Effects of salinity and temperature on long-term survival of the eel pathogen Vibrio vulnificus biotype 2 (serovar E).
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Vibrio vulnificus biotype 2 (serovar E) is a primary eel pathogen. In this study, we performed long-term survival experiments to investigate whether the aquatic ecosystem can be a reservoir for this bacterium. We have used microcosms containing water of different salinities (ranging from 0.3 to 3.8%) maintained at three temperatures (12, 25, and 30 degrees C). Temperature and salinity significantly affected long-term survival: (i) the optimal salinity for survival was 1.5%; (ii) lower salinities reduced survival, although they were nonlethal; and (ii) the optimal temperature for survival was dependent on the salinity (25 degrees C for microcosms at 0.3 and 0.5% and 12 degrees C for microcosms at 1.5 to 3.8%). In the absence of salts, culturability dropped to zero in a few days, without evidence of cellular lysis. Under optimal conditions of salinity and temperature, the bacterium was able to survive in the free-living form for at least 3 years. The presence of a capsule on the bacterial cell seemed to confer an advantage, since the long-term survival rate of opaque variants was significantly higher than that of translucent ones. Long-term-starved cells maintained their infectivity for eels (as determined by both intraperitoneal and immersion challenges) and mice. Examination under the microscope showed that (i) the capsule was maintained, (ii) the cell size decreased, (iii) the rod shape changed to coccuslike along the time of starvation, and (iv) membrane vesicles and extracellular material were occasionally produced. In conclusion, V. vulnificus biotype 2 follows a survival strategy similar to that of biotype 1 of this species in response to starvation conditions in water. Moreover, the aquatic ecosystem is one of its reservoirs. (+info)
Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan.
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The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance. (+info)
Identification by differential display of a hypertonicity-inducible inward rectifier potassium channel highly expressed in chloride cells.
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By using differential mRNA display to monitor the molecular alterations associated with adaptation of euryhaline eels to different salinities, we identified a cDNA fragment strongly induced in seawater eel gills. Cloning of a full-length cDNA and its expression in COS-7 cells indicated that the clone codes for an inward rectifier K+ channel (eKir) of 372 amino acid residues, which has two transmembrane segments and a typical pore-forming region (H5). Only low sequence similarities are present, except the H5 region, compared with other members of the inward rectifier K+ channel family (Kir). Consistent with this divergence in the amino acid sequence, a phylogenetic analysis indicated early divergence and independent evolution of eKir from other members; it is only distantly related to the Kir5.0 subfamily members. RNase protection analysis showed that eKir is highly expressed in the seawater eel gill, kidney, and posterior intestine but very weakly in freshwater eels. Immunohistochemistry of gill sections revealed dense localization of eKir in the chloride cells. Immunoelectron microscopy indicated that eKir is mainly present in the microtubular system in the chloride cell. This location and its salt-inducible nature suggest that the eKir channel cloned here is a novel member of the Kir5.0 subfamily of the Kir family and is implicated in osmoregulation. (+info)
The anticancer prodrug CPT-11 is a potent inhibitor of acetylcholinesterase but is rapidly catalyzed to SN-38 by butyrylcholinesterase.
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Patients treated with high doses of CPT-11 rapidly develop a cholinergic syndrome that can be alleviated by atropine. Although CPT-11 was not a substrate for acetylcholinesterase (AcChE), in vitro assays confirmed that CPT-11 inhibited both human and electric eel AcChE with apparent K(i)s of 415 and 194 nM, respectively. In contrast, human or equine butyryl-cholinesterase (BuChE) converted CPT-11 to SN-38 with K(m)s of 42.4 and 44.2 microM for the human and horse BuChE, respectively. Modeling of CPT-11 within the predicted active site of AcChE and BuChE corroborated experimental results indicating that, although the drug was oriented correctly for activation, the constraints dictated by the active site gorge were such that CPT-11 would be unlikely to be activated by AcChE. (+info)
Indole-positive Vibrio vulnificus isolated from disease outbreaks on a Danish eel farm.
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Vibrio vulnificus was isolated in 1996 from 2 disease outbreaks on a Danish eel farm which used brackish water. A characteristic clinical sign was extensive, deep muscle necrosis in the head region. V. vulnificus was isolated from kidney, mucus, spleen, gill and intestine of diseased eels. Thirty-two isolates were examined phenotypically and serologically for pathogenicity to eels and for correlation to ribotype and plasmid profile. Biochemically, the isolates showed properties similar to those described previously for eel-pathogenic strains of V. vulnificus, with the exception of indole production. Virulence was evaluated by LD50 (the 50% lethal dose), which ranged from < 9.4 x 10(3) to 2.3 x 10(5) CFU (colony-forming units) per fish. The isolates which were lethal for eels showed identical ribotypes and serotypes. A relationship between certain plasmids and virulence was not found. A serotyping system based on lipopolysaccharide (LPS)-associated O antigen type and on carbohydrate capsule antigens showed that the eel-virulent isolates shared a common LPS-based homogeneous O serogroup and a capsule antigen. V. vulnificus serovar O4 and capsule type 9 was identical serologically to the Japanese isolate ATCC 33149 and was the agent responsible for the disease outbreaks that occurred on the Danish eel farm. Despite absence of antibiotic resistance, treatment had little effect and disease reoccurred. (+info)
Determination of the solution structure of the N-domain plus linker of Antarctic eel pout antifreeze protein RD3.
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RD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypeptide divided into homologous N-terminal (residues Asn(1)-Glu(64)) and C-terminal (residues Ser(74)-Glu(134)) domains, each of which has a high sequence identity with Type III AFP. A 9-residue linker (-D(65)GTTSPGLK(73)-) connects these two domains in tandem and is thought to play a significant role in defining the nature of the intact molecule. The present paper shows for the first time the solution structure and preliminary (15)N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-Nl: residues 1-73) by employing homo- and heteronuclear multidimensional NMR spectroscopy. Forty converged structures of RD3-Nl were successfully calculated by using a total of 958 NMR-derived structural restraints. It was found that the N-domain of RD3-Nl has a globular form comprising six beta-strands, three type III turns, and several loops, which stabilize a flat, ice-binding site formed on one side of this domain. Further, the linker portion appears to have a definitive structure, which is independent of the globular N-domain. This definitive linker is roughly divided into two short strands, -D(65)GTTSP(70)- and -G(71)LK(73)-, which are bent around -T(67)TSPG(71)- at an angle of approximately 60 degrees. This bending motif of the linker may function to orient the two ice-binding sites of the N- and C-domains of RD3 in the same direction, leading to their simultaneous interactions with the ice crystal surface. (+info)
Accelerated evolution in the protein-coding region of galectin cDNAs, congerin I and congerin II, from skin mucus of conger eel (Conger myriaster).
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Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5' and 3' untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS = 2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel. (+info)
Random amplified polymorphic DNA analysis of eel genome.
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Eel family is a huge one, in which many kinds of eels especially some migratory eels, bear strong resemblance to each other, and are therefore difficult to be identified. In this study 29 random primers were used to make RAPD analysis for Japanese eel (Anguilla japonica), European eel (Anguilla anguilla) and Pike eel (Muraenesox cinereus). And totally 299 fragments were counted. Shared or specific fragments were counted and genetic similarity or genetic distance were calculated. The genetic similarity between Japanese eel and Pike eel is 0.68 and the genetic distance between them is 0.32; those between European eel and Pike eel are 0.72 and 0.28 respectively, and between Japanese eel and European eel are 0.74 and 0.25 respectively. The method has been shown to be suitable to molecular identification of eels. It provides an alternative approach to determine the relationship between species. (+info)