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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↗
PubMed2003

Development and use of an ELISA test to detect IgE antibody to Cry9c following possible exposure to bioengineered corn.

BACKGROUND: Starlink(TM), a variety of corn genetically engineered to contain the insecticidal protein Cry9c, had not been approved for human consumption because it possessed some characteristics associated with allergenic proteins. However, in the fall of 2000 CRY9C DNA was detected in several corn-containing products, suggesting that Starlink corn had entered the human food supply. Subsequently, consumers, following consumption of corn products, reported a number of adverse health events, possibly consistent with allergic reaction. METHODS: To investigate the possibility of allergic reactions due to Cry9c in these consumers an ELISA test was developed for the purpose of detecting IgE antibodies to Cry9c and blood samples were taken from a total of 18 people who self-reported allergic reactions. Sera collected prior to the 1996 development of Starlink were used as negative controls. RESULTS: None of the adverse event sera were found to be reactive with recombinant Cry9c antigen, based on comparison with normal controls. Although a known human positive control serum containing IgE specific for Cry9c was not available, other controls were incorporated into the ELISA protocol, including the use of sera from subjects allergic to other allergens and their homologous antigens (cat, grass, peanut) to validate the IgE detection reagents. CONCLUSIONS: While the results do not support the likely occurrence of allergic reactions to Cry9c, such reactions cannot be ruled out, nor can the possibility that sera might react with unique glycosylated epitopes of Cry9c that may be expressed in the corn plant/seed.

Bacillus thuringiensis Toxins↗
PubMed2006

Effects of feeding calves genetically modified corn bt11: a clinico-biochemical study.

Genetically modified corn Bt11 is insect-resistant and expresses Cry1Ab toxin, an insecticidal protein, in kernels. Although Bt11 corn is considered safe based on animal performance, there are no reports available on the clinico-biochemical effects of feeding it to cattle. In this study, we evaluated the effects of feeding Bt11 to calves, using blood and ruminal clinico-biochemical parameters. Our three-month-long feeding experiment demonstrated that calves (n=6), fed with a ration containing 43.3% of Bt11 corn kernels as dry matter, did not develop any discernible clinical, hematological, biochemical, or ruminal abnormalities as compared with control calves (n=6) fed non-Bt11 corn. The results suggest that the transgenic Bt11 has no negative clinico-biochemical effects on calves.

Alanine Transaminase↗
PubMed2002

Oviposition on and mining in bolls of Bt and non-Bt cotton by resistant and susceptible pink bollworm (Lepidoptera: Gelechiidae).

Transgenic cotton that produces insecticidal crystal protein Cry1Ac of Bacillus thuringiensis (Bt) has been effective in controlling pink bollworm, Pectinophora gossypiella (Saunders). We compared responses to bolls of Bt cotton and non-Bt cotton by adult females and neonates from susceptible and Cry1Ac-resistant strains of pink bollworm. In choice tests on caged cotton plants in the greenhouse, neither susceptible nor resistant females laid fewer eggs on Bt cotton bolls than on non-Bt cotton bolls, indicating that the Bt toxin did not deter oviposition. Multiple regression revealed that the number of eggs laid per boll was negatively associated with boll age and positively associated with boll diameter. Females also laid more eggs per boll on plants with more bolls. The distribution of eggs among bolls of Bt cotton and non-Bt cotton was clumped, indicating that boll quality rather than avoidance of previously laid eggs was a primary factor in oviposition preference. Parallel to the results from oviposition experiments, in laboratory no-choice tests with 10 neonates per boll, the number of entrance holes per boll did not differ between Bt cotton and non-Bt cotton for susceptible and resistant neonates. Also, like females, neonates preferred younger bolls and larger bolls. Thus, acceptance of bolls by females for oviposition and by neonates for mining was affected by boll age and diameter, but not by Bt toxin in bolls. The lack of discrimination between Bt and non-Bt cotton bolls by pink bollworm from susceptible and resistant strains indicates that oviposition and mining initiation are independent of susceptibility to Cry1Ac.

Animals↗
PubMed2003

Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7-11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.

Animals↗
PubMed2004

Relative concentration of Cry1A in maize leaves and cotton bolls with diverse chlorophyll content and corresponding larval development of fall armyworm (Lepidoptera: Noctuidae) and southwestern corn borer (Lepidoptera: Crambidae) on maize whorl leaf profiles.

To manage insect resistance to transgenic crops that express insecticidal proteins from Bacillus thuringiensis (Bt) Berliner, the U.S. Environmental Protection Agency recommends a refuge-based insect resistance management strategy where a percentage of non-Bt (refuge) crop is grown in proximity to a Bt-expressing crop. An important requirement for this strategy is that the toxin exists at a high effective dose for control of the target pest(s), so that heterozygous individuals in the population do not reach adulthood. Factors that cause reduced levels of toxin in the plant are a threat to this strategy. We quantified Cry1Ab from different areas of the maize, Zea mays L., leaf. In general, the distal tip of the V7 maize leaf had a higher concentration of Cry1Ab compared with the middle section of the V7 leaf, and the middle section of the developing V9 leaf had the lowest concentration of Cry1Ab. When these sections of maize tissue were fed to fall armyworm, Spodoptera frugiperda (J.E. Smith), and southwestern corn borer, Diatraea grandiosella Dyar, there was not a reduction in development or an increase in mortality with tissue that had higher concentrations of toxin. Another study tested the relative concentration of Cry1Ab between the white-yellow, yellow-green, and green portions of the developing ninth leaf within the maize whorl. There were differences in Cry1Ab concentration among these leaf areas. The green tissue had the highest concentration of toxin followed by the yellow-green and white-yellow tissues. Correlations between concentration of Cry1Ab and 5-d fall armyworm larval weights among the three leaf color profiles were all significant and negative, i.e., decreased concentration of Cry1Ab in the leaf tissue resulted in increased 5-d larval weights. There was 100% mortality to the southwestern corn borer larvae fed Cry1Ab maize leaf tissue. Differences in the amount of Cry1Ab in the developing V9 leaf profiles did not alter the absolute susceptibility of the southwestern corn borer to the toxin. In cotton, Gossypium hirsutum L., the amount of Cry1Ac was significantly lower in boll tips where flowers had remained attached compared with normal boll tips. Boll tips where the flowers remained attached are often the site where corn earworms, Helicoverpa zea (Boddie), penetrate Bt cotton bolls. This study demonstrated that, in two diverse plant species, tissue that has low chlorophyll content does not fully express Cry1A. Photosynthesis regulating factors related to mRNA transcription and translation should be studied for their effect on Cry1A production and insect control.

Animals↗
PubMed2005

Abundance of Coleomegilla maculata (Coleoptera: Coccinellidae) in corn rootworm-resistant Cry3Bb1 maize.

Lady beetles (Coleoptera: Coccinellidae) are important polyphagous predators in maize, Zea mays L., fields. Transgenic Cry3Bb1 maize hybrids express a coleopteran-specific insecticidal protein derived from Bacillus thuringiensis (Berliner) subsp. kumamotoensis that is targeted at corn rootworm larvae. This study evaluated impacts of Cry3Bb1 protein-expressing maize, tefluthrin-treated maize, and untreated controls on lady beetle abundance at preanthesis, anthesis, and postanthesis maize-developmental periods near Brookings in eastern South Dakota during 2001 and 2002. The dominant lady beetle species captured on Pherocon AM sticky traps was Coleomegilla maculata De Geer. It comprised 73.5 and 69.9% of all adult Coccinellidae caught in 2001 and 2002, respectively. Numbers of C. maculata captured in Cry3Bb1 maize were not significantly different from those in untreated plots during preanthesis, and adults were more abundant in Cry3Bb1 maize than in tefluthrin-treated and untreated plots during anthesis and postanthesis. Whole-plant sampling confirmed C. maculata predominance with the species representing 89.2 and 91.4% of all adult lady beetles observed in 2001 and 2002, respectively. Whole-plant sampling also indicated a lack of negative effects from Cry3Bb1 maize on abundance of lady beetle eggs, larvae, pupae, or adults. Overall, these findings indicate that Cry3Bb1-expressing hybrids are not likely to impose harmful effects on C. maculata, a species common to maize production systems in the northern Great Plains. This research further suggests that Cry3Bb1 maize has the potential for conservation of these beneficial coccinellids in maize production systems.

Animals↗
PubMed2004

Characterization of melanin produced by a wild-type strain of Bacillus thuringiensis.

Bt L-7601 is a UV resistant wild-type strain, which belongs to Bacillus thuringiensis subsp. dendrolimus serotype H4a4b. It was isolated from nature, and produced a dark brown pigment during the exponential phase of growth. Bt L-7601 had the ability to produce pigment in a general nutrition-abundant medium, which had no L-tyrosine. The pigment was identified as melanin based on chemical testing, its light absorbance, and FT-IR analysis. Bt L-7601 has a strong resistance to UV light. After 30 min irradiation its survival rate was 17 times higher than that of the strain B. thuringiensis subsp. colmeri 15A3, which had no pigment. Results of the bioassays of residual insecticidal activity of Bt formulation with and without pigment produced by Bt L-7601 against larvae of Helicoverpa armigera and Spodoptera exigua after exposure to UV irradiation showed that the pigment is an excellent UV protective agent for the insecticidal proteins.

Animals↗
PubMedDate not supplied

The ecological risks of transgenic plants.

Biotechnologies have been utilized "ante litteram" for thousands of years to produce food and drink and genetic engineering techniques have been widely applied to produce many compounds for human use, from insulin to other medicines. The debate on genetically modified (GM) organisms broke out all over the world only when GM crops were released into the field. Plant ecologists, microbiologists and population geneticists carried out experiments aimed at evaluating the environmental impact of GM crops. The most significant findings concern: the spread of transgenes through GM pollen diffusion and its environmental impact after hybridisation with closely related wild species or subspecies; horizontal gene transfer from transgenic plants to soil microbes; the impact of insecticide proteins released into the soil by transformed plants on non-target microbial soil communities. Recent developments in genetic engineering produced a technology, dubbed "Terminator", which protects patented genes introduced in transgenic plants by killing the seeds in the second generation. This genetic construct, which interferes so heavily with fundamental life processes, is considered dangerous and should be ex-ante evaluated taking into account the data on "unexpected events", as here discussed, instead of relying on the "safe until proven otherwise" claim. Awareness that scientists, biotechnologists and genetic engineers cannot answer the fundamental question "how likely is that transgenes will be transferred from cultivated plants into the natural environment?" should foster long-term studies on the ecological risks and benefits of transgenic crops.

Biotechnology↗
PubMed2004

[Study on the teratogenicity effects of genetically modified rich which expressed cowpea trypsin inhibitor on rats].

OBJECTIVE: Rats were fed by transgenic rice which expressed insecticidal protein CpTI (cowpea trypsin inhibitor) to study if the transgenic rice possessed potential teratogenicity effects. METHODS: Weanling Wistar rats were randomly divided into four groups: transgenic rice group, non transgenic rice group, negative control group and positive control group. The diet of transgenic rice group contained 78.3% transgenic rice. The diet of non transgenic rice group contained 74.7% non transgenic rice which was the parent line of the transgenic one. The diet formula of the two control groups was AIN93G. The marco- and micronutrient content were equal in three diets. When the sexual maturation period of rats arrived, conventional teratogenicity test was performed. RESULTS: The body weight gain of pregnant rats and body weight, body length and tail length of fetal rats in transgenic rice group were significant higher than those in positive control group (P < 0.01). The malformation rate of fetus in transgenic rice group was significant lower than that in positive control group (P < 0.01). CONCLUSION: There were no significant differences of all indexes among transgenic rice group, non transgenic rice group and negative control group (P > 0.05). Judging from these results, the transgenic rice modified with CpTI was considered to have no maternal toxicity, embryotoxicity and teratogenicity effects.

Animals↗
PubMed2004

[Study on food safety of genetically modified rice which expressed cowpea trypsin inhibitor by 90 day feeding test on rats].

OBJECTIVE: Rats were fed by transgenic rice which expressed insecticidal protein CPTI (cowpea trypsin inhibitor) to study if the transgenic rice possessed potential toxic or adverse effects. METHODS: Weanling Wistar rats were randomly divided into three groups: T, N and C group. The diet of T group contained 78.3% transgenic rice. The diet of N group contained 74.7% non transgenic rice which was the parent line of the transgenic one. The diet formula of C group was AIN93G. The macro- and micronutrient content were equal in three diets. The rats were fed for 90 days. Food intakes were weight every day, body-weight were weight and body-length were measured every week. In the middle and at the end of feeding period, haematological value and clinical chemistry parameters were measured, at the end of the 90th day, post-mortem organ coefficient were measured, organ tissues analysis was performed and bone density was measured. RESULTS: In most situation, there were no significant differences among the three groups(P > 0.05) and no histopathological damage were detected. At the end of the 1st month, the male rats' body length of the T group was longer than the other two groups and at the end of the test period, the male rats' blood glucose and ALT were lower than the other two groups. In the middle of the test period, the female rats' red blood cell number and hemoglobin were higher than the other two groups and at the end of the test period, the female rats' monocyte number was higher than the other two groups (P < 0.05). But all of the results were in the normal range which had been reported before. CONCLUSION: From the results of the 90 days feeding test of transgenic rice on rats there did not reveal any signs of toxic and adverse effects.

Animal Feed↗
PubMed2006

[Transformation of embryogenic Calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) mediated by agrobacterium].

Formation of embryogenic calli of Siberian wildrye grass (Elymus sibiricus L. cv. Chuancao No.2) was induced from mature seeds as explants, and proliferated on MS medium containing 2,4-D 5.0 mg/L and KT 0.05 mg/L. An effective and stable callus regeneration system was established by optimizing the culture conditions (Tables 1, 2 and Fig.2). After the calli were subcultured 8 weeks, selected the whitish-yellow-coloured compact nodular calli that transformed with plasmid pCAMBIA1304 carrying hygromycin resistance gene (hptII) and Pseudomonas pseudoalcaligenes insecticidal protein gene (ppIP), which was mediated by an Agrobacterium strain EHA105. Resistant plants were obtained after hygromycin selection (Figs.3, 4). Some important factors that affect the transformation efficiency were studied, which included selection pressure, time of embryogenic calli proliferation, OD value of Agrobacterium suspension, temperature, medium and time of co-cultivation, and concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformation plant regeneration. This research is the first successful genetic transformation of Elymus sibiricus L. cv. Chuancao No.2 mediated by Agrobacterium.

Culture Media↗
PubMed1994

A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro.

CryIA(c) delta-endotoxin, a member of the CryI family of Bacillus thuringiensis insecticidal proteins, specifically recognizes and binds with high affinity to target proteins in the midgut of susceptible insects. Protein blots of Manduca sexta brush-border membranes probed with 125I-CryIA(c) identify a major binding protein of 120 kDa and a minor binding protein of 65 kDa. Monoclonal antibodies were raised against the 120-kDa toxin binding protein. Using isoelectric focusing and monoclonal antibodies (2B3, 8G1, and 12B8) 120- and 65-kDa brush-border proteins were isolated. Labeled CryIA(c) and monoclonal antibodies probed to blots of the affinity-selected proteins recognized the 120- and 65-kDa proteins. When reconstituted into phospholipid vesicles, antibody-selected proteins increased toxin binding (35%) and enhanced toxin-induced 86Rb+ release up to 1000-fold. The 120-kDa protein was identified as aminopeptidase N (EC 3.4.11.2). A CryIA(c)-sensitive phosphatase was also present in the 120/65-kDa protein mixture. These findings provide the first identification of B. thuringiensis toxin binding proteins, although confirmation is needed in vivo.

Amino Acid Sequence↗
PubMed1993

Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.

The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms.

Animals↗
PubMed1996

Insecticidal activity of the protein encoded by the cryV gene of Bacillus thuringiensis kurstaki INA-02.

A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.

Animals↗
PubMed2005

Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B.

The sensitivity of acetylcholinesterase (AChE) biosensors for insecticide detection could be increased substantially by engineering AChE B of Nippostrongylus brasiliensis. The introduction of 10 single and 4 double mutations into the AChE peptide chain led to an increase in sensitivity to 10 of the 11 insecticides tested. The combination of three mutants with the wild-type enzyme in a multienzyme biosensor array enabled the detection of 11 out of the 14 most important organophosphates and carbamates at concentrations below 10 microg/kg, the maximum residue limit of infant food. The detection limit for pirimiphos methyl could be reduced from 10 microg/L to a value as low as 1 ng/L (3.5 x 10(-)(12) mol/L). The newly created biosensors exhibited an extraordinary high storage stability. There was no loss of sensitivity of N. brasiliensis AChE B, immobilized on screen-printed, disposable electrodes, even after 17-month storage at room temperature.

Acetylcholinesterase↗