabolism leading to alanine formation; (3) detoxify ammonia to glutamine; (4) tolerate very high ammonia levels in its tissues; (5) get rid of ammonia as NH(3) gas and, probably, (6) possesses background K(+) channels that are impermeable to NH(4)(+). The effects of extracellular ammonia on the contraction performance of the heart from this fish were found to be the same as in rainbow trout, an ammonia-sensitive fish. It suggests that the hearts of most, if not all, fish species are protected against ammonia. MK-801, an NMDA receptor blocker, was found to have a protective effect against ammonia intoxication in the oriental weatherloach, which suggests that the NMDA receptor, as in mammals, is involved in ammonia toxicity.

While recent evidence indicates that the generation of reactive oxygen species (ROS) and associated oxidative damage are frequently observed in fish populations with exposure to pulp and paper mill effluents, the potential for ROS generation from municipal sewage effluents has not been addressed. This study investigates the utility of measures of oxidative stress in delineating the effects of both pulp and paper mill and municipal sewage discharges. Longnose sucker (Catostomus catostomus) were collected below three pulp and paper mill and two municipal sewage effluent discharges over a 3-year period within the Wapiti and Athabasca River systems in northern Alberta. Biochemical responses in longnose sucker varied between the two rivers systems, with more pronounced changes occurring within the Wapiti River. Of the suite of biochemical parameters examined, fatty acyl-CoA oxidase (FAO) activity was the most sensitive indicator of pulp and paper mill exposure, but was only infrequently induced with exposure to municipal sewage effluent. Hepatic and gonadal 2-thiobarbituric acid reactive substances, lipid hydroperoxides, and hepatic free iron were less consistently elevated with exposure to pulp and paper mill effluent than FAO activity, and were also only infrequently altered with sewage effluent exposure. Hepatic ascorbic acid, liver somatic index, and condition factor were consistently altered with exposure to both sewage and pulp and paper mill effluents. While specific biochemical and organismal responses varied with effluents and time, the collective suite of oxidative stress endpoints proved to be useful tools in identifying relative influences of municipal sewage and pulp and paper mill effluent on fish populations in adjacent receiving waters.

NO is an important modulator of the brain serotoninergic function as it is co-localized with 5-hydroxytryptamine in the neurons of raphe nuclei in the brain of all the higher vertebrates. In the brain of fishes this property of NO is poorly studied. The purpose of this work was to study the neuromorphological peculiarities and distribution of NO-ergic neurons in raphe nuclei of Teleost species--carps and perch-like fishes. Using standard neuromorphological methods and the histochemical reaction demonstrating NADPH-diaphorase activity, the comparative analysis of NO-ergic neurons was performed and their localization was studied in the raphe complex of five teleost species. Within the raphe complex in fishes two main nuclei--nucleus raphe superior and nucleus raphe inferior were demonstrated. NADPH-diaphorase activity was observed in practically all the neurons of raphe niclei in species examined. Some intergroup morpho-adaptational neurochemical differences were found in the of the ventro-medial reticular formation adjacent to the raphe complex.

The effect of endogenous ammonia produced in dead fish or ammonia in ammonium bicarbonate solutions was evaluated on the viability of Clonorchis sinensis metacercariae. Viability was determined by worm recovery in rats infected with metacercariae exposed to ammonia in decaying fish or in vitro solutions. The recovery rates were 58%, 48%, 44% and 2% in fish that had been allowed to decay for 5, 10, 20 and 30 days at 4 degrees C, respectively, and this rate ( Y) was inversely correlated with the ammonia concentration ( X) in fish muscle extract ( Y=80.62-0.0667 X, r(2)=0.905, P=0.049). C. sinensis metacercariae barely survived in ammonia solutions with concentrations exceeding 1 g N/l. Our results indicate that the ammonia produced in dead fish is lethal to the viability of C. sinensis metacercariae.

Recent evidence indicates that contaminant-stimulated free radical production and resulting oxidative damage may be an important mechanism of toxicity in organisms exposed to water-borne contaminants. This study tested the hypothesis that increases in oxidative stress and associated biochemical alterations would be present in white sucker (Catostomus commersoni) living in an environment receiving pulp mill effluent. Consistent increases in liver TBARS and frequent increases in gonadal TBARS were observed immediately downstream of the pulp mill effluent discharge; observable effects were attenuated with distance until they were not significantly different from reference values. Increases detected with the TBARS assay were commensurate with increases detected using the lipid hydroperoxides assay, a mechanistically independent technique. Fish exposed to pulp mill effluent also exhibited significant increases in hepatic free iron and ascorbic acid and a reduced free radical scavenging capacity in the livers of fish downstream of the effluent discharge relative to reference fish. Increases in oxidative stress are not necessarily dependent on increases in lipid substrate or related to reductions in ascorbic acid. TBARS values similar to those observed in fish resident below pulp mill effluent discharges were observed in white sucker 2 h after intraperitoneal injection using 15 mg/kg body weight ferric nitrilotriacetate (Fe(3+) NTA) as a positive control. This study indicates oxidative stress could be a mechanism of toxicity in fish exposed to pulp mill effluent and demonstrates the utility of TBARS in delineating zones of exposure to pulp mill effluent.

A membrane chromatographic method was developed for the rapid purification of vitellogenin (Vtg) from the plasma of 17beta-estradiol induced loach (Misgurnus angaillicaud atus) and carp (Cyprinus carpio). The time required for the proposed procedure is less then 10 min at a flow-rate of 5 ml/min of the mobile phase, and 0.5 ml of fish plasma could be separated in one cycle. Multistep gradient elution was more suitable for the separation than linear gradient elution. Under optimized conditions, a single Vtg peak can be obtained and its identity was confirmed by SDS-PAGE and gel-permeation chromatography assessment. This method is rapid and easy to operate compared to conventional HPLC and FPLC columns for Vtg separation., (Copyright 2002 Elsevier Science B.V.)

The objective of this study was to characterize the effects of pulp mill effluent on the properties of the sex steroid binding protein (SBP) in the plasma of white sucker (Catostomus commersoni) and longnose sucker (C. catostomus). SBPs which specifically bind estradiol and testosterone with high affinity (k(D) approximately 3 nM) and low capacity (B(max) approximately 73-81 nM) were identified in both species. Subsequent studies determined if the properties of the SBP in white sucker exposed to bleached kraft mill effluent (BKME) at Terrace Bay, ON, and in longnose sucker exposed to BKME at Grande Prairie, AB. differed from appropriate reference fish. There were no effects of BKME exposure on the binding affinity (k(D)) of the SBP in either species, but there was a significant increase in the binding capacity (B(max)) of longnose sucker SBP exposed to BKME. The livers of nai;ve white sucker exposed to effluent at Terrace Bay or a bleached sulfite/groundwood mill in Edmundston, NB, rapidly accumulated compounds of differing hydrophobicity that bound to both the white sucker and goldfish (Carassius auratus) SBP. Conversely, there was reduced accumulation of SBP ligands in the bile of effluent-exposed fish. We have demonstrated that constituents present within pulp mill effluent bind to both the white sucker and goldfish SBP, and that native species residing downstream of pulp mill effluents may experience modifications in the properties of their SBP.

Elevated selenium concentrations documented in water, sediment, and biota in irrigation drain water studies by U.S. Department of the Interior agencies and academia have raised concerns that selenium may be adversely affecting endangered fish in the upper Colorado River basin. The objective of the study was to determine the effects on endangered razorback sucker (Xyrauchen texanus) larvae from exposure to selenium and other trace elements in water and zooplankton collected from sites adjacent to the Colorado River near Grand Junction, CO. A 30-day study was initiated with 5-day-old larvae exposed in a 4 x 4 factor experiment with four food and four water treatments, and the biological endpoints measured were survival, growth, development, and whole-body residues of selenium. Mean selenium concentration in reference water (24-Road) was <0.7 microg/l, in reference food (brine shrimp) was 3.2 microg/g, at Horsethief was 1.6 microg/l in water and 6.0 microg/g in zooplankton, at Adobe Creek was 3.4 microg/l in water and 32 microg/g in zooplankton, and at Walter Walker was 13 microg/l in water and 52 microg/g in zooplankton. Although there were differences in concentrations of inorganic elements in water and biota among the three sites, selenium was apparently the only element elevated to concentrations of concern. Effects on survival were more prominent from dietary exposure compared to waterborne exposure. Selenium concentrations of >or=4.6 microg/g in food organisms adversely affected the survival of razorback sucker larvae. The onset of mortality in larvae exposed to food and water from Walter Walker seemed delayed compared to mortality in larvae exposed to food and water from Horsethief, which has been observed in two other studies. Elevated arsenic in one food source seemed to interact with selenium to reduce the toxic effects of selenium.

Transgenic mud loaches (Misgurnus mizolepis), in which the entire transgene originated from the same species, have been generated by microinjecting the mud loach growth hormone (mlGH) gene fused to the mud loach beta-actin promoter. Out of 4,100 eggs injected, 7.5% fish derived from the injected eggs showed dramatically accelerated growth, with a maximum of 35-fold faster growth than their non-transgenic siblings. Many fast-growing transgenic individuals showed extraordinary gigantism: their body weight and total length (largest fish attained to 413 g and 41.5 cm) were larger and longer than even those of 12-year-old normal broodstock (maximum size reached to 89 g and 28 cm). Of 46 transgenic founders tested, 30 individuals transmitted the transgene to next generation with a wide range of germ-line transmission frequencies ranging from 2% to 33%. The growth performance of the subsequent generation (F1) was also dramatically accelerated up to 35-fold, although the levels of enhanced growth were variable among transgenic lines. Three transgenic germ-lines up to F4 were established, showing the expected Mendelian inheritance of the transgene. Expression of GH mRNA in many tissues was detected by RT-PCR analyses. The time required to attain marketable size (10 g) in these transgenic lines was only 30-50 days after fertilization, while at least 6 months in non-transgenic fish. Besides growth enhancement, significantly improved feed-conversion efficiency up to 1.9-fold was also observed.

The interaction of DNA polymerase delta purified from eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes with single-strand gaps of 1-13 nucleotides was studied. In the absence of template-restricting DNA, the enzyme elongated primers on single-stranded DNA templates in a distributive manner. However, in the presence of the proximal 5;-terminus restricting the template, the enzyme activity significantly increased. In this case, the enzyme was capable of processive synthesis by filling gaps of 5-9 nucleotides in DNA duplexes. These data indicate that DNA polymerase delta can interact with both the 3;- and 5;-termini located upstream and downstream from the gap. Analysis of the complexes formed by DNA polymerase delta and different DNA substrates by electrophoretic mobility shift assay confirmed the assumption that this enzyme can interact with the proximal 5;-terminus restricting the gap. DNA polymerase delta displayed much higher affinity in duplexes with gaps of approximately 10 nucleotides compared to the standard template-primer complexes. Maximal affinity was observed in experiments with DNA substrates containing unpaired 3;-tails in primers. The results of this study suggest that DNA polymerase delta exerts high activity in the cell nuclei during repair of DNA intermediates with single-strand gaps and unpaired 3;-termini.

The loach Misgurnus anguillicaudatus inhabits rice fields in Southern China. It encounters drought during summer and ammonia loading during agricultural fertilization. In the laboratory, aerial exposure led to decreases in its ammonia and urea excretion. Ammonia accumulated to very high levels in the muscle and liver. Urea synthesis through the ornithine-urea cycle was not involved in ammonia detoxification in M. anguillicaudatus. However, M. anguillicaudatus was capable of partial amino acid catabolism leading to the accumulation of alanine in the first 24 h of aerial exposure. This was apparently coupled to a possible decrease in protein/amino acid catabolism. These are not detoxification mechanisms but mechanisms that avoid internal fouling by ammonia. Misgurnus anguillicaudatus was also capable of detoxifying internally produced ammonia in part to glutamine, which appears to be an important adaptation after 24 h of aerial exposure. However, unlike the case of the marble goby (Oxyeleotris marmoratus), there was no alteration to the kinetic properties of the hepatic glutamine synthetase. During dry seasons, M. anguillicaudatus moves actively on land until it encounters soft mud in which it can bury itself through several strong wriggling actions of the body. Hence, it is possible that M. anguillicaudatus uses partial amino acid catabolism to fuel its short period of activities on land and switches to the formation of glutamine to detoxify internally produced ammonia when it remains relatively inactive in the mud.

It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc.

We studied the interaction of DNA polymerase delta (pol delta) purified from the eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes containing single-stranded gaps of 1-13 nucleotides (nt). In the absence of processivity factors (PCNA, RF-C, and ATP), pol delta elongated primers on single-stranded DNA templates in a distributive manner. However, the enzyme was capable of processive synthesis by filling gaps of 5-9 nt in DNA duplexes. These data suggest that, upon filling a small gap, pol delta interacts with the 5'-terminus downstream of the gap as well as with the 3'-terminus of the primer. Interaction of pol delta with the proximal 5'-terminus restricting the gap was confirmed by electrophoretic mobility shift assay. Analysis of the enzyme binding to DNA duplexes containing gaps of various sizes showed a much higher affinity of pol delta for duplexes with gaps of about 10 nt than for DNA substrates with primers annealed to single-stranded templates. The most efficient pol delta binding was observed in experiments with DNA substrates containing unpaired 3'-tails in primers. The data obtained suggest that DNA molecules with small gaps and single-stranded tails may serve as substrates for direct action of pol delta in the course of DNA repair.