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Jo Ann M. Burkholder - One of the best experts on this subject based on the ideXlab platform.
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Microfluorimetric Analysis of a Purinergic Receptor (P2X 7) in GH 4C 1 Rat Pituitary Cells: Effects of a Bioactive Substance Produced by Pfiesteria piscicida
2013Co-Authors: Ana Clara Melo, Jo Ann M. Burkholder, Peter D R Moeller, Howard Glasgow, John S RamsdellAbstract:Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH 4C 1 rat pituitary cells. Extracellular adenosine 5’-triphosphate (ATP) acting on P2X 7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH 4C 1 rat pituitary cells express functional P2X 7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2´-3´-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6azophenyl-2´-4´-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH 4C 1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH 4C 1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH 4C 1 cells express a purinoceptor with characteristics consistent with the P2X 7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP. Key words: GH 4C 1, Pfiesteria, purinergic receptors, P2X 7, toxin. — Environ Health Perspect 109(suppl 5):731–737 (2001)
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Use of Molecular Probes to Assess Geographic Distribution of Pfiesteria Species
2013Co-Authors: Parke A. Rublee, Jo Ann M. Burkholder, David W. Oldach, Torstein Tengs, Jason W. Kempton, Eric F. Schaefer, Coy Allen, Janera Harris, Holly Bowers, H. B. GlasgowAbstract:We have developed multiple polymerase chain reaction (PCR)-based methods for the detection of Pfiesteria sp. in cultures and environmental samples. More than 2,100 water and sediment samples from estuarine sites of the U.S. Atlantic and gulf coasts were assayed for the presence of Pfiesteria piscicida Steidinger & Burkholder and Pfiesteria shumwayae Glasgow & Burkholder by PCR probing of extracted DNA. Positive results were found in about 3 % of samples derived from routine monitoring of coastal waters and about 8 % of sediments. The geographic range of both species was the same, ranging from New York to Texas. Pfiesteria spp. are likely common and generally benign inhabitants of coastal areas, but their presence maintains a potential for fish and human health problems. Key words: molecular probes, PCR, Pfiesteria, toxic dinoflagellates. — Enviro
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Current Progress in Isolation and Characterization of Toxins Isolated from Pfiesteria piscicida
2013Co-Authors: Peter D R Moeller, Jo Ann M. Burkholder, Nora J. Deamer-melia, Steve L. Morton, Brad A. Mitchell, Scott K. Sivertsen, Elizabeth R. Fairey, Tina M. Mikulski, Howard Glasgow, John S RamsdellAbstract:The isolation and partial purification of toxic substances derived from Pfiesteria piscicida Steidinger & Burkholder extracts is described. Four distinct bioassay systems were used to monitor bioactivity of the P. piscicida extracts, including a high throughput cell cytotoxicity assay and a reporter gene assay as well as assays using brine shrimp and fish. Using these bioassays to guide fractionation, we have isolated two distinct, active fractions from Pfiesteria culture medium and cell mass extracts on the basis of their solubility characteristics. We have identified and characterized a bioactive lipophilic substance from Pfiesteria-derived extracts as di(2-ethylhexyl)phthalate, a commonly used plasticizer. The source of this typically man-made substance has been identified as originating from Instant Ocean (Aquarium Systems, Mentor, OH, USA), a commercially available seawater salt mixture used to prepare our mass culture growth medium. We have developed chromatographic methodology to isolate a bioactive polar compound isolated from extracts of Pfiesteria culture and presently report the characterization of the activity of this substance. The molecular structural analysis of the polar active component(s) using mass spectrometry and nuclear magnetic resonance spectroscopy is currently under way. Key words: assay, chromatography, GH 4C 1, Pfiesteria piscicida, toxin bioassay. — Environ Health Perspect 109(suppl 5):739–743 (2001)
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Articles Reporter Gene Assay for Fish-Killing Activity Produced by Pfiesteria piscicida
2013Co-Authors: Peter R. Moeller, Jo Ann M. Burkholder, John S RamsdellAbstract:Collaborative studies were peormed to develop a finctional assay for fish-killing activity produced by If ia piscida Eight cell lines were used to screen organic fractions and residual water fraton by using a 3-[4,5-dimethyliazol-(2-4)1-diphenyltaolium bromide cytotoxicity asay. Diethyl ether and a residual water fraction were cytoc to several cell lines icl-uding rat pituitary (GH4C1) cells. Residual water as wdl as pre acted culture water continin P. piscicida cels induced c-fiwlucifre xr in GH4C, cells with a rapid time course of induction and sensitiv detection. The reporter gene assay detet activity in toxic isolates of P. pisciida from seral North Carolina estuaries in 1997 and 1998 and may also be suitable for detg toxic activity in human and serum. Key wordve assay c-for, GH4C1, Pfiestria pscici4da, pituitary, toxin. Enuron Heah Pmpc*t 107:711-714 (1999). [Online 28 July 1999] bttp://dpnetld. niehs.nibgwv/lds/l999/107p711-714fai"r/absstracthtml Pfiesteria piscicida is a toxic dinoflagellate that has been a causative agent of fish epizootics and mortalities in estuaries of th
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grazing activity of Pfiesteria piscicida dinophyceae and susceptibility to ciliate predation vary with toxicity status
Harmful Algae, 2006Co-Authors: Bonnie M. Willis, Matthew W. Parrow, Alan J. Lewitus, Jo Ann M. Burkholder, Michael S Wetz, Howard B GlasgowAbstract:Abstract Variability has been reported in the toxicity potential of Pfiesteria piscicida that is partly a function of the history of exposure to live fish. Grazing properties of P. piscicida and its susceptibility to ciliate predation were compared in three functional types or toxicity states of this species: actively toxic cultures, cultures with temporary loss of demonstrable toxicity, and cultures with no demonstrable toxicity. Pronounced differences in predator–prey interactions were found between actively toxic cultures and cultures with reduced toxicity. When grown with Rhodomonas sp. (Cryptophyceae) prey, specific growth rates were relatively low in actively toxic cultures under both relatively high and low irradiances. In the cultures with reduced toxicity, prey chloroplast material was apparent in nearly 100% of dinoflagellate cells 3 h after feeding, while chloroplast inclusions were found in
John S Ramsdell - One of the best experts on this subject based on the ideXlab platform.
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Microfluorimetric Analysis of a Purinergic Receptor (P2X 7) in GH 4C 1 Rat Pituitary Cells: Effects of a Bioactive Substance Produced by Pfiesteria piscicida
2013Co-Authors: Ana Clara Melo, Jo Ann M. Burkholder, Peter D R Moeller, Howard Glasgow, John S RamsdellAbstract:Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH 4C 1 rat pituitary cells. Extracellular adenosine 5’-triphosphate (ATP) acting on P2X 7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH 4C 1 rat pituitary cells express functional P2X 7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2´-3´-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6azophenyl-2´-4´-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH 4C 1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH 4C 1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH 4C 1 cells express a purinoceptor with characteristics consistent with the P2X 7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP. Key words: GH 4C 1, Pfiesteria, purinergic receptors, P2X 7, toxin. — Environ Health Perspect 109(suppl 5):731–737 (2001)
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Articles Reporter Gene Assay for Fish-Killing Activity Produced by Pfiesteria piscicida
2013Co-Authors: Peter R. Moeller, Jo Ann M. Burkholder, John S RamsdellAbstract:Collaborative studies were peormed to develop a finctional assay for fish-killing activity produced by If ia piscida Eight cell lines were used to screen organic fractions and residual water fraton by using a 3-[4,5-dimethyliazol-(2-4)1-diphenyltaolium bromide cytotoxicity asay. Diethyl ether and a residual water fraction were cytoc to several cell lines icl-uding rat pituitary (GH4C1) cells. Residual water as wdl as pre acted culture water continin P. piscicida cels induced c-fiwlucifre xr in GH4C, cells with a rapid time course of induction and sensitiv detection. The reporter gene assay detet activity in toxic isolates of P. pisciida from seral North Carolina estuaries in 1997 and 1998 and may also be suitable for detg toxic activity in human and serum. Key wordve assay c-for, GH4C1, Pfiestria pscici4da, pituitary, toxin. Enuron Heah Pmpc*t 107:711-714 (1999). [Online 28 July 1999] bttp://dpnetld. niehs.nibgwv/lds/l999/107p711-714fai"r/absstracthtml Pfiesteria piscicida is a toxic dinoflagellate that has been a causative agent of fish epizootics and mortalities in estuaries of th
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Current Progress in Isolation and Characterization of Toxins Isolated from Pfiesteria piscicida
2013Co-Authors: Peter D R Moeller, Jo Ann M. Burkholder, Nora J. Deamer-melia, Steve L. Morton, Brad A. Mitchell, Scott K. Sivertsen, Elizabeth R. Fairey, Tina M. Mikulski, Howard Glasgow, John S RamsdellAbstract:The isolation and partial purification of toxic substances derived from Pfiesteria piscicida Steidinger & Burkholder extracts is described. Four distinct bioassay systems were used to monitor bioactivity of the P. piscicida extracts, including a high throughput cell cytotoxicity assay and a reporter gene assay as well as assays using brine shrimp and fish. Using these bioassays to guide fractionation, we have isolated two distinct, active fractions from Pfiesteria culture medium and cell mass extracts on the basis of their solubility characteristics. We have identified and characterized a bioactive lipophilic substance from Pfiesteria-derived extracts as di(2-ethylhexyl)phthalate, a commonly used plasticizer. The source of this typically man-made substance has been identified as originating from Instant Ocean (Aquarium Systems, Mentor, OH, USA), a commercially available seawater salt mixture used to prepare our mass culture growth medium. We have developed chromatographic methodology to isolate a bioactive polar compound isolated from extracts of Pfiesteria culture and presently report the characterization of the activity of this substance. The molecular structural analysis of the polar active component(s) using mass spectrometry and nuclear magnetic resonance spectroscopy is currently under way. Key words: assay, chromatography, GH 4C 1, Pfiesteria piscicida, toxin bioassay. — Environ Health Perspect 109(suppl 5):739–743 (2001)
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Learning impairment caused by a toxin produced by Pfiesteria piscicida infused into the hippocampus of rats.
Neurotoxicology and teratology, 2003Co-Authors: Edward D. Levin, Jo Ann M. Burkholder, Peter D R Moeller, Howard B Glasgow, W.paul Blackwelder, John S RamsdellAbstract:Pfiesteria piscicida, an estuarine dinoflagellate, which has been shown to kill fish, has also been associated with neurocognitive deficits in humans. With a rat model, we have demonstrated the cause-and-effect relationship between Pfiesteria exposure and learning impairment. In several studies, we have replicated the finding in Sprague-Dawley rats that exposure to fixed acute doses of Pfiesteria cells or filtrates caused radial-arm maze learning impairment. Recently, this finding of Pfiesteria-induced learning impairment in rats has been independently replicated in another laboratory as well. We have demonstrated significant Pfiesteria-induced learning impairment in both the win-shift and repeated-acquisition tasks in the radial-arm maze and in reversal learning in a visual operant signal detection task. These learning impairments have been seen as long as 10 weeks after a single acute exposure to Pfiesteria. In the current study, we used a hydrophilic toxin isolated from clonal P. piscicida cultures (PfTx) and tested its effect when applied locally to the ventral hippocampus on repeated acquisition of rats in the radial-arm maze. Toxin exposure impaired choice accuracy in the radial-arm maze repeated acquisition procedure. The PfTx-induced impairment was seen at the beginning of the session and the early learning deficit was persistent across 6 weeks of testing after a single administration of the toxin. Eventually, with enough practice, in each session, the PfTx-exposed rats did learn that session's problem as did control rats. This model has demonstrated the cause-and-effect relationship between exposure to a hydrophilic toxin produced by P. piscicida and learning impairment, and specifically that the ventral hippocampus was critically involved.
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microfluorimetric analysis of a purinergic receptor p2x7 in gh4c1 rat pituitary cells effects of a bioactive substance produced by Pfiesteria piscicida
Environmental Health Perspectives, 2001Co-Authors: Ana Clara Melo, Jo Ann M. Burkholder, Peter D R Moeller, Howard B Glasgow, John S RamsdellAbstract:Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP.
Howard B Glasgow - One of the best experts on this subject based on the ideXlab platform.
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grazing activity of Pfiesteria piscicida dinophyceae and susceptibility to ciliate predation vary with toxicity status
Harmful Algae, 2006Co-Authors: Bonnie M. Willis, Matthew W. Parrow, Alan J. Lewitus, Jo Ann M. Burkholder, Michael S Wetz, Howard B GlasgowAbstract:Abstract Variability has been reported in the toxicity potential of Pfiesteria piscicida that is partly a function of the history of exposure to live fish. Grazing properties of P. piscicida and its susceptibility to ciliate predation were compared in three functional types or toxicity states of this species: actively toxic cultures, cultures with temporary loss of demonstrable toxicity, and cultures with no demonstrable toxicity. Pronounced differences in predator–prey interactions were found between actively toxic cultures and cultures with reduced toxicity. When grown with Rhodomonas sp. (Cryptophyceae) prey, specific growth rates were relatively low in actively toxic cultures under both relatively high and low irradiances. In the cultures with reduced toxicity, prey chloroplast material was apparent in nearly 100% of dinoflagellate cells 3 h after feeding, while chloroplast inclusions were found in
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identical ribosomal dna sequence data from Pfiesteria piscicida dinophyceae isolates with different toxicity phenotypes
Environmental Research, 2003Co-Authors: Torstein Tengs, Jo Ann M. Burkholder, David W. Oldach, Howard B Glasgow, Holly A. BowersAbstract:Complete small subunit ribosomal RNA, internal transcribed spacer 1 and 2, 5.8S, and partial large subunit ribosomal RNA gene sequences were generated from multiple isolates of Pfiesteria piscicida. Sequences were derived from isolates that have been shown to be ichthyotoxic as well as isolates that have no history of toxic behavior. All of the sequences generated were identical for the different cultures, and we therefore conclude that differences in toxicity seen between isolates of P. piscicida are linked to factors other than genetic strain variation detectable by ribosomal gene sequence analyses.
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Learning impairment caused by a toxin produced by Pfiesteria piscicida infused into the hippocampus of rats.
Neurotoxicology and teratology, 2003Co-Authors: Edward D. Levin, Jo Ann M. Burkholder, Peter D R Moeller, Howard B Glasgow, W.paul Blackwelder, John S RamsdellAbstract:Pfiesteria piscicida, an estuarine dinoflagellate, which has been shown to kill fish, has also been associated with neurocognitive deficits in humans. With a rat model, we have demonstrated the cause-and-effect relationship between Pfiesteria exposure and learning impairment. In several studies, we have replicated the finding in Sprague-Dawley rats that exposure to fixed acute doses of Pfiesteria cells or filtrates caused radial-arm maze learning impairment. Recently, this finding of Pfiesteria-induced learning impairment in rats has been independently replicated in another laboratory as well. We have demonstrated significant Pfiesteria-induced learning impairment in both the win-shift and repeated-acquisition tasks in the radial-arm maze and in reversal learning in a visual operant signal detection task. These learning impairments have been seen as long as 10 weeks after a single acute exposure to Pfiesteria. In the current study, we used a hydrophilic toxin isolated from clonal P. piscicida cultures (PfTx) and tested its effect when applied locally to the ventral hippocampus on repeated acquisition of rats in the radial-arm maze. Toxin exposure impaired choice accuracy in the radial-arm maze repeated acquisition procedure. The PfTx-induced impairment was seen at the beginning of the session and the early learning deficit was persistent across 6 weeks of testing after a single administration of the toxin. Eventually, with enough practice, in each session, the PfTx-exposed rats did learn that session's problem as did control rats. This model has demonstrated the cause-and-effect relationship between exposure to a hydrophilic toxin produced by P. piscicida and learning impairment, and specifically that the ventral hippocampus was critically involved.
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THE LIFE CYCLE AND TOXICITY OF Pfiesteria piscicida REVISITED1
Journal of Phycology, 2002Co-Authors: Jo Ann M. Burkholder, Howard B GlasgowAbstract:Despite use of excellent molecular techniques, Litaker et al. (2002) cannot provide insights about the life history of toxic Pfiesteria piscicida because they showed no data in support of having used toxic strains; ratherthey presented evidence that they used non-inducible strains. Litaker et al. did not find amoeboid stages or a chrysophyte-like cyst stage in several cultures and unequivocally concluded that the stages do not exist in all P. piscicida strains. Thus, they did not consider the tenet that absence of evidence does not constitute proof of absence. Apparent discrepancies between the research by Litaker et al. and previous research on Pfiesteria can be resolved as follows: First, Litaker et al. did not use toxic strains. We have reported findings (similar to Litaker et al.) showing few amoeboid transformations in non-inducible strains, which manifest some but not all of the forms that have been documented in some toxic strains. We . and others, have documented active toxicity to fish, transformations to amoebae, and chrysophyte-like cysts in some clonal toxic strains. Second, the data from several recent publications, which were available but not mentioned by Litaker et al. or by Coats (2002) in accompanying commentary, have verified P. piscicida amoebae, chrysophyte-like cysts, and other stages in some toxic strains through a combination of approaches including PCR data from clonal cultures.
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grazing by microzooplankton on Pfiesteria piscicida cultures with different histories of toxicity
Aquatic Microbial Ecology, 2002Co-Authors: Diane K. Stoecker, Matthew W. Parrow, Jo Ann M. Burkholder, Howard B GlasgowAbstract:Susceptibility of actively toxic (TOX-A) zoospores, temporarily non-toxic (TOX-B) zoospores, and zoospores non-inducible to toxicity (NON-IND) of Pfiesteria piscicida to microzoo- plankton grazing was compared in a laboratory experiment. Zoospores from all cultures were ingested by microzooplankton, but community grazing coefficients for TOX-A were < 20% of those for TOX-B or NON-IND zoospores in 6 h incubations. Tintinnids and strobilidiid ciliates that fed on P. piscicida declined in incubations containing TOX-A zoospores. There was no decline in a strom- bidiid ciliate or heterotrophic dinoflagellate populations that fed on TOX-A zoospores. These data suggest that, although microzooplankton grazing on non-toxic zoospores can be a significant source of mortality to planktonic populations of P. piscicida, grazing on toxic or very recently toxic zoospores is relatively low.
Senjie Lin - One of the best experts on this subject based on the ideXlab platform.
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Mitogen-activated protein kinase in Pfiesteria piscicida and its growth raterelated expression
2015Co-Authors: Senjie Lin, Huan ZhangAbstract:A full-length cDNA (1,434 bp) of mitogen-activated protein kinase (MAPK), a key molecule of a signal transduction cascade, was isolated from the estuarine heterotrophic dinoflagellate Pfiesteria piscicida. This cDNA (Ppmapk1) encoded a protein (PpMAPK1) of 428 amino acid residues that shared about 30 to 40 % amino acid similarity with MAPKs in other organisms. Phylogenetic analysis indicated that PpMAPK1 was tightly clustered with MAPK3 in protozoans. Using reverse transcription-PCR, expression of this gene was evaluated for P. piscicida cultures grown under different conditions. While salinity shock, heat shock, starvation, and a subsequent encounter with prey did not appear to affect expression of this gene, Ppmapk1 expression level was correlated with growth rate, suggesting involvement of this gene in the regulation of cell proliferation in the organism. Pfiesteria piscicida Steidinger et Burkholder is an ambush predator dinoflagellate implicated in major fish kills in North Carolina and Maryland estuaries (5, 6). First discovered in 1988 (6), this organism constitutes a novel genus (34) whose phylogenetic affiliation has only recently been established (24, 32). Over the last decade, P. piscicida and closely related spe
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low abundance distribution of Pfiesteria piscicida in pacific and western atlantic as detected by mtdna 18s rdna real time polymerase chain reaction
Journal of Plankton Research, 2006Co-Authors: Senjie Lin, Huan Zhang, Angela DuboisAbstract:Information on the abundance of Pfiesteria piscicida in the natural environment is needed for understanding the ecological roles of this dinoflagellate. In this study, a real-time polymerase chain reaction (PCR) assay was developed using mitochondrial cytochrome b upstream region and 18S rDNA (PpmtDNA and Ppl8S), and the geographic and temporal distribution of P. piscicida was investigated in several locations. Both PpmtDNA and Ppl8S generally gave similar results, indicating that P. piscicida was present at all studied regions along the American coast (from Maine to North Carolina along the US Atlantic coast and Los Lagos along the Chilean Pacific coast). Despite its widespread distribution, P. piscicida was only detected in 36% of the 431 water samples analyzed, and its abundance was generally low (<1.0-1.5 cells mL -1 ). Populations detected at the jive stations in the Nuse River (North Carolina) and two stations in Chesapeake Bay (Maryland) were genetically homogeneous, whereas those from other locations appeared to be genetically diverse. It can be concluded that (i) the PpmtDNA-Pp18S real-time PCR assay is sensitive and specific for detecting and quantifying P. piscicida in the natural environment and (ii) P. piscicida is widespread along the American coasts, but normally only as a minor component of plankton even in the high-risk estuaries (Neuse River, Chesapeake Bay).
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isolation and characterization of proliferating cell nuclear antigen from the dinoflagellate Pfiesteria piscicida
Journal of Eukaryotic Microbiology, 2006Co-Authors: Huan Zhang, Yubo Hou, Senjie LinAbstract:Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases delta and epsilon, is essential for DNA replication and repair. Understanding the structure and expression characteristics of this gene in dinoflagellates would enable us to gain insights into how the cell cycle in these enigmatic eukaryotes is regulated and whether this gene can be a growth marker of these ecologically important organisms. We analyzed pcna and its encoded protein from Pfiesteria piscicida (Ppi_PCNA). Using reverse transcription-polymerase chain reaction (RT-PCR) and RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) methods, Ppi_pcna cDNA was isolated; it contained a coding region for 258 amino acid residues (aa) preceded by various 5'- and 3'-untranslated ends. The deduced protein length was similar to that of typical vertebrate and plant PCNA. PCR using genomic DNA as the template yielded multiple products whose sequences revealed multiple copies of pcna in tandem repeats separated by an unknown sequence. Using real-time PCR, we estimated 41+/-7 copies of this gene in each P. piscicida cell. Reverse transcription real-time PCR indicated a similar pcna mRNA level between the exponential and the stationary growth phases. Western blot analysis revealed a slightly higher PCNA level (<2-fold) in the exponential than in the stationary growth phases. We conclude that (1) P. piscicida possesses a typical eukaryote PCNA; (2) unlike in other eukaryotes, pcna in P. piscicida occurs in multiple copies arranged in tandem; and (3) regulation of P. piscicida PCNA probably lies in post-translational modification.
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intense grazing and prey dependent growth of Pfiesteria piscicida dinophyceae 1
Journal of Phycology, 2004Co-Authors: Senjie Lin, Huan Zhang, Timothy N Feinstein, Margaret R Mulholland, Frank J Jochem, Edward J CarpenterAbstract:Grazing and growth of Pfiesteria piscicida (Pfiest) were investigated using batch and cyclostat cultures with Rhodomonas sp. (Rhod) as prey. Observed maximum growth rates (1.4 d - 1 ) and population densities (2 × 10 5 cells . mL - 1 ) corresponded to values predicted by Monod functions (1.76 d - 1 ; 1.4 × 10 5 cells.mL - 1 ). In batch cultures under a range of prey-to-predator ratios (0.1:1 to 180:1) and prey concentrations (1000-71,000 cells . mL - 1 ), Rhodomonas sp. was always depleted rapidly and P. piscicida concentrations increased briefly. The rate of Rhodomonas sp. depletion and the magnitude of P. piscicida population maxima depended on the prey-to-predator ratio and prey concentration. Starvation resulted in cell cycle arrest at G1 and G2 + M and ultimately the demise of both P. piscicida and Rhodomonas sp. populations, demonstrating the dependence of P. piscicida on the supply of appropriate prey. The depletion of Rhodomonas sp. populations could be attributed directly to grazing, because P. piscicida did not exert detectable inhibitory effects on the growth of Rhodomonas sp. but grazed intensely, with maximum grazing rates > 10 Rhod . Pfiest - 1 . d - 1 and with no apparent threshold prey abundance for grazing. The results suggest that 1) the abundance of appropriate prey may be an important factor regulating P. piscicida abundance in nature, 2) P. piscicida may control prey population, and 3) high growth and grazing potentials of P. piscicida along with cell cycle arrest may confer survival advantages.
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development of an immunofluorescence technique for detecting Pfiesteria piscicida
Harmful Algae, 2003Co-Authors: Senjie Lin, Huan Zhang, Timothy N Feinstein, Edward J CarpenterAbstract:Abstract While several DNA-based methods have been developed for the putatively toxic dinoflagellate Pfiesteria piscicida Burkholder et Steidinger, an independent detection method such as immunofluorescence can be a useful alternative. In this study, P. piscicida-specific antisera were developed, and an immunofluorescence (IF) procedure was optimized. A total of six antisera were raised using whole cells (WCA) and the insoluble cellular fraction (ICF) as antigens, respectively, and their titer and specificity were examined using dot blot analysis and whole cell IF. Results showed that the two antisera produced from the ICF antigen had a markedly higher titer (1500) than the other four yielded from the WCA (200). In addition, the two ICF-derived antisera exhibited much higher species specificity, showing no cross-reaction with P. shumwayae, Cryptoperidiniopsis sp., Karlodinium micrum, and other more distant algae tested, and very low background for field collected samples. In evaluation of the IF technique using a P. piscicida-specific polymerase chain reaction (PCR) technique, results from both methods generally agreed well for both field samples (from eastern Long Island Sound) spiked with cultured P. piscicida and those containing naturally occurring P. piscicida (from Chesapeake Bay tributaries).
Edward D. Levin - One of the best experts on this subject based on the ideXlab platform.
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A Rat Model of the Cognitive Impairment from Pfiesteria piscicida Exposure
2013Co-Authors: Edward D. Levin, Pfiesteria Piscicida SteidingerAbstract:Pfiesteria piscicida Steidinger & Burkholder, an estuarine dinoflagellate known to kill fish, has also been associated with neurocognitive deficits in humans. We have developed a rat model to determine the cause-and-effect relationship between exposure to Pfiesteria-containing water and cognitive impairment and to determine the neurobehavioral mechanisms underlying the Pfiesteria effect. The rat model of Pfiesteria toxicity can also provide important information concerning the toxin or toxins responsible for neurocognitive deficits resulting from Pfiesteria exposure. With the rat model we have repeatedly documented a Pfiesteria-induced choice accuracy impairment during radial-arm maze learning. The Pfiesteria-induced impairment was relatively specific to the acquisition phase of training. When rats were pretrained, Pfiesteria treatment did not affect performance. However, when these same rats were retrained on another task, the Pfiesteria-induced impairment became evident. Pfiesteria-induced effects were also seen in a locomotor activity test in the figure-8 apparatus and selected components of the functional observational battery. Pfiesteria effects on choice accuracy in the radial-arm maze in rats constitute a critical component of the model of Pfiesteria toxicity, as the hallmark of Pfiesteria toxicity in humans is cognitive dysfunction. Our finding that analysis of the first six sessions of radial-arm maze testing is sufficient for determining the effect means that this test will be useful as a rapid screen for identifying the critical neurotoxin(s) of Pfiesteria in future studies. Key words: cognition, functional observational battery, learning, locomotor activity, Pfiesteria piscicida, radial-arm maze, rat model. — Environ Healt
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Learning impairment caused by a toxin produced by Pfiesteria piscicida infused into the hippocampus of rats.
Neurotoxicology and teratology, 2003Co-Authors: Edward D. Levin, Jo Ann M. Burkholder, Peter D R Moeller, Howard B Glasgow, W.paul Blackwelder, John S RamsdellAbstract:Pfiesteria piscicida, an estuarine dinoflagellate, which has been shown to kill fish, has also been associated with neurocognitive deficits in humans. With a rat model, we have demonstrated the cause-and-effect relationship between Pfiesteria exposure and learning impairment. In several studies, we have replicated the finding in Sprague-Dawley rats that exposure to fixed acute doses of Pfiesteria cells or filtrates caused radial-arm maze learning impairment. Recently, this finding of Pfiesteria-induced learning impairment in rats has been independently replicated in another laboratory as well. We have demonstrated significant Pfiesteria-induced learning impairment in both the win-shift and repeated-acquisition tasks in the radial-arm maze and in reversal learning in a visual operant signal detection task. These learning impairments have been seen as long as 10 weeks after a single acute exposure to Pfiesteria. In the current study, we used a hydrophilic toxin isolated from clonal P. piscicida cultures (PfTx) and tested its effect when applied locally to the ventral hippocampus on repeated acquisition of rats in the radial-arm maze. Toxin exposure impaired choice accuracy in the radial-arm maze repeated acquisition procedure. The PfTx-induced impairment was seen at the beginning of the session and the early learning deficit was persistent across 6 weeks of testing after a single administration of the toxin. Eventually, with enough practice, in each session, the PfTx-exposed rats did learn that session's problem as did control rats. This model has demonstrated the cause-and-effect relationship between exposure to a hydrophilic toxin produced by P. piscicida and learning impairment, and specifically that the ventral hippocampus was critically involved.
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A rat model of the cognitive impairment from Pfiesteria piscicida exposure.
Environmental health perspectives, 2001Co-Authors: Edward D. LevinAbstract:Pfiesteria piscicida Steidinger & Burkholder, an estuarine dinoflagellate known to kill fish, has also been associated with neurocognitive deficits in humans. We have developed a rat model to determine the cause-and-effect relationship between exposure to Pfiesteria-containing water and cognitive impairment and to determine the neurobehavioral mechanisms underlying the Pfiesteria effect. The rat model of Pfiesteria toxicity can also provide important information concerning the toxin or toxins responsible for neurocognitive deficits resulting from Pfiesteria exposure. With the rat model we have repeatedly documented a Pfiesteria-induced choice accuracy impairment during radial-arm maze learning. The Pfiesteria-induced impairment was relatively specific to the acquisition phase of training. When rats were pretrained, Pfiesteria treatment did not affect performance. However, when these same rats were retrained on another task, the Pfiesteria-induced impairment became evident. Pfiesteria-induced effects were also seen in a locomotor activity test in the figure-8 apparatus and selected components of the functional observational battery. Pfiesteria effects on choice accuracy in the radial-arm maze in rats constitute a critical component of the model of Pfiesteria toxicity, as the hallmark of Pfiesteria toxicity in humans is cognitive dysfunction. Our finding that analysis of the first six sessions of radial-arm maze testing is sufficient for determining the effect means that this test will be useful as a rapid screen for identifying the critical neurotoxin(s) of Pfiesteria in future studies.
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Specificity of cognitive impairment from Pfiesteria piscicida exposure in rats: attention and visual function versus behavioral plasticity.
Neurotoxicology and teratology, 2001Co-Authors: Amir H. Rezvani, Jo Ann M. Burkholder, Howard B Glasgow, Philip J. Bushnell, Edward D. LevinAbstract:Pfiesteria piscicida is a toxic dinoflagellate that has caused massive fish kills in estuaries along the East Coast of the United States, and exposure of humans to toxic Pfiesteria has been associated with cognitive impairment. A visual signal detection task was used to determine the possible importance of attentional and visual processes in Pfiesteria effects on cognitive function. Adult female rats were trained to perform the signal detection task. After training, the rats were injected subcutaneously with fish culture water containing toxic Pfiesteria (35,600 or 106,800 cells of Pfiesteria/kg of rat body weight) or with (control) fish culture water containing no Pfiesteria. Effects of toxic Pfiesteria on maintenance of signal detection behavior were assessed for 2 weeks after treatment. Then, the signal-response contingencies were reversed. After the discrimination was reestablished on the reversed levers, the rats received a second dose of toxic Pfiesteria. The rats were again tested for 2 weeks, after which a second reversal was imposed. Pfiesteria did not affect behavior in the signal detection task during 2 weeks of prereversal testing after either exposure. However, a significant Pfiesteria-induced deficit emerged when the signal-response contingencies were reversed. These findings suggest that Pfiesteria-induced deficits emerge during periods of behavioral transition and not during performance of previously learned tasks.
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Rapid neurobehavioral analysis of Pfiesteria piscicida effects in juvenile and adult rats.
Neurotoxicology and teratology, 2000Co-Authors: Edward D. Levin, Jo Ann M. Burkholder, Howard B Glasgow, Amir H. Rezvani, Nadine C. Christopher, Nora J. Deamer-melia, Virginia C. Moser, Karl F. JensenAbstract:The estuarine dinoflagellate Pfiesteria piscicida is known to kill fish and has been associated with neurocognitive deficits in humans. We have developed a rat model to demonstrate that exposure to Pfiesteria causes significant learning impairments. This has been repeatedly seen as a choice accuracy impairment during radial-arm maze learning. Pfiesteria-induced effects were also seen in a locomotor activity test in the figure-8 apparatus. The current studies used the short-term radial-arm maze acquisition, the figure-8 activity test, and the functional observational battery (FOB) to assess Pfiesteria-induced neurobehavioral effects in adult and juvenile rats. In study 1, the neurobehavioral potency of three different Pfiesteria cultures (Pf 113, Pf 728, and Pf Vandermere) was assessed. Ninety-six (12 per group) adult female Sprague-Dawley rats were injected subcutaneously with a single dose of Pfiesteria taken from aquarium-cultured Pfiesteria (35,600 or 106,800 Pfiesteria cells per kilogram of rat body weight). One control group (N = 12) was injected with saline and one (N = 12) with aquarium water not containing Pfiesteria. All three of the Pfiesteria samples (p < 0.05) impaired choice accuracy over the first six sessions of training. At the time of the radial-arm maze choice accuracy impairment, no overt Pfiesteria-related effects were seen using an FOB, indicating that the Pfiesteria-induced choice accuracy deficit was not due to generalized debilitation. In the figure-8 apparatus, Pfiesteria treatment caused a significant decrease in mean locomotor activity. In study 2, the neurobehavioral effects of the Pf 728 sample type were assessed in juvenile rats. Twenty-four day-old male and female rats were injected with 35,600 or 106,800 Pf-728 Pfiesteria cells per kilogram of rat body weight. As with adult females, the juvenile rats showed a significant impairment in radial-arm maze choice accuracy. No changes in locomotor activity or the FOB were detected in the juvenile rats. Furthermore, there were no differences between male and female rats in the Pfiesteria-induced choice accuracy impairment. Pfiesteria effects on choice accuracy in the radial-arm maze in rats constitute a critical component of the model of Pfiesteria toxicity, because the hallmark of Pfiesteria toxicity in humans is cognitive dysfunction. Our finding that analysis of the first six sessions of radial-arm maze testing is sufficient for determining the effect means that this test will be useful as a rapid screen for identifying the critical neurotoxin(s) of Pfiesteria in future studies.