Structure and sequence conservation of a putative hypoxia response element in the lactate dehydrogenase-B gene of Fundulus.
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Many aquatic habitats are characterized by periodic or sustained episodes of low oxygen concentration, or hypoxia, and organisms that survive in these habitats do so by utilizing a suite of behavioral, physiological and biochemical adjustments to low oxygen (1-3). In the killifish Fundulus heteroclitus, one response to prolonged exposure to hypoxia is an increase in the activity of lactate dehydrogenase-B (LDH-B), the terminal enzyme of anaerobic glycolysis, in liver tissue (4). An increase in glycolytic enzyme activity also occurs in mammalian cells during hypoxia, a process due, in part, to increased rates of gene transcription mediated by the hypoxia-inducible transcription factor, HIF-1 (5). Given that a homolog of HIF-1 has been identified in fish (6), we hypothesized that HIF might be involved in the observed up-regulation of LDH-B in F. heteroclitus. Herein, we describe the presence of DNA elements in intron 2 of the Ldh-B gene from F. heteroclitus that resemble hypoxia response elements (HRE) describedfor mammalian genes (7-10). Specifically, over a region of approximately 50 base pairs we identified two consensus HIF-1 binding sites, as well as DNA elements that may bind other transcription factors (e.g., cyclic AMP response elements; CRE). We found that these sites were perfectly conserved among geographically diverse populations of F. heteroclitus, as well as being highly conserved among multiple species in the genus Fundulus. The spacing, orientation, and sequence conservation of these putative regulatory elements suggest that they may be functionally involved in the hypoxic regulation of Ldh-B in these fish. (+info)
A novel 14-3-3 gene is osmoregulated in gill epithelium of the euryhaline teleost Fundulus heteroclitus.
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We have cloned and analyzed the full-length coding sequence and 3' untranslated region (UTR) of a unique 14-3-3 gene of the euryhaline teleost Fundulus heteroclitus, which we named 14-3-3.a. Phylogenetic analysis of the deduced amino acid sequence revealed that the 14-3-3.a gene product is most similar to vertebrate 14-3-3 zeta and beta, yet it displays considerable divergence to known classes of vertebrate 14-3-3 isoforms. The N and C termini of 14-3-3.a are the most unique regions, whereas the amino acid residues forming the amphipathic ligand-binding groove are highly conserved. F. heteroclitus 14-3-3.a mRNA expression is high in gill epithelium, moderate in intestine and brain, and low in gonads, white muscle and heart. Because 14-3-3 proteins are important molecular scaffolds and cofactors for phosphoproteins and signaling complexes, the high level of 14-3-3.a expression in gill epithelium of the euryhaline teleost F. heteroclitus suggests that it is crucial for signal transduction in gill epithelial cells. We provide evidence that 14-3-3.a is involved in osmosensory signal transduction by showing that its mRNA and protein levels in gill epithelium, but not in any other tissue analyzed, increase two- to fourfold within 24h of salinity transfer of fish from sea water to fresh water. These data are clear evidence for an important role of 14-3-3.a in the remodeling of gill epithelium during transition of euryhaline fish between plasma-hyperosmotic and plasma-hypoosmotic environments. (+info)
Nucleoside phosphonate interactions with multiple organic anion transporters in renal proximal tubule.
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The interactions of two antiviral, acyclic nucleoside phosphonates, adefovir and cidofovir, with xenobiotic transporters was studied in intact killifish (Fundulus heteroclitus) renal proximal tubules by using fluorescent substrates, confocal microscopy, and quantitative image analysis. Both drugs reduced in a concentration-dependent manner the transport of fluorescein on the classical organic anion system and transport of fluorescein-methotrexate on multidrug resistance-associated protein 2 (Mrp2). Neither drug inhibited transport of a fluorescent cyclosporin A derivative on P-glycoprotein. Inhibition of Mrp2-mediated transport was abolished by 50 microM p-aminohippurate, indicating that adefovir and cidofovir entered the cells at the basolateral membrane on the classical organic anion transport system (OAT1). Comparison of the inhibitory potencies of the nucleoside phosphonates with other substrates and inhibitors showed them to be moderate inhibitors of OAT1- and Mrp2-mediated transport. (+info)
Killifish metallothionein messenger RNA expression following temperature perturbation and cadmium exposure.
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Metallothionein (MT), a cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals. Induction of MT in mammalian and nonmammalian tissues following heavy metal exposure may serve as a defense mechanism and a biomarker of environmental exposure to chemical stressors such as toxic metals. In this study, MT messenger RNA (mRNA) expression was characterized in male and female nonspawning and spawning killifish (Fundulus heteroclitus) following an 8-day exposure to specific sublethal stressors, which included temperature perturbation (26 degrees C or 10 degrees C) and/or 6 ppb of waterborne cadmium chloride (CdCl2). Hepatic, gill, and intestinal MT mRNA, expressed as copy number per microgram of total RNA, was assessed by reverse transcriptase-polymerase chain reaction and electrochemiluminescence using winter flounder (Pleuronectes americanus) MT complementary DNA primers. Liver, gill, and intestine MT mRNA expression was significantly (P < 0.05) increased in nonspawning killifish exposed to 26 degrees C compared with those exposed to 19 degrees C (control). In addition, a significant (P < 0.05) increase in gill MT mRNA induction was observed in nonspawning killifish exposed to 6 ppb of waterborne CdCl2 compared with controls. The results of this study demonstrate significant MT mRNA induction in nonspawning killifish following short-term exposure to physiological and chemical stressors. Thus, further research may be necessary before the use of killifish MT mRNA induction as a biomarker of environmental chemical stress exposure alone. (+info)
Nitrogen metabolism and excretion in the mangrove killifish Rivulus marmoratus I. The influence of environmental salinity and external ammonia.
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At a field site in Belize, mangrove killifish Rivulus marmoratus inhabit hypersaline waters (up to 48 per thousand containing approximately 1 mmol l(-1) ammonia. We tested the hypotheses that R. marmoratus modify their nitrogen metabolism and excretion (i) by accumulating free amino acids (FAAs) and urea in the tissues during hyperosmotic stress and (ii) by shifting to ureotelism and accumulating FAAs during hyperammonia stress. Urea excretion (J(Urea)) (but not ammonia excretion, J(Amm)) displayed a diurnal pattern, with significantly less (75 %) urea excreted at night than during the day in both laboratory-reared clones and wild-caught killifish. When fish were exposed to hypersaline conditions (45 per thousand sea water), J(Urea) was significantly reduced and tissue urea and FAA levels were elevated compared with those of control fish (15 per thousand sea water). When R. marmoratus were exposed to 0, 1, 2, 5 and 10 mmol l(-1) NH(4)Cl (pH 8) for 48 h, no differences were found in J(Urea). Remarkably, prolonged exposure (10 days) to 5 mmol l(-1) NH(4)Cl (pH 8) did not result in an elevation of tissue ammonia levels. In addition, tissue urea and total FAA levels did not differ between control and ammonia-exposed fish after > or =4 days. We propose that the euryhaline R. marmoratus retain urea and FAAs within their tissues in response to extreme osmotic stress. In contrast to many ammonia-tolerant fishes, R. marmoratus do not shift to ureotelism during prolonged hyperammonia stress, nor do they convert nitrogenous wastes into FAAs. The data suggest that killifish continue to eliminate ammonia despite an unfavourable blood-to-water gradient, thereby avoiding accumulation of ammonia. (+info)
Nitrogen metabolism and excretion in the mangrove killifish Rivulus marmoratus II. Significant ammonia volatilization in a teleost during air-exposure.
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The mangrove killifish Rivulus marmoratus can tolerate prolonged periods of air-exposure (>1 month). During these periods of emersion, we hypothesized that R. marmoratus would convert potentially toxic ammonia into urea and free amino acids (FAAs). In air-exposed fish, both ammonia (J(Amm)) and urea (J(Urea)) excretion continued at approximately 57 % and 39 %, respectively, of submerged rates. Remarkably, approximately 42 % of the total ammonia excreted during air-exposure was through NH(3) volatilization. Ammonia did not accumulate in whole-body tissues of air-exposed fish, but levels of both urea and some FAAs (primarily alanine and glutamine) were up to twofold higher after 10 days. The activities of the ornithine-urea cycle enzymes carbamoyl phosphate synthetase III and ornithine transcarbamylase increased (by approximately 30 % and 36 %, respectively) in whole-body tissues of air-exposed fish, while levels of arginase remained unchanged. The activities of enzymes involved in amino acid and oxidative metabolism were not significantly different between control and air-exposed fish. Partitioning of the anterior and posterior ends of immersed fish revealed that just over half (57 %) of the total nitrogen (ammonia+urea) was excreted through the anterior end of the fish, presumably via the branchial tissues, while emersed fish increased excretion via the posterior end (kidney+skin). R. marmoratus do not undergo a shift towards ureotelism during air-exposure. Rather, we propose that R. marmoratus are able to survive on land for extended periods without significant ammonia accumulation because they continuously release ammonia, partially by NH(3) volatilization. (+info)
Role of NO in endothelin-regulated drug transport in the renal proximal tubule.
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We previously demonstrated in intact killifish renal proximal tubules that endothelin (ET), acting through an ET(B) receptor and protein kinase C (PKC), reduced transport mediated by multidrug resistance-associated protein 2 (Mrp2), i.e., luminal accumulation of fluorescein methotrexate (FL-MTX) (Masereeuw R, Terlouw SA, Van Aubel RAMH, Russel FGM, and Miller DS. Mol Pharmacol 57: 59-67, 2000). In the present study, we used confocal microscopy and quantitative image analysis to measure Mrp2-mediated transport of FL-MTX in killifish tubules as an indicator of the status of this ET-fired, intracellular signaling pathway. Exposing tubules to sodium nitroprusside (SNP), a nitric oxide (NO) donor, signaled a reduction in luminal accumulation of FL-MTX, which suggested pathway activation. N(G)-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, blocked the action of ET-1 on transport. Because SNP effects on transport were blocked by bisindoylmaleide, a PKC-selective inhibitor, but not by RES-701-1, an ET(B)-receptor antagonist, generation of NO occurred after ET(B) receptor signaling but before PKC activation. NO generation was implicated in the actions of several nephrotoxicants, i.e., diatrizoate, gentamicin, amikacin, HgCl(2), and CdCl(2), each of which decreased Mrp2-mediated transport by activating ET signaling. For each nephrotoxicant, decreased FL-MTX transport was prevented when tubules were exposed to L-NMMA. ET-1 and each nephrotoxicant stimulated NO production by the tubules, as determined by a fluorescence-based assay. Together, the data show that NO generation follows ET binding to the basolateral ET(B) receptor and that, in activating the ET-signaling pathway, nephrotoxicants produce NO, a molecule that could contribute to subsequent toxic effects. (+info)
Evolution of Sp transcription factors.
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The Sp family of transcription factors binds GC-rich DNA sequences. The ubiquitously expressed Sp1 and Sp3 have been well characterized in mammals. Presented here is the characterization of the only Sp protein expressed in the liver or heart tissue of the teleost fish Fundulus heteroclitus. This protein, fSp3, is most similar to and homologous with mammalian Sp3 proteins. The evolution of the Sp transcription family is described, with Sp1 and Sp3 representing the most recent duplication of the Sp family. Sp4 appears to be the most ancestral member. Sp1, Sp3, and Sp4 form a monophyletic group without Sp2. Sp2 is the least similar of the Sp family and is more similar to the non-Sp transcription factors. These results suggest that Sp2 should not be considered a member of the Sp family. Only two domains (zinc fingers and B domain) share similarity outside the Sp family. The zinc fingers are homologous to other GC-binding domains, yet the B domain is homologous to protein-protein interacting domains in the CCAAT-binding/NF-Y transcription factor families. These results suggest that these different domains have different evolutionary histories. (+info)