Biomedical subjects

N D Denslow

Publications and source records attributed to N D Denslow.

At least 19 recordsLinked to original sources

PubMed2006

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.

Amino Acid Sequence↗

PubMed2003

Differential gene expression analysis in fish exposed to endocrine disrupting compounds.

This review discusses various methodologies that can be used to understand, at the gene level, the consequences to fish upon exposure to endocrine disrupting compounds (EDCs). Several approaches for measuring expression of gene transcripts are discussed, including directed approaches, such as Northern blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) as well as open-ended approaches, such as differential display RT-PCR, subtractive hybridizations, and gene arrays. Each of these systems has advantages and disadvantages, strengths and weaknesses. Conducting experiments with each of these methods provides important information about the molecular mechanisms that result from exposure to EDCs, information which can be used in risk assessment of polluted sites found in the environment.

Animals↗

PubMed2003

Analysis of gene expression profiles in largemouth bass exposed to 17-beta-estradiol and to anthropogenic contaminants that behave as estrogens.

Novel molecular based methods are being developed to study changes in gene expression in wildlife exposed to anthropogenic chemicals. Gene arrays, in particular, are useful tools that can be used to simultaneously monitor hundreds to thousands of genes within a single experiment, giving an investigator the ability to determine how exposure affects multiple metabolic pathways. These methods are thought to be both sensitive and able to reveal biochemical mechanisms of action. A largemouth bass (LMB) array containing 132 genes has been designed to study the impact of gene expression in male fish exposed to 17-beta estradiol or to the compounds 4-nonylphenol (4-NP) or 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p,p'-DDE). The results of these experiments demonstrate distinct gene expression patterns in LMB exposed to these compounds.

Animals↗

PubMed2002

Gene expression analysis of largemouth bass exposed to estradiol, nonylphenol, and p,p'-DDE.

The purpose of this study was to determine the specific expression profile of 132 genes, some of which are estrogen responsive, in largemouth bass (LMB) following exposure to estradiol (E(2)), or to two hormonally active agents, 4-nonylphenol (4-NP) and 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p,p'-DDE), using gene array technology. The results of these experiments show that LMB exposed to E(2) and 4-NP had similar, but not identical genetic signatures for the genes examined, some of which are known to be estrogen-responsive genes. The differences suggest that 4-NP may have additional modes of action that are independent of the estrogen receptor (ER). We have also shown that exposure of male LMB to p,p'-DDE results in an increase in some estrogen-responsive genes. But in female LMB, the observed changes were a down-regulation of the normally up-regulated estrogen responsive genes. Other genes were also down-regulated. These results suggest that p,p'-DDE may affect regulation of genes differently in male and female LMB. This study further suggests that gene arrays have the potential to map out the gene activation pathways of hormonally active compounds.

Animals↗

PubMed2001

Induction of gene expression in sheepshead minnows (Cyprinodon variegatus) treated with 17beta-estradiol, diethylstilbestrol, or ethinylestradiol: the use of mRNA fingerprints as an indicator of gene regulation.

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variegatus) exposed in vivo to the natural and pharmaceutical estrogens 17beta-estradiol, ethinylestradiol, and diethylstilbestrol. This method used differential display reverse transcriptase polymerase chain reaction assays to compare the expression of individual mRNAs from control and estrogen-exposed fish. Forty-eight differentially expressed cDNAs were isolated by this method, including cDNAs for vitelline envelope proteins and vitellogenin. The mRNA expression patterns for fish injected with a pharmacological dose of estradiol (5 mg/kg) were identical to those obtained in fish receiving constant aqueous exposure to 212 ng estradiol/liter. Further, the cDNA "fingerprint" pattern observed in the estradiol-treated fish also matched that obtained in fish receiving continuous-flow aqueous exposures to 192 ng ethinyl estradiol/liter and a nominal concentration of 200 ng diethylstilbestrol/liter. The results demonstrate a characteristic expression pattern for genes upregulated by exposure to a variety of natural and anthropogenic estrogens and suggest this approach may be valuable to examine the potential effects of environmental contaminants on other endocrine-mediated pathways of reproduction, growth, and development.

Amino Acid Sequence↗

PubMed2001

Identification of lysine decarboxylase as a mammalian cell growth inhibitor in Eikenella corrodens: possible role in periodontal disease.

The pathogenesis of inflammatory periodontal disease was studied by examining the mechanism of HeLa and HL60 cell growth inhibition by cell-free saline-soluble extracts of Eikenella corrodens and bacterial plaque. Previous studies identified a protein (p80) as causing growth inhibition by E. corrodens extracts. After purification by two-dimensional SDS-PAGE, p80 was digested with protease lysC. Amino acid sequences were obtained and backtranslated for use as PCR primers. A 5840 nucleotide sequence containing a lysine decarboxylase gene was obtained from a Sau3 A1 genomic library of E. corrodens DNA. Lysine decarboxylase activity was present at physiologic pH in the E. corrodens extracts containing p80, and also in bacterial plaque. Both extracts caused growth inhibition by depleting lysine from cell culture media through conversion to cadaverine. Adding lysine, or immune goat IgG to a peptide derived from the active site sequence of E. corrodens lysine decarboxylase, retarded lysine depletion and growth inhibition. epsilon-Amino caproic acid specifically enhanced lysine decarboxylase activity at the low lysine concentration in HL60 cell culture media, and also increased the growth inhibition. Thus, lysine decarboxylases such as p80 inhibit growth by removing lysine from mammalian cell culture media. A new role for lysine decarboxylase activity in the microbial aetiology of periodontal disease is discussed.

Carboxy-Lyases↗

PubMed2001

Altered serum sex steroids and vitellogenin induction in walleye (Stizostedion vitreum) collected near a metropolitan sewage treatment plant.

Several recent in situ studies have reported that domestic and mixed domestic/industrial sewage effluents contain one or more natural or anthropogenic estrogenic substances. Those studies examined caged or feral fish for the presence of the egg yolk precursor protein, vitellogenin (VTG), in the blood of male fish. We have previously reported that male, feral carp (Cyprinus carpio) obtained from the effluent channel of a major sewage treatment plant (STP) exhibited depressed serum testosterone (T) concentrations, as well as detectable levels of VTG. The present study examines male and female walleye (Stizostedion vitreum), a native species with a different life history and feeding habits, collected from the same Mississippi River locations below the St. Paul metropolitan STP. All male and female walleye collected from the effluent channel contained measurable levels of VTG in their blood. Males from that location also exhibited depressed serum T concentrations and elevated serum estradiol-17beta (E2) concentrations compared with males from the Snake River reference site. Males obtained from Mississippi River Navigational Pool #2 (MRP-2), 3-20 miles downstream of the STP also exhibited reduced serum T concentrations, but showed no alterations in E2 concentrations or the presence of VTG in the serum. Females collected at the STP site had greatly elevated serum E2 concentrations, but serum T concentrations were not different from females collected in the Snake River. Our results demonstrate that the St. Paul metropolitan STP continues to release an estrogenic effluent, capable of inducing VTG production and altering normal serum sex steroid concentrations in a commercially valuable, native fish, the walleye. Additional studies will be required to determine whether these observations portend long-term population level effects.

Animals↗

PubMed2001

Assessment of reproductive effects in largemouth bass (Micropterus salmoides) exposed to bleached/unbleached kraft mill effluents.

This study evaluated the potential effects of different concentrations of bleached/unbleached kraft mill effluent (B/UKME) on several reproductive endpoints in adult largemouth bass (Micropterus salmoides). The kraft mill studied produces a 50/50 mix of bleached/unbleached market pulp with an estimated release of 36 million gal of effluent/day. Bleaching sequences were C90d10EopHDp and CEHD for softwood (pines) and hardwoods (mainly tupelo, gums, magnolia, and water oaks), respectively. Bass were exposed to different effluent concentrations (0 [controls, exposed to well water], 10, 20, 40, or 80%) for either 28 or 56 days. At the end of each exposure period, fish were euthanized, gonads collected for histological evaluation and determination of gonadosomatic index (GSI), and plasma was analyzed for 17beta-estradiol, 11-ketotestosterone, and vitellogenin (VTG). Largemouth bass exposed to B/UKME responded with changes at the biochemical level (decline in sex steroids in both sexes and VTG in females) that were usually translated into tissue/organ-level responses (declines in GSI in both sexes and in ovarian development in females). Although most of these responses occurred after exposing fish to 40% B/UKME concentrations or greater, some were observed after exposures to 20% B/UKME. These threshold concentrations fall within the 60% average yearly concentration of effluent that exists in the stream near the point of discharge (Rice Creek), but are above the <10% effluent concentration present in the St. Johns River. The chemical(s) responsible for such changes as well as their mode(s) of action remain unknown at this time.

Animals↗

PubMed2001

Vitellogenin-induced pathology in male summer flounder (Paralichthys dentatus).

Male summer flounder (Paralichthys dentatus) were given two injections (initially and 2 weeks later) of 17beta-estradiol (E2) totaling 0.2 (2 x 0.1), 2.0 (2 x 1.0) or 20.0 (2 x 10.0) mg E2/kg body weight. Blood and tissue samples were collected 4, 6 and 8 weeks after the initial injection in the (2 x 0.1) mg/kg treatment, 4, 6, 8, and 15 weeks after the first injection in the (2 x 1.0) mg/kg treatment and at 4 weeks only in the (2 x 10.0) mg/kg treatment. Five of the 12 fish injected twice with 10.0 mg/kg were moribund before the first sampling period. Circulating levels of vitellogenin (VTG) in the blood of all E2-injected fish from all treatments were comparable with those concentrations found in the blood of wild male carp (Cyprinus carpio) and walleye (Stezostedion vitreum) previously collected near a sewage treatment plant (0.1-10.0 mg VTG/ml plasma). Excessive hyalin material accumulated in the livers, kidneys and testes of the treated fish. A portion of that material was identified as VTG by immunohistochemistry. The accumulation of VTG, and possibly other estrogen-inducible proteins, resulted in hepatocyte hypertrophy, disruption of spermatogenesis, and obstruction or rupture of renal glomeruli.

Animals↗

PubMed2001

Multiple responses in gene expression in fish treated with estrogen.

During the last decade there has been a significant body of research conducted on environmental estrogens. These include industrial, agricultural and pest-control chemicals that bind to the estrogen receptor and induce biological changes during development or reproduction. Most of these changes are probably due to modified gene expression, since estrogen receptors function at this level. We have mapped qualitative gene expression responses (by differential display reverse transcriptase polymerase chain reaction, DD) in adult male sheepshead minnows (Cyprinidon variegatus) receiving high dose injections (5 mg/kg), or constant flow-through aquatic exposures to environmentally relevant concentrations (100 ng/l) of estradiol-17beta, and found them nearly identical. We have observed both up-regulation and down-regulation of transcripts, which fit into known responses to estradiol. Among the genes up-regulated are vitellogenin and several vitelline envelope proteins indicating that genes for proteins involved in egg development and maturation are susceptible to environmental estrogen exposure. While physiological changes caused by estradiol treatment are not totally explained by changes at the mRNA level, those changes can nevertheless be used as fingerprints to characterize an in vivo estrogenic response.

Animals↗

PubMed2001

Effects of p-nonylphenol, methoxychlor, and endosulfan on vitellogenin induction and expression in sheepshead minnow (Cyprinodon variegatus).

Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3, and 42.7 micrograms/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1, and 18.4 micrograms/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590, and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 microliters/L) and 17 beta-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 35, and 42 of exposure, and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p-nonylphenol at concentrations of 5.4 micrograms/L or greater and to methoxychlor at concentrations of 2.5 micrograms/L or greater. Exposure to 0.64 microgram/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Animals↗

PubMed2001

Assessment of the estrogenicity and toxicity of a domestic wastewater effluent flowing through a constructed wetland system using biomarkers in male fathead minnows (Pimephales promelas rafinesque, 1820).

The toxicity and estrogenicity of a final treated municipal effluent was examined while flowing through a constructed wetland in north-central Texas, USA. Fish data were collected, and a baseline wetland characterization was performed to assess wetland treatment potential for these effluent properties. Vitellogenin (VTG), gonadosomatic index (GSI), hepatosomatic index (HSI), and secondary sexual characteristics were biomarkers used in fish models to assess aqueous estrogenicity. Biological indicators used to assess overall fish health included hematocrit and condition factor. Estrogenic nature of final sewage treatment works effluent was screened, concurrent with a three-week fish exposure, via gas chromatography/mass spectrometry for target estrogenic compounds, including 17beta-estradiol, ethynylestradiol, bisphenol A, nonylphenolic compounds, phthalates, and DDT. The VTG in Pimephales promelas was measured after exposure at four sites in a treatment wetland and was significantly elevated (p < 0.0001) in fish exposed at the inflow site. The GSIs were significantly less (alpha = 0.001) at the inflow site. At wetland sites closest to the inflow, secondary sexual characteristics, tubercle numbers, and fatpad thickness were less (alpha = 0.0001) than in laboratory controls. The HSIs and density of male breeding stripes were not significantly different from those of laboratory controls. However, elevated HSIs were found at the inflow site. Hematocrit and condition factors were both less (alpha = 0.001) in effluent-exposed fish at wetland sites closer to the inflow than in control fish or fish further downstream.

Animals↗

PubMed2001

Soluble scute proteins of healthy and ill desert tortoises (Gopherus agassizii).

OBJECTIVES: To characterize protein composition of shell scute of desert tortoises and to determine whether detectable differences could be used to identify healthy tortoises from tortoises with certain illnesses. ANIMALS: 20 desert tortoises. PROCEDURES: Complete postmortem examinations were performed on all tortoises. Plastron scute proteins were solubilized, scute proteins were separated by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were analyzed, using densitometry. Two-dimensional immobilized pH gradient-PAGE (2D IPG-PAGE) and immunoblot analysis, using polyclonal antisera to chicken-feather beta keratin and to alligator-scale beta keratin, were conducted on representative samples. The 14-kd proteins were analyzed for amino acid composition. RESULTS: The SDS-PAGE and densitometry revealed 7 distinct bands, each with a mean relative protein concentration of > 1 %, ranging from 8 to 47 kd, and a major protein component of approximately 14 kd that constituted up to 75% of the scute protein. The 2D IPG-PAGE revealed additional distinct 62- and 68-kd protein bands. On immunoblot analysis, the 14-, 32-, and 45-kd proteins reacted with both antisera. The 14-kd proteins had an amino acid composition similar to that of chicken beta keratins. There was a substantial difference in the percentage of the major 14-kd proteins from scute of ill tortoises with normal appearing shells, compared with 14-kd proteins of healthy tortoises. CONCLUSIONS AND CLINICAL RELEVANCE: The major protein components of shell scute of desert tortoises have amino acid composition and antigenic features of beta keratins. Scute protein composition may be altered in tortoises with certain systemic illnesses.

Animal Diseases↗

PubMed2000

Contrasting effects of estrogen on transthyretin and vitellogenin expression in males of the marine fish, Sparus aurata.

A partial cDNA encoding for the C-terminus of vitellogenin (VTG) was cloned from liver of Sparus aurata male treated with 17beta-estradiol (E(2)). E(2) treatment of S. aurata males resulted in increased synthesis and secretion of VTG protein into the plasma, determined by a specific enzyme-linked immunosorbent assay (ELISA) in a time-dependent manner. While VTG mRNA was induced by E(2) treatment, transthyretin (TTR) mRNA levels were reduced. These data provide the first demonstration that estrogen exhibits contrasting effect on VTG and on TTR gene expression in teleosts.

Amino Acid Sequence↗

PubMed2000

Epstein-Barr virus nuclear antigen-1 (EBNA-1) associated oligoclonal bands in patients with multiple sclerosis.

Oligoclonal bands (OCBs) are frequently observed in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS), but the target antigens of these antibodies remain unknown. We used antigen specific immunoblotting to determine whether Epstein Barr virus nuclear antigen-1 (EBNA-1) was a target of the OCBs in the CSF of patients with MS. Antibody indices (AIs) were measured by ELISA and calculated by the formula of Reiber and Lange which includes correction factors for both breakdown of the blood brain barrier and intrathecal polyclonal IgG synthesis. A distinctive oligoclonal antigen specific banding pattern for EBNA-1 was observed in 5/15 MS patients, but 0/12 controls (P=0.037, Fisher's Exact Probability). AIs in this EBNA-l positive subgroup were extremely high, comparable with levels observed in viral CNS infections. In one patient with EBNA-1 specific OCBs, EBNA-1 and a peptide 'equivalent', p62, were able to absorb a component of the total IgG. Our results suggest that in a subset of MS patients, EBNA-1 may be a major target of selected OCBs.

Adult↗

PubMed2000

Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus).

Many environmentally persistent xenobiotic chemicals appear to disrupt normal endocrine function by acting as ligands for endogenous steroid receptors, including the estrogen receptor. Xenobiotics that bind to the estrogen receptor may elicit several effects, one of which is activating estrogen-responsive genes, such as vitellogenin (Vtg). Primers to vitellogenin mRNA have been used to amplify a portion of the coding sequence in sheepshead minnow (SHM) (Cyprinodon variegatus). Two Vtg cDNA fragments from SHM were isolated exhibiting 72% sequence homology and corresponding to the two Vtg genes identified in the mummichog, Fundulus heteroclitus. Using these Vtg cDNA fragments as sensitive genetic probes, we evaluated the initial estrogenic response of fish exposed to natural or anthropogenic chemicals. These probes were used to study in vivo gene induction in SHM exposed to 17beta-estradiol (E(2)) and ethinylestradiol (EE(2)) under controlled laboratory conditions. Hepatic Vtg mRNA was upregulated and plasma Vtg synthesis in estrogen-induced SHM was assessed. Two in vivo time-course experiments were conducted; a single injection of E(2) followed over 72 h and a double E(2) injection examined for 12 days. These two protocols provided evidence for differential hepatic Vtg mRNA regulation resulting from a single or a double injection. In a separate experiment using an aqueous flowthrough system, constant exposures to low doses of E(2) (200 ng/L) and EE(2) (100 ng/L) induced hepatic Vtg mRNA and plasma Vtg to levels comparable with the E(2) injections. Larger aqueous exposure doses (2000 ng/L E(2) or 1000 ng/L EE(2)) in the flowthrough experiment resulted in greater responses of hepatic Vtg mRNA and plasma Vtg at 7 days. Constant aqueous exposure to E(2) (2000 ng/L) or EE(2) (1000 ng/L) may thus be more effective than a single large-dose injection (5 mg/kg) to stimulate Vtg gene activation and synthesis.

Amino Acid Sequence↗

PubMed1999

Mammalian mitochondrial ribosomal proteins (2). Amino acid sequencing, characterization, and identification of corresponding gene sequences.

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.

Amino Acid Sequence↗

PubMed1999

Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds.

The egg yolk precursor protein, vitellogenin (VTG), was purified from blood plasma of 17beta-estradiol (E2)-treated male fathead minnows (Pimephales promnelas) by anion-exchange chromatography on DEAE-agarose. A rabbit antiserum was raised against their blood plasma and then adsorbed with plasma from untreated (control) males to render the antiserum specific to VTG. The adsorbed antiserum was used to detect fathead minnow VTG (fVTG) in Western and dot blotting experiments and in an enzyme-linked immunosorbent assay (ELISA). The antiserum recognised fVTG as a approximately 156 kDa protein in plasma from vitellogenic females and E2-injected males but not untreated males. Its identity was confirmed by analysis of: (1) amino acid composition; (2) an internal amino acid sequence; (3) reactivity to the homologous antiserum; and (4) recognition by monoclonal antibodies prepared against the VTG from common carp (Cyprinus carpio) and brown bullhead (Ameiurus nebulosus). Specificity of the homologous antiserum to fVTG was confirmed by Western blotting of serially diluted plasma from vitellogenic females. Utility of the antiserum and purified fVTG for detecting exposure of male fathead minnows to estrogenic compounds was verified using a dot blotting immunoassay of fVTG and detected by chemiluminescence. Adult male fish were exposed to various concentrations of E2 (10(-8), 10(-9) and 10(-10) M) in their rearing water and plasma assayed for the presence of VTG at different time points (2, 7, 14 and 21 days). A competitive, antibody-capture, quantitative ELISA was then developed based on the purified fVTG and its respective antiserum. The ELISA was validated by demonstrating parallel binding slopes of dilution curves prepared with plasma from E2-injected males, vitellogenic females, and aqueous egg extracts as compared with purified fVTG standard. Plasma concentrations of VTG as low as 3 ng ml(-1) were detected in the ELISA, for which inter- and intra-assay coefficients of variation were both less than 5%. Furthermore, plasma from control males was unreactive with the fVTG antiserum. The VTG ELISA could be useful for the detection of estrogenic properties associated with certain compounds and could be easily incorporated into standard laboratory toxicity assays using this species.

Amino Acid Sequence↗