Early phosphoproteomic changes for adverse outcome pathway development in the fathead minnow (Pimephales promelas) brain.

Adverse outcome pathways (AOPs) are conceptual frameworks that organize and link contaminant-induced mechanistic molecular changes to adverse biological responses at the individual and population level. AOPs leverage molecular and high content mechanistic information for regulatory decision-making, but most current AOPs for hormonally active agents (HAAs) focus on nuclear receptor-mediated effects only despite the overwhelming evidence that HAAs also activate membrane receptors. Activation of membrane receptors triggers non-genomic signaling cascades often transduced by protein phosphorylation leading to phenotypic changes. We utilized label-free LC-MS/MS to identify proteins differentially phosphorylated in the brain of fathead minnows (Pimephales promelas) aqueously exposed for 30 minutes to two HAAs, 17α-ethinylestradiol (EE2), a strong estrogenic substance, and levonorgestrel (LNG), a progestin, both components of the birth control pill. EE2 promoted differential phosphorylation of proteins involved in neuronal processes such as nervous system development, synaptic transmission, and neuroprotection, while LNG induced differential phosphorylation of proteins involved in axon cargo transport and calcium ion homeostasis. EE2 and LNG caused similar enrichment of synaptic plasticity and neurogenesis. This study is the first to identify molecular changes in vivo in fish after short-term exposure and highlights transduction of rapid signaling mechanisms as targets of HAAs, in addition to nuclear receptor-mediated pathways.

Evaluation of water quality threats to the endangered Okaloosa darter (Etheostoma okaloosae) in East Turkey Creek on Eglin Air Force Base.

The threatened Okaloosa darter (Etheostoma okaloosae) is found almost exclusively on the Eglin Air Force Base in the Choctawhatchee Bay watershed of Florida. Portions of this limited habitat are threatened with soil erosion, altered hydrology, and impaired water quality. In the present study, general water quality parameters (i.e., dissolved oxygen, specific conductance, pH, temperature, relative turbidity, and primary productivity) were characterized in East Turkey Creek, which is a body of water potentially impacted by treated wastewater sprayfields, and Long Creek, an adjacent reference stream that does not border the sprayfields. Water quality was assessed during a 30-day exposure using passive samplers for both non-polar and polar effluent parameters. Because the Okaloosa darter was listed as endangered at the time of sampling we chose a closely related species from the same creeks, the sailfin shiner (Pteronotropis hypseleotris) in which to measure metal body burdens. Additionally, fathead minnows (Pimephales promelas) were used for microarray analysis on gonad and liver tissues after 48 h exposures to water collected from the two creeks and brought into the laboratory. Waters from all sites, including reference sites, affected the expression of genes related to various biological processes including transcription and translation, cell cycle control, metabolism, and signaling pathways, suggesting that the sum of anthropogenic compounds in the site waters may cause a generalized stress response in both liver and testis, an effect that could be related to the generally low populations of the Okaloosa darter. Furthermore, effects of site waters on fish gene expression may be related to the impact of human activities other than the wastewater sprayfields, as nearby areas are closed to the public for military testing, training, and administrative activities and due to ordnance contamination.

The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.

Biomarkers of contaminant exposure in Northern Pike (Esox lucius) from the Yukon River Basin, Alaska.

As part of a larger investigation, northern pike (n = 158; Esox lucius) were collected from ten sites in the Yukon River Basin (YRB), Alaska, to document biomarkers and their correlations with organochlorine pesticide (total p,p'-DDT, total chlordane, dieldrin, and toxaphene), total polychlorinated biphenyls (PCBs), and elemental contaminant (arsenic, cadmium, copper, lead, total mercury, selenium, and zinc) concentrations. A suite of biomarkers including somatic indices, hepatic 7-ethoxyresorufin O-deethylase (EROD) activity, vitellogenin concentrations, steroid hormone (17B- ustradiol and 16-kebtestosteront) concentrations, splenic macrophage aggregates (MAs), oocyte atresia, and other microscopic anomalies in various tissues were documented in YRB pike. Mean condition factor (0.50 to 0.68), hepatosomatic index (1.00% to 3.56%), and splenosomatic index (0.09% to 0.18%) were not anomalous at any site nor correlated with any contaminant concentration. Mean EROD activity (0.71 to 17.51 pmol/min/mg protein) was similar to basal activity levels previously measured in pike and was positively correlated with selenium concentrations (r = 0.88, P < 0.01). Vitellogenin concentrations in female (0.09 to 5.32 mg/mL) and male (<0.0005 to 0.097 mg/mL) pike were not correlated with any contaminant, but vitellogenin concentrations >0.01 mg/mL in male pike from multiple sites indicated exposure to estrogenic compounds. Mean steroid hormone concentrations and percent oocyte atresia were not anomalous in pike from any YRB site. Few site differences were significant for mean MA density (1.86 to 6.42 MA/mm(2)), size (812 to 1481 microm(2)), and tissue occupied (MA-%; 0.24% to 0.75%). A linear regression between MA-% and total PCBs was significant, although PCB concentrations were generally low in YRB pike (< or =63 ng/g), and MA-% values in female pike (0.24% to 0.54%) were lower than in male pike (0.32% to 0.75%) at similar PCB concentrations. Greater numbers of MAs were found as zinc concentrations increased in YRB female pike, but it is unlikely that this is a causative relationship. Histological abnormalities observed in gill, liver, spleen, and kidney tissues were not likely a result of contaminant exposure but provide information on the general health of YRB pike. The most common histologic anomalies were parasitic infestations in various organs and developing nephrons and nephrocalcinosis in posterior kidney tissues. Overall, few biomarker responses in YRB pike were correlated with chemical contaminant concentrations, and YRB pike generally appeared to be healthy with no site having multiple anomalous biomarker responses.

ABRF ESRG 2006 study: Edman sequencing as a method for polypeptide quantitation.

The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Chemical cleavage of proteins on membranes.

Described in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins bound to membranes. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS-skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid-proline (Asp-Pro) peptide bonds; hydroxylamine cleaves at asparagine-glycine (Asn-Gly) peptide bonds, and 2-nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides. In addition, Alternate Protocol an describes CNBr cleavage of PVDF-bound protein previously analyzed by Edman degradation. Finally, a Support Protocol discusses preferred methods of separating and analyzing peptide fragments generated by the chemical cleavage reactions described in the basic protocols.