Crop varieties with multiple GM events combined by conventional breeding have become important in... more Crop varieties with multiple GM events combined by conventional breeding have become important in global agriculture. The regulatory requirements in different countries for such products vary considerably, placing an additional burden on regulatory agencies in countries where the submission of additional data is required and delaying the introduction of innovative products to meet agricultural needs. The process of conventional plant breeding has predictably provided safe food and feed products both historically and in the modern era of plant breeding. Thus, previously approved GM events that have been combined by conventional plant breeding and contain GM traits that are not likely to interact in a manner affecting safety should be considered to be as safe as their conventional counterparts. Such combined GM event crop varieties should require little, if any, additional regulatory data to meet regulatory requirements.
A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin I... more A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin III (ATIII) with thrombin bound to the surface of the microvascular endothelium. Addition of ATIII to the system after thrombin is equilibrated with its binding sites results in inhibition of the enzyme as measured by disappearance of thrombin enzymatic activity from the circulation or by appearance of 125I-thrombin-ATIII complexes. The rate of inhibition of thrombin as reflected by either method is independent of the bound state of thrombin. Comparable results are obtained with ATIII modified at a single tryptophan residue. This modification does not alter the reaction rate of ATIII with thrombin but abolishes the capacity of heparin or heparan sulfate to enhance the reaction rate. From the kinetics and structural studies and the fit of the kinetics to a theoretical model relating binding equilibrium to thrombin inhibition, it is concluded that glycosaminoglycans are not involved in the ...
Poster Session P1. Allergy sensitisation lected for preliminary clinical trial study in compariso... more Poster Session P1. Allergy sensitisation lected for preliminary clinical trial study in comparison with gel base. The results showed severe allergic reactions in 55% and 85% of patients which received topical gel containing chamomile (3.5%)-myrrh (1%) and chamomile (3.5%), respectively. The inflammation, erythema and itching were the main allergic reactions in investigated volunteers. These side effects were observed in 10% and 5% of patients, which received myrrh 2% and 1% topical gel, respectively. The results showed that the preparation containing 1% myrrh had the fewer side effects and the most effects on treatment of Paederus dermatitis.
hosted an expert workshop 22-24 February 2005 in Mallorca, Spain, to review the state-of-the-scie... more hosted an expert workshop 22-24 February 2005 in Mallorca, Spain, to review the state-of-the-science for conducting a sequence homology/bioinformatics evaluation in the context of a comprehensive allergenicity assessment for novel proteins, to obtain consensus on the value and role of bioinformatics in evaluating novel proteins, and to discuss the utility and methods of allergenspecific IgE testing in the diagnosis of food allergy. The workshop participants included over forty international experts from academia, industry, and government. The workshop was hosted by the HESI Protein Allergenicity Technical committee, which has established a long-term program whose mission is to advance the scientific understanding of the relevant parameters for characterizing the allergenic potential of novel proteins.
Proceedings of the National Academy of Sciences, 1991
A laboratory-selected colony of Heliothis virescens displaying a 20-to 70-fold level of resistanc... more A laboratory-selected colony of Heliothis virescens displaying a 20-to 70-fold level of resistance to Bacilus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains ofH. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2-to 4-fold reduction in binding affinity was accompanied by a 4-to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryMA, nor did these proteins compete with the CryLA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins.
The host range and relative efficacy of three purified Bacillus thuringiensis insect control prot... more The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. rhuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kursiaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. renebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (L&J varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. ihuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC,). This extremely sensitive assay enabled detection of B. rhuringiensis ssp. kurstaki HD-73 levels as low as 1 rig/ml. 8 1990 academic press, IIIC. KEY WORDS: Bacillus thuringiensis ssp. lenebrionis; Bacillus rhuringiensis ssp. kursraki; insecticidal spectrum; purified 8-endotoxin.
The International Life Science Institute&... more The International Life Science Institute's Health and Environmental Sciences Institute's Protein Allergenicity Technical Committee hosted an international workshop October 23-25, 2007, in Nice, France, to review and discuss existing and emerging methods and techniques for improving the current weight-of-evidence approach for evaluating the potential allergenicity of novel proteins. The workshop included over 40 international experts from government, industry, and academia. Their expertise represented a range of disciplines including immunology, chemistry, molecular biology, bioinformatics, and toxicology. Among participants, there was consensus that (1) current bioinformatic approaches are highly conservative; (2) advances in bioinformatics using structural comparisons of proteins may be helpful as the availability of structural data increases; (3) proteomics may prove useful for monitoring the natural variability in a plant's proteome and assessing the impact of biotechnology transformations on endogenous levels of allergens, but only when analytical techniques have been standardized and additional data are available on the natural variation of protein expression in non-transgenic bred plants; (4) basophil response assays are promising techniques, but need additional evaluation around specificity, sensitivity, and reproducibility; (5) additional research is required to develop and validate an animal model for the purpose of predicting protein allergenicity.
One component of the safety assessment of agricultural products produced through biotechnology is... more One component of the safety assessment of agricultural products produced through biotechnology is evaluation of the safety of newly expressed proteins. The ILSI International Food Biotechnology Committee has developed a scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology. Recommendations draw upon knowledge of the biological and chemical characteristics of proteins and testing methods for evaluating potential intrinsic hazards of chemicals. Tier I (potential hazard identification) includes an assessment of the biological function or mode of action and intended application of the protein, history of safe use, comparison of the amino acid sequence of the protein to other proteins, as well as the biochemical and physico-chemical properties of the proteins. Studies outlined in Tier II (hazard characterization) are conducted when the results from Tier I are not sufficient to allow a determination of safety (reasonable certainty of no harm) on a case-by-case basis. These studies may include acute and repeated dose toxicology studies and hypothesis-based testing. The application of these guidelines is presented using examples of transgenic proteins applied for agricultural input and output traits in genetically modified crops along with recommendations for future research considerations related to protein safety assessment.
The safety assessment of genetically modified crops includes the evaluation for potential allerge... more The safety assessment of genetically modified crops includes the evaluation for potential allergenicity. The current 'state-of-the-science' utilizes a weight of evidence approach, as outlined by the Codex Alimentarius commission (Alinorm 03/34 A), recognizing no single endpoint is predictive of the allergenic potential of a novel protein. This approach evaluates: whether the gene source is allergenic, sequence similarity to known allergens, and protein resistance to pepsin in vitro. If concerns are identified, serological studies may be necessary to determine if a protein has IgE binding similar to known allergens. Since there was a lack of standardized/validated methods to conduct the allergenicity assessment, a committee was assembled under the International Life Sciences Institute Health and Environmental Sciences Institute to address this issue. Over the last eight years, the Protein Allergenicity Technical Committee has convened workshops and symposia with allergy experts and government authorities to refine methods that underpin the assessment for potential protein allergenicity. This publication outlines this ongoing effort, summarizing workshops and formal meetings, referencing publications, and highlighting outreach activities. The purpose is to (1) outline 'the state-of-the-science' in predicting protein allergenicity in the context of current international recommendations for novel protein safety assessment, and (2) identify approaches that can be improved and future research needs.
Biochemical and Biophysical Research Communications, 1994
Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of t... more Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.
Regulatory toxicology and pharmacology : RTP, 2004
Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modifie... more Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulo...
Crop varieties with multiple GM events combined by conventional breeding have become important in... more Crop varieties with multiple GM events combined by conventional breeding have become important in global agriculture. The regulatory requirements in different countries for such products vary considerably, placing an additional burden on regulatory agencies in countries where the submission of additional data is required and delaying the introduction of innovative products to meet agricultural needs. The process of conventional plant breeding has predictably provided safe food and feed products both historically and in the modern era of plant breeding. Thus, previously approved GM events that have been combined by conventional plant breeding and contain GM traits that are not likely to interact in a manner affecting safety should be considered to be as safe as their conventional counterparts. Such combined GM event crop varieties should require little, if any, additional regulatory data to meet regulatory requirements.
A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin I... more A recirculating perfused rabbit heart preparation is used to study the reaction of antithrombin III (ATIII) with thrombin bound to the surface of the microvascular endothelium. Addition of ATIII to the system after thrombin is equilibrated with its binding sites results in inhibition of the enzyme as measured by disappearance of thrombin enzymatic activity from the circulation or by appearance of 125I-thrombin-ATIII complexes. The rate of inhibition of thrombin as reflected by either method is independent of the bound state of thrombin. Comparable results are obtained with ATIII modified at a single tryptophan residue. This modification does not alter the reaction rate of ATIII with thrombin but abolishes the capacity of heparin or heparan sulfate to enhance the reaction rate. From the kinetics and structural studies and the fit of the kinetics to a theoretical model relating binding equilibrium to thrombin inhibition, it is concluded that glycosaminoglycans are not involved in the ...
Poster Session P1. Allergy sensitisation lected for preliminary clinical trial study in compariso... more Poster Session P1. Allergy sensitisation lected for preliminary clinical trial study in comparison with gel base. The results showed severe allergic reactions in 55% and 85% of patients which received topical gel containing chamomile (3.5%)-myrrh (1%) and chamomile (3.5%), respectively. The inflammation, erythema and itching were the main allergic reactions in investigated volunteers. These side effects were observed in 10% and 5% of patients, which received myrrh 2% and 1% topical gel, respectively. The results showed that the preparation containing 1% myrrh had the fewer side effects and the most effects on treatment of Paederus dermatitis.
hosted an expert workshop 22-24 February 2005 in Mallorca, Spain, to review the state-of-the-scie... more hosted an expert workshop 22-24 February 2005 in Mallorca, Spain, to review the state-of-the-science for conducting a sequence homology/bioinformatics evaluation in the context of a comprehensive allergenicity assessment for novel proteins, to obtain consensus on the value and role of bioinformatics in evaluating novel proteins, and to discuss the utility and methods of allergenspecific IgE testing in the diagnosis of food allergy. The workshop participants included over forty international experts from academia, industry, and government. The workshop was hosted by the HESI Protein Allergenicity Technical committee, which has established a long-term program whose mission is to advance the scientific understanding of the relevant parameters for characterizing the allergenic potential of novel proteins.
Proceedings of the National Academy of Sciences, 1991
A laboratory-selected colony of Heliothis virescens displaying a 20-to 70-fold level of resistanc... more A laboratory-selected colony of Heliothis virescens displaying a 20-to 70-fold level of resistance to Bacilus thuringiensis proteins was evaluated to identify mechanism(s) of resistance. Brush-border membrane vesicles were isolated from larval midgut epithelium from the susceptible and resistant strains ofH. virescens. Two B. thuringiensis proteins, CryIA(b) and CryIA(c), were iodinated and shown to specifically bind to brush-border membrane vesicles of both insect strains. Multiple changes in the receptor-binding parameters were seen in the resistant strain as compared with the susceptible strain. A 2-to 4-fold reduction in binding affinity was accompanied by a 4-to 6-fold increase in binding-site concentration for both proteins. Although these two B. thuringiensis proteins competed for the same high-affinity binding site, competition experiments revealed different receptor specificity toward these proteins in the resistant H. virescens line. The H. virescens strains were not sensitive to a coleopteran-active protein, CryMA, nor did these proteins compete with the CryLA proteins for binding. Complexity of the mechanism of resistance is consistent with the complex mode of action of B. thuringiensis proteins.
The host range and relative efficacy of three purified Bacillus thuringiensis insect control prot... more The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. rhuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kursiaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. renebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (L&J varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. ihuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC,). This extremely sensitive assay enabled detection of B. rhuringiensis ssp. kurstaki HD-73 levels as low as 1 rig/ml. 8 1990 academic press, IIIC. KEY WORDS: Bacillus thuringiensis ssp. lenebrionis; Bacillus rhuringiensis ssp. kursraki; insecticidal spectrum; purified 8-endotoxin.
The International Life Science Institute&... more The International Life Science Institute's Health and Environmental Sciences Institute's Protein Allergenicity Technical Committee hosted an international workshop October 23-25, 2007, in Nice, France, to review and discuss existing and emerging methods and techniques for improving the current weight-of-evidence approach for evaluating the potential allergenicity of novel proteins. The workshop included over 40 international experts from government, industry, and academia. Their expertise represented a range of disciplines including immunology, chemistry, molecular biology, bioinformatics, and toxicology. Among participants, there was consensus that (1) current bioinformatic approaches are highly conservative; (2) advances in bioinformatics using structural comparisons of proteins may be helpful as the availability of structural data increases; (3) proteomics may prove useful for monitoring the natural variability in a plant's proteome and assessing the impact of biotechnology transformations on endogenous levels of allergens, but only when analytical techniques have been standardized and additional data are available on the natural variation of protein expression in non-transgenic bred plants; (4) basophil response assays are promising techniques, but need additional evaluation around specificity, sensitivity, and reproducibility; (5) additional research is required to develop and validate an animal model for the purpose of predicting protein allergenicity.
One component of the safety assessment of agricultural products produced through biotechnology is... more One component of the safety assessment of agricultural products produced through biotechnology is evaluation of the safety of newly expressed proteins. The ILSI International Food Biotechnology Committee has developed a scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology. Recommendations draw upon knowledge of the biological and chemical characteristics of proteins and testing methods for evaluating potential intrinsic hazards of chemicals. Tier I (potential hazard identification) includes an assessment of the biological function or mode of action and intended application of the protein, history of safe use, comparison of the amino acid sequence of the protein to other proteins, as well as the biochemical and physico-chemical properties of the proteins. Studies outlined in Tier II (hazard characterization) are conducted when the results from Tier I are not sufficient to allow a determination of safety (reasonable certainty of no harm) on a case-by-case basis. These studies may include acute and repeated dose toxicology studies and hypothesis-based testing. The application of these guidelines is presented using examples of transgenic proteins applied for agricultural input and output traits in genetically modified crops along with recommendations for future research considerations related to protein safety assessment.
The safety assessment of genetically modified crops includes the evaluation for potential allerge... more The safety assessment of genetically modified crops includes the evaluation for potential allergenicity. The current 'state-of-the-science' utilizes a weight of evidence approach, as outlined by the Codex Alimentarius commission (Alinorm 03/34 A), recognizing no single endpoint is predictive of the allergenic potential of a novel protein. This approach evaluates: whether the gene source is allergenic, sequence similarity to known allergens, and protein resistance to pepsin in vitro. If concerns are identified, serological studies may be necessary to determine if a protein has IgE binding similar to known allergens. Since there was a lack of standardized/validated methods to conduct the allergenicity assessment, a committee was assembled under the International Life Sciences Institute Health and Environmental Sciences Institute to address this issue. Over the last eight years, the Protein Allergenicity Technical Committee has convened workshops and symposia with allergy experts and government authorities to refine methods that underpin the assessment for potential protein allergenicity. This publication outlines this ongoing effort, summarizing workshops and formal meetings, referencing publications, and highlighting outreach activities. The purpose is to (1) outline 'the state-of-the-science' in predicting protein allergenicity in the context of current international recommendations for novel protein safety assessment, and (2) identify approaches that can be improved and future research needs.
Biochemical and Biophysical Research Communications, 1994
Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of t... more Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.
Regulatory toxicology and pharmacology : RTP, 2004
Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modifie... more Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulo...
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