The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for pr... more 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% ...
Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish,... more Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies.
Biochemical and Biophysical Research Communications, 1979
ABSTRACT Salt-washed ribosomes from bovine mitochondria and E. coli require homologous factor pre... more ABSTRACT Salt-washed ribosomes from bovine mitochondria and E. coli require homologous factor preparations for activity in poly (U)-directed systems for phenylalanine polymerization, contrary to the widespread notion that bacterial and mitochondrial elongation factors are functionally interchangeable. The elongation factor EF-G of E. coli and its equivalent in bovine mitochondria cannot substitute for each other in the EF-G-dependent GTPase reaction of bacterial and mitochondrial ribosomes.
Using monoclonal antibodies (mAbs) specific for fathead minnow (FHM) Vtg, as well as a purified F... more Using monoclonal antibodies (mAbs) specific for fathead minnow (FHM) Vtg, as well as a purified FHM Vtg standard, we have developed a highly sensitive, simple and robust quantitative ELISA for Vtg in this species. The assay has been validated both in-house ...
Environmental toxicology and chemistry, Oct 18, 2017
Fundamental questions remain about the application of omics in environmental risk assessments, su... more Fundamental questions remain about the application of omics in environmental risk assessments, such as the consistency of data across laboratories. The objective of this study was to determine the congruence of transcript data across six independent laboratories. Male fathead minnows (FHM) were exposed to a measured concentration of 15.8 ng /L EE2 for 96 h. Livers were divided equally and sent to the participating laboratories for transcriptomic analysis using the same FHM microarray. Each laboratory was free to apply bioinformatics pipelines of their choice. There were 12,491 transcripts that were identified by one or more of the laboratories as responsive to EE2. Of these, 587 transcripts (4.7%) were detected by all laboratories. Mean overlap for differentially expressed genes among laboratories was ∼50%, which improved to ∼59.0% using a standardized analysis pipeline. Dynamic range of fold change estimates were variable between laboratories, but ranking transcripts by their relat...
... Chris Crompton, Orange County Watersheds Jim Colston, Orange County Sanitation District ... D... more ... Chris Crompton, Orange County Watersheds Jim Colston, Orange County Sanitation District ... Dr. Geoffrey Scott and Jan Carson of NOAA along with Dr. Michael Crosby and Erin Pulster of the Mote Marine Laboratory are acknowledged for hosting Panel meetings. ...
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and ... more 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 as...
In recent years, concerns have been raised that low concentrations of chemicals may alter the nor... more In recent years, concerns have been raised that low concentrations of chemicals may alter the normal functions of the endocrine system, resulting in potentially significant adverse effects on growth, reproduction, and/or development. For domestic wastewater discharges to surface water bodies, estrogenic activity of effluents has been suggested by chemical analysis, biomarkers, and/or in vitro assays. This two-year WERF project explored approaches to evaluate the potential for biomarker formation as a result of effluent exposures, and the subsequent relevance of the emerging assays and physiological measurements on potential adverse impacts to individuals or populations of fish in the receiving streams. The endocrine system is complex, and many factors can influence the physiological measurements, including methods, sex, age, reproductive status, seasonal and circadian rhythms, diet, temperature, etc. and produce transient changes in physiology but no significant effect on the individual. This study highlights issues in the measurement and interpretation of biomarkers based on laboratory and field studies. Key observations made during this study include the following: This title belongs to WERF Research Report Series ISBN: 9781843396505 (Print) ISBN: 9781780403120 (eBook)
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and ... more 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 as...
In recent years, concerns have been raised that low concentrations of chemicals may alter the nor... more In recent years, concerns have been raised that low concentrations of chemicals may alter the normal functions of the endocrine system, resulting in potentially significant adverse effects on growth, reproduction, and/or development. For domestic wastewater discharges to surface water bodies, estrogenic activity of effluents has been suggested by chemical analysis, biomarkers, and/or in vitro assays. This two-year WERF project explored approaches to evaluate the potential for biomarker formation as a result of effluent exposures, and the subsequent relevance of the emerging assays and physiological measurements on potential adverse impacts to individuals or populations of fish in the receiving streams. The endocrine system is complex, and many factors can influence the physiological measurements, including methods, sex, age, reproductive status, seasonal and circadian rhythms, diet, temperature, etc. and produce transient changes in physiology but no significant effect on the individual. This study highlights issues in the measurement and interpretation of biomarkers based on laboratory and field studies. Key observations made during this study include the following: This title belongs to WERF Research Report Series ISBN: 9781843396505 (Print) ISBN: 9781780403120 (eBook)
Quantum dots with different surface chemistries display a sex-specific tissue distribution follow... more Quantum dots with different surface chemistries display a sex-specific tissue distribution following oral exposure in fathead minnows, an agastric fish.
Quantum dots with different surface chemistries display a sex-specific tissue distribution follow... more Quantum dots with different surface chemistries display a sex-specific tissue distribution following oral exposure in fathead minnows, an agastric fish.
The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for pr... more 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% ...
Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish,... more Endocrine-disrupting chemicals are exogenous substances that can impact the reproduction of fish, potentially by altering circulating concentrations of 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT). Common methods to measure steroids in plasma samples include radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs). The present study examines variability in E2, T, and 11-KT across 8 laboratories measuring reference and pulp mill effluent-exposed white sucker (Catostomus commersoni) plasma. We examine the contribution of assay type (RIA vs ELISA), standardized hormone extraction, location of values on the standard curve (upper and lower limits), and other variables on the ability to distinguish hormone levels between reference and exposed fish and the impact of these variables on quantitation of hormones in different laboratories. Of the 8 participating laboratories, 7 of 8 and 7 of 7 identified differences between sites for female E2 and female T, respectively, and 7 of 7 and 4 of 5 identified no differences between male T and male 11-KT. Notably, however, the ng/mL concentration of steroids measured across laboratories varied by factors of 10-, 6-, 14-, and 10-fold, respectively. Within laboratory intra-assay variability was generally acceptable and below 15%. Factors contributing to interlaboratory variability included calculation errors, assay type, and methodology. Based on the interlaboratory variability detected, we provide guidelines and recommendations to improve the accuracy and precision of steroid measurements in fish ecotoxicology studies.
Biochemical and Biophysical Research Communications, 1979
ABSTRACT Salt-washed ribosomes from bovine mitochondria and E. coli require homologous factor pre... more ABSTRACT Salt-washed ribosomes from bovine mitochondria and E. coli require homologous factor preparations for activity in poly (U)-directed systems for phenylalanine polymerization, contrary to the widespread notion that bacterial and mitochondrial elongation factors are functionally interchangeable. The elongation factor EF-G of E. coli and its equivalent in bovine mitochondria cannot substitute for each other in the EF-G-dependent GTPase reaction of bacterial and mitochondrial ribosomes.
Using monoclonal antibodies (mAbs) specific for fathead minnow (FHM) Vtg, as well as a purified F... more Using monoclonal antibodies (mAbs) specific for fathead minnow (FHM) Vtg, as well as a purified FHM Vtg standard, we have developed a highly sensitive, simple and robust quantitative ELISA for Vtg in this species. The assay has been validated both in-house ...
Environmental toxicology and chemistry, Oct 18, 2017
Fundamental questions remain about the application of omics in environmental risk assessments, su... more Fundamental questions remain about the application of omics in environmental risk assessments, such as the consistency of data across laboratories. The objective of this study was to determine the congruence of transcript data across six independent laboratories. Male fathead minnows (FHM) were exposed to a measured concentration of 15.8 ng /L EE2 for 96 h. Livers were divided equally and sent to the participating laboratories for transcriptomic analysis using the same FHM microarray. Each laboratory was free to apply bioinformatics pipelines of their choice. There were 12,491 transcripts that were identified by one or more of the laboratories as responsive to EE2. Of these, 587 transcripts (4.7%) were detected by all laboratories. Mean overlap for differentially expressed genes among laboratories was ∼50%, which improved to ∼59.0% using a standardized analysis pipeline. Dynamic range of fold change estimates were variable between laboratories, but ranking transcripts by their relat...
... Chris Crompton, Orange County Watersheds Jim Colston, Orange County Sanitation District ... D... more ... Chris Crompton, Orange County Watersheds Jim Colston, Orange County Sanitation District ... Dr. Geoffrey Scott and Jan Carson of NOAA along with Dr. Michael Crosby and Erin Pulster of the Mote Marine Laboratory are acknowledged for hosting Panel meetings. ...
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and ... more 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 as...
In recent years, concerns have been raised that low concentrations of chemicals may alter the nor... more In recent years, concerns have been raised that low concentrations of chemicals may alter the normal functions of the endocrine system, resulting in potentially significant adverse effects on growth, reproduction, and/or development. For domestic wastewater discharges to surface water bodies, estrogenic activity of effluents has been suggested by chemical analysis, biomarkers, and/or in vitro assays. This two-year WERF project explored approaches to evaluate the potential for biomarker formation as a result of effluent exposures, and the subsequent relevance of the emerging assays and physiological measurements on potential adverse impacts to individuals or populations of fish in the receiving streams. The endocrine system is complex, and many factors can influence the physiological measurements, including methods, sex, age, reproductive status, seasonal and circadian rhythms, diet, temperature, etc. and produce transient changes in physiology but no significant effect on the individual. This study highlights issues in the measurement and interpretation of biomarkers based on laboratory and field studies. Key observations made during this study include the following: This title belongs to WERF Research Report Series ISBN: 9781843396505 (Print) ISBN: 9781780403120 (eBook)
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and ... more 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 as...
In recent years, concerns have been raised that low concentrations of chemicals may alter the nor... more In recent years, concerns have been raised that low concentrations of chemicals may alter the normal functions of the endocrine system, resulting in potentially significant adverse effects on growth, reproduction, and/or development. For domestic wastewater discharges to surface water bodies, estrogenic activity of effluents has been suggested by chemical analysis, biomarkers, and/or in vitro assays. This two-year WERF project explored approaches to evaluate the potential for biomarker formation as a result of effluent exposures, and the subsequent relevance of the emerging assays and physiological measurements on potential adverse impacts to individuals or populations of fish in the receiving streams. The endocrine system is complex, and many factors can influence the physiological measurements, including methods, sex, age, reproductive status, seasonal and circadian rhythms, diet, temperature, etc. and produce transient changes in physiology but no significant effect on the individual. This study highlights issues in the measurement and interpretation of biomarkers based on laboratory and field studies. Key observations made during this study include the following: This title belongs to WERF Research Report Series ISBN: 9781843396505 (Print) ISBN: 9781780403120 (eBook)
Quantum dots with different surface chemistries display a sex-specific tissue distribution follow... more Quantum dots with different surface chemistries display a sex-specific tissue distribution following oral exposure in fathead minnows, an agastric fish.
Quantum dots with different surface chemistries display a sex-specific tissue distribution follow... more Quantum dots with different surface chemistries display a sex-specific tissue distribution following oral exposure in fathead minnows, an agastric fish.
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