Isolation of viral hemorrhagic septicemia virus from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap.
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Viral hemorrhagic septicemia virus (VHSV) was isolated from apparently healthy Greenland halibut Reinhardtius hippoglossoides caught in the Flemish Cap, a deep fishing ground in the North Atlantic Ocean in international waters near Newfoundland. The identity of the virus was confirmed by electron microscopy, immunodot, seroneutralization and reverse transcriptasepolymerase chain reaction. In the serology assays, all isolates reacted in the immunodot assay with a polyclonal antiserum against the European VHSV Type Strain F1, and were neutralized by the same antiserum, although most of the strains showed low or moderate neutralization titers. None of the isolates were detected by immunofluorescence using a specific monoclonal antibody against a nucleocapsid-related protein of VHSV F1. This is the first report of VHSV isolated from wild Greenland halibut, which represents a new host species for the virus, and it is also the first evidence of VHSV in a location close to the Atlantic coast of North America. This isolation indicates that VHSV is more widely distributed than has been thought, and appears to support a marine origin of this virus. (+info)
Intestinal bicarbonate secretion by marine teleost fish--why and how?
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Intestinal fluids of most marine teleosts are alkaline (pH 8.4-9.0) and contain high levels of HCO(3)(-) equivalents (40-130 mM) which are excreted at a significant rate (>100 microEq kg(-1) h(-1)). Recent research reveals the following about this substantial HCO(3)(-) secretion: (1) It is not involved in acid-base regulation or neutralisation of stomach acid, but increases in parallel with drinking rate at elevated ambient salinities suggesting a role in osmoregulation; (2) In species examined so far, all sections of the intestine can secrete bicarbonate; (3) The secretion is dependent on mucosal Cl(-), sensitive to mucosal DIDS, and immuno-histochemistry indicates involvement of an apical Cl(-)/HCO(3)(-) exchanger. In addition, hydration of CO(2) via carbonic anhydrase in combination with proton extrusion appears to be essential for bicarbonate secretion. The mode of proton extrusion is currently unknown but potential mechanisms are discussed. One consequence of the luminal alkalinity and high bicarbonate concentrations is precipitation of calcium and magnesium as carbonate complexes. This precipitation is hypothesised to reduce the osmolality of intestinal fluids and thus play a potential role in water absorption and osmoregulation. The present studies on European flounder reveal that elevated luminal calcium (but not magnesium) concentrations stimulate intestinal bicarbonate secretion both acutely and chronically, in vitro and in vivo. At the whole animal level, the result of this elevated bicarbonate secretion was increased calcium precipitation with an associated reduction in the osmolality of rectal fluids and plasma. These observations suggest direct functional links between intestinal bicarbonate secretion, divalent cation precipitation and osmoregulation in marine teleost fish. (+info)
Transport of cimetidine by flounder and human renal organic anion transporter 1.
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The H(2)-receptor antagonist cimetidine is efficiently excreted by the kidneys. In vivo studies indicated an interaction of cimetidine not only with transporters for basolateral uptake of organic cations but also with those involved in excretion of organic anions. We therefore tested cimetidine as a possible substrate of the organic anion transporters cloned from winter flounder (fROAT) and from human kidney (hOAT1). Uptake of [(3)H]cimetidine into fROAT-expressing Xenopus laevis oocytes exceeded uptake into control oocytes. At -60-mV clamp potential, 1 mM cimetidine induced an inward current, which was smaller than that elicited by 0.1 mM PAH. Cimetidine concentrations exceeding 0.1 mM decreased PAH-induced inward currents, indicating interaction with the same transporter. At pH 6.6, no current was seen with 0.1 mM cimetidine, whereas at pH 8.6 a current was readily detectable, suggesting preferential translocation of uncharged cimetidine by fROAT. Oocytes expressing hOAT1 also showed [(3)H]cimetidine uptake. These data reveal cimetidine as a substrate for fROAT/hOAT1 and suggest that organic anion transporters contribute to cimetidine excretion in proximal tubules. (+info)
Heteroplasmy and evidence for recombination in the mitochondrial control region of the flatfish Platichthys flesus.
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The general assumption that mitochondrial DNA (mtDNA) does not undergo recombination has been challenged recently in invertebrates. Here we present the first direct evidence for recombination in the mtDNA of a vertebrate, the flounder Platichthys flesus. The control region in the mtDNA of this flatfish is characterized by the presence of a variable number of tandem repeats and a high level of heteroplasmy. Two types of repeats were recognized, differing by two C-T point mutations. Most individuals carry a pure "C" or a pure "T" array, but one individual showed a compound "CT" array. Such a compound array is evidence for recombination in the mtDNA control region from the flounder. (+info)
Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney.
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Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent P(i) cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family. (+info)
Freezing of a fish antifreeze protein results in amyloid fibril formation.
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Amyloid is associated with a number of diseases including Alzheimer's, Huntington's, Parkinson's, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the alpha-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils. (+info)
The role of N and C termini in the antifreeze activity of winter flounder (Pleuronectes americanus) antifreeze proteins.
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Antifreeze proteins (AFPs) are found in many marine fish and have been classified into five biochemical classes: AFP types I-IV and the antifreeze glycoproteins. Type I AFPs are alpha-helical, partially amphipathic, Ala-rich polypeptides. The winter flounder (Pleuronectes americanus) produces two type I AFP subclasses, the liver-type AFPs (wflAFPs) and the skin-type AFPs (wfsAFPs), that are encoded by distinct gene families with different tissue-specific expression. wfsAFPs and wflAFPs share a high level of identity even though the wfsAFPs have approximately half the activity of the wflAFPs. Synthetic polypeptides based on two representative wflAFPs and wfsAFPs were generated to examine the role of the termini in antifreeze activity. Through systematic exchange of N and C termini between wflAFP-6 and wfsAFP-2, the termini were determined to be the major causative agents for the variation in activity levels between the two AFPs. Furthermore, the termini of wflAFP-6 possessed greater helix-stabilizing ability compared with their wfsAFP-2 counterparts. The observed 50% difference in activity between wflAFP-6 and wfsAFP-2 can be divided into approximately 20% for differences at each termini and approximately 10% for differences in the core. Furthermore, the N terminus was determined to be the most critical component for antifreeze activity. (+info)
Inhibition of chemiluminescent response of olive flounder Paralichthys olivaceus phagocytes by the scuticociliate parasite Uronema marinum.
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Experiments were conducted to evaluate the in vitro capacity of the scuticociliatian parasite Uronema marinum to inhibit chemiluminescence (CL) of olive flounder Paralichthys olivaceus phagocytes. Luminol-enhanced CL was used to measure the production of reactive oxygen intermediates (ROIs) generated by respiratory bursts of phagocytes using zymosan as a stimulant. Cytotoxic and antioxidative activities of excretory-secretory (ES) products of the parasite were evaluated as well. Live U. marinum and its ES products had a negative and dose-dependent effect on luminol-enhanced CL responses of zymosan-stimulated phagocytes of olive flounder. After CL assay, the number of phagocytes showing viability was significantly reduced in the cells incubated with live U. marinum at ratios of 2:1 and 1:1 phagocytes:ciliates or ES products with 0.3 mg protein ml(-1) compared to controls. Lysis of phagocytes by exposure to ES products was observed also. ES products from U. marinum showed considerably high activities of superoxide dismutase (SOD) and catalase. The results of this study suggest that U. marinum can protect itself against host's phagocytes mediated oxidative damage by destroying phagocytes and scavenging ROIs. (+info)