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James D Astwood

Publications and source records attributed to James D Astwood.

10 recordsLinked to original sources
PubMed2006

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation.

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.

Arabidopsis↗
PubMed2006

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 2. Assessing natural variability.

Proteomics is currently tested as a complementary tool for the safety assessment of genetically modified (GM) crops. Understanding the natural variability of the proteome is crucial for the interpretation of biological differences between transgenic and nontransgenic parental lines. The natural variation of seed protein profiles among a set of 12 Arabidopsis thaliana ecotypes was determined by utilizing two-dimensional electrophoresis (2DE). The total number of different resolved protein spots found among the 12 ecotypes was 931 with a range of 573 (Mt-0) to 653 (Condara) in any one ecotype. Although the ecotypes were grown side-by-side in an environmentally controlled growth chamber, almost half of the resolved spots varied with respect to their presence/absence, and 95% of the spots present in all ecotypes varied in spot quantity (2-53-fold). In the evaluation of unintended effects of genetic modification, it is concluded that the experimental design must account for existing natural variability, which, in the case of the expressed proteome, can be substantial.

Arabidopsis↗
PubMed2006

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of gentically modified crops. 3. Assessing unintended effects.

The current procedures to assess the safety of food and feed derived from modern biotechnology include the investigation of possible unintended effects. To improve the probability of detecting unintended effects, profiling techniques such as proteomics are currently tested as complementary analytical tools to the existing safety assessment. An optimized two-dimensional gel electrophoresis (2DE) method was used as a proteomics approach to investigate insertional and pleiotropic effects on the proteome due to genetic engineering. Twelve transgenic Arabidopsis thaliana lines were analyzed by 2DE, and their seed proteomes were compared to that of their parental line as well as to 12 Arabidopsis ecotype lines. The genetic modification of the Arabidopsis lines, using three different genes and three different promoters, did not cause unintended changes to the analyzed seed proteome. Differences in spot quantity between transgenic and nontransgenic lines fell in the range of values found in the 12 Arabidopsis ecotype lines or were related to the introduced gene.

Arabidopsis↗
PubMed2005

Natural variability of metabolites in maize grain: differences due to genetic background.

Understanding the impact of genetic diversity on crop biochemical composition is a prerequisite to the interpretation and potential relevance of biochemical differences experimentally observed between genotypes. This is particularly important in the context of comparative safety assessments for crops developed by new technologies such as genetic engineering. To interrogate the natural variability of biochemical composition, grain from seven maize hybrids grown at four geographically distinct sites in Europe was analyzed for levels of proximates (fat, protein, moisture, ash, and carbohydrates), fiber, amino acids, fatty acids, four vitamins, nine minerals, and secondary metabolites. Statistical evaluation of the compositional data at the p < 0.05 level compared each hybrid against every other hybrid (head-to-head) for all analytes at each site and then across all sites to understand the factors contributing to variability. Of the 4935 statistical comparisons made in this study, 40% (1986) were found to be significant. The magnitude of differences observed, as a percent, ranged between 0.84 and 149% when all individual sites and the combined sites were considered. The large number of statistically significant differences in the levels of these analytes between seven commercial hybrids emphasizes the importance of genetic background and environment as determinants of the biochemical composition of maize grain, reflects the inherent natural variability in those analytes across a representative sampling of maize hybrids, and provides a baseline of the natural range of these nutritional and antinutritional components in maize for comparative compositional assessments.

Algorithms↗
PubMed2004

Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to that of conventional corn (Zea mays l.).

Insect-protected corn hybrids containing event MON 863 protect corn plants against feeding damage from corn rootworm (Diabrotica), a major North American insect pest. Corn event MON 863 contains a gene that expresses an amino acid sequence variant of the wild-type Cry3Bb1 insecticidal protein from Bacillus thuringiensis. The purpose of this study was to compare the composition of corn containing event MON 863 with that of conventional nontransgenic corn. Compositional analyses were conducted to measure proximates, fiber, amino acids, fatty acids, minerals, folic acid, thiamin, riboflavin, vitamin E, antinutrients, and certain secondary metabolites in grain and proximates and fiber content in forage collected from a total of eight field sites in the U.S. and Argentina. Compositional analyses demonstrated that the grain and forage of event MON 863 are comparable in their nutritional content to the control corn hybrid and conventional corn. These comparisons, together with the history of the safe use of corn as a common component of animal feed and human food, support the conclusion that corn event MON 863 is compositionally equivalent to, and as safe and nutritious as, conventional corn hybrids grown commercially today.

Amino Acids↗
PubMed2004

The composition of grain and forage from glyphosate tolerant wheat MON 71800 is equivalent to that of conventional wheat (Triticum aestivum L.).

Glyphosate tolerant wheat MON 71800, simply referred to as MON 71800, contains a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein from Agrobacterium sp. strain CP4 (CP4 EPSPS) that has a reduced affinity for glyphosate as compared to the endogenous plant EPSPS enzyme. The purpose of this work was to evaluate the compositional equivalence of MON 71800 to its nontransgenic parent as well as to conventional wheat varieties. The compositional assessment evaluated the levels of proximates, amino acids, fatty acids, minerals, vitamins, secondary metabolites, and antinutrients in wheat forage and grain grown during two field seasons across a total of eight sites in the United States and Canada. These data demonstrated that with respect to these important nutritional components, the forage and grain from MON 71800 were equivalent to those of its nontransgenic parent and commercial wheat varieties. These data, together with the previously established safety of the CP4 EPSPS protein, support the conclusion that glyphosate tolerant wheat MON 71800 is as safe and nutritious as commercial wheat varieties.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2003

Workshop overview: approaches to the assessment of the allergenic potential of food from genetically modified crops.

There is a need to assess the safety of foods deriving from genetically modified (GM) crops, including the allergenic potential of novel gene products. Presently, there is no single in vitro or in vivo model that has been validated for the identification or characterization of potential food allergens. Instead, the evaluation focuses on risk factors such as source of the gene (i.e., allergenic vs. nonallergenic sources), physicochemical and genetic comparisons to known allergens, and exposure assessments. The purpose of this workshop was to gather together researchers working on various strategies for assessing protein allergenicity: (1) to describe the current state of knowledge and progress that has been made in the development and evaluation of appropriate testing strategies and (2) to identify critical issues that must now be addressed. This overview begins with a consideration of the current issues involved in assessing the allergenicity of GM foods. The second section presents information on in vitro models of digestibility, bioinformatics, and risk assessment in the context of clinical prevention and management of food allergy. Data on rodent models are presented in the next two sections. Finally, nonrodent models for assessing protein allergenicity are discussed. Collectively, these studies indicate that significant progress has been made in developing testing strategies. However, further efforts are needed to evaluate and validate the sensitivity, specificity, and reproducibility of many of these assays for determining the allergenicity potential of GM foods.

Animals↗
PubMed2002

Comparison of the nutritional profile of glyphosate-tolerant corn event NK603 with that of conventional corn (Zea mays L.).

The composition of glyphosate-tolerant (Roundup Ready) corn event NK603 was compared with that of conventional corn grown in the United States in 1998 and in the European Union in 1999 to assess compositional equivalence. Grain and forage samples were collected from both replicated and nonreplicated field trials, and compositional analyses were performed to measure proximates, fiber, amino acids, fatty acids, vitamin E, nine minerals, phytic acid, trypsin inhibitor, and secondary metabolites in grain as well as proximates and fiber in forage. Statistical analysis of the data was conducted to assess statistical significance at the p < 0.05 level. The values for all of the biochemical components assessed for corn event NK603 were similar to those of the nontransgenic control or were within the published range observed for nontransgenic commercial corn hybrids. In addition, the compositional profile of Roundup Ready corn event NK603 was compared with that of traditional corn hybrids grown in Europe by calculating a 99% tolerance interval to describe compositional variability in the population of traditional corn varieties in the marketplace. These comparisons, together with the history of the safe use of corn as a common component of animal feed and human food, support the conclusion that Roundup Ready corn event NK603 is compositionally equivalent to, and as safe and nutritious as, conventional corn hybrids grown commercially today.

Amino Acids↗
PubMed2002

Bioinformatic methods for allergenicity assessment using a comprehensive allergen database.

BACKGROUND: A principal aim of the safety assessment of genetically modified crops is to prevent the introduction of known or clinically cross-reactive allergens. Current bioinformatic tools and a database of allergens and gliadins were tested for the ability to identify potential allergens by analyzing 6 Bacillus thuringiensis insecticidal proteins, 3 common non-allergenic food proteins and 50 randomly selected corn (Zea mays) proteins. METHODS: Protein sequences were compared to allergens using the FASTA algorithm and by searching for matches of 6, 7 or 8 contiguous identical amino acids. RESULTS: No significant sequence similarities or matches of 8 contiguous amino acids were found with the B. thuringiensis or food proteins. Surprisingly, 41 of 50 corn proteins matched at least one allergen with 6 contiguous identical amino acids. Only 7 of 50 corn proteins matched an allergen with 8 contiguous identical amino acids. When assessed for overall structural similarity to allergens, these 7 plus 2 additional corn proteins shared >or=35% identity in an overlap of >or=80 amino acids, but only 6 of the 7 were similar across the length of the protein, or shared >50% identity to an allergen. CONCLUSIONS: An evaluation of a protein by the FASTA algorithm is the most predictive of a clinically relevant cross-reactive allergen. An additional search for matches of 8 amino acids may provide an added margin of safety when assessing the potential allergenicity of a protein, but a search with a 6-amino-acid window produces many random, irrelevant matches.

Algorithms↗