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Sample records for delta endotoxins cry1ac

  1. Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N.

    PubMed

    de Maagd, R A; Bakker, P L; Masson, L; Adang, M J; Sangadala, S; Stiekema, W; Bosch, D

    1999-01-01

    Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.

  2. Screening of the Bacillus thuringiensis Cry1Ac delta-endotoxin on the artificial phospholipid monolayer incorporated with brush border membrane vesicles of Plutella xylostella by optical biosensor technology.

    PubMed

    Okumura, S; Akao, T; Mizuki, E; Ohba, M; Inouye, K

    2001-02-26

    The binding of Cry1Ac, an insecticidal protein of Bacillus thuringiensis, to a brush border membrane (BBM) isolated from midguts of the diamondback moth Plutella xylostella was examined by surface plasmon resonance (SPR)-based biosensor. BBM was mixed with 1,3-ditetradecylglycero-2-phosphocholine (PC14), a neutral charged artificial lipid, and was reconstructed to a monolayer on a hydrophobic chip for the biosensor. The binding of Cry1Ac to the reconstructed monolayer was analyzed by a two-state binding model, and it was shown that Cry1Ac bound to the monolayer in the first step with an affinity constant (K(1)) of 508 nM, followed by the second uni-molecular step with an equilibrium constant (K(2)) of 0.472. The overall affinity constant K(d) was determined to be 240 nM. The binding was markedly inhibited by N-acetyl-D-galactosamine (K(i)=8 mM). The monolayer was shown to retain a high affinity to Cry1Ac, providing an insect-free system for rapid and large-scale screening of B. thuringiensis insecticidal proteins by the SPR-based biosensor technology.

  3. Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics.

    PubMed

    Rawat, Preeti; Singh, Amarjeet Kumar; Ray, Krishna; Chaudhary, Bhupendra; Kumar, Sanjeev; Gautam, Taru; Kanoria, Shaveta; Kaur, Gurpreet; Kumar, Paritosh; Pental, Deepak; Burma, Pradeep Kumar

    2011-06-01

    High levels of expression of the cry1Ac gene from Bacillus thuringiensis cannot be routinely achieved in transgenic plants despite modifications made in the gene to improve its expression. This has been attributed to the instability of the transcript in a few reports. In the present study, based on the genetic transformation of cotton and tobacco, we show that the expression of the Cry1Ac endotoxin has detrimental effects on both the in vitro and in vivo growth and development of transgenic plants. A number of experiments on developing transgenics in cotton with different versions of cry1Ac gene showed that the majority of the plants did not express any Cry1Ac protein. Based on Southern blot analysis, it was also observed that a substantial number of lines did not contain the cry1Ac gene cassette although they contained the marker gene nptII. More significantly, all the lines that showed appreciable levels of expression were found to be phenotypically abnormal. Experiments on transformation of tobacco with different constructs expressing the cry1Ac gene showed that in vitro regeneration was inhibited by the encoded protein. Further, out of a total of 145 independent events generated with the different cry1Ac gene constructs in tobacco, only 21 showed expression of the Cry1Ac protein, confirming observations made in cotton that regenerants that express high levels of the Cry1Ac protein are selected against during regeneration of transformed events. This problem was circumvented by targeting the Cry1Ac protein to the chloroplast, which also significantly improved the expression of the protein.

  4. Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

    PubMed Central

    Angelucci, Constanza; Barrett-Wilt, Gregory A.; Hunt, Donald F.; Akhurst, Raymond J.; East, Peter D.; Gordon, Karl H.J.; Campbell, Peter M.

    2010-01-01

    Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) δ-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA. PMID:18549954

  5. Susceptibilities of Geographic Populations of Helicoverpa zea (Lepidoptera: Noctuidae) in Mexico to Bt ∂-Endotoxins Cry1Ac and Cry2Ab: An 18-Yr Study.

    PubMed

    Aguilar-Medel, Sotero; Rodríguez, J Concepción; Martínez-Carrillo, José L; Silva-Aguayo, Gonzalo

    2017-09-02

    An insect resistance monitoring program was developed for Mexico to accommodate the commercial introduction and stewardship of Bt cotton. Between 1998 and 2015, field-collected geographic populations of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) were evaluated against Cry1Ac and Cry2Ab proteins of Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) to establish baseline susceptibility data before the commercial use of Bollgard (Cry1Ac) and Bollgard II (Cry1Ac and Cry2Ab) cotton. An annual monitoring program was subsequently established in which a single diagnostic concentration of each Bt protein was used in a diet overlay bioassay. The diagnostic concentration represented the concentration where larvae, evaluated in baseline studies, were reduced in weight by ≥97% relative to untreated controls or failed to molt to third instar after 5 d. In the monitoring study, populations were tested against Cry1Ac from 1998 through 2015, and against Cry2Ab from 2002 through 2004 and again from 2007 through 2015. None of the Cry1Ac-exposed larvae tested during the 18-yr period reached the third larval instar after an exposure of 5 d, and weight reduction relative to untreated control larvae was uniform at about 98-99%. For the 12 yr of Cry2Ab monitoring, no larvae reached third instar, and weight reduction was uniform at >97% relative to controls. These results indicate that H. zea susceptibility to Cry1Ac and Cry2Ab has not changed during the period Bollgard and Bollgard II have been cultivated in Mexico. © The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America.

  6. Potential factors impacting season-long expression of Cry1Ac in 13 commercial varieties of Bollgard® cotton

    PubMed Central

    Adamczyk, John J.; Sumerford, Douglas V.

    2001-01-01

    Thirteen commercial varieties of transgenic Cry1Ac Bacillus thuringiensis Berliner (Bt) cotton were examined across two sites in 2000 for potential factors that impact endotoxin expression. In all cases, two varieties (NuCOTN 33B and DP 458B/RR, Delta & Pineland Co., Scott, MS) expressed more Cry1Ac than the other 11 varieties in various plant structures. These two varieties share the same parental background (DP 5415). Furthermore, when the next generation of plants were tested in the greenhouse, the same varietal patterns were exhibited. These data strongly suggest that factors such as parental background had a stronger impact on the expression of Cry1Ac than the environment. PMID:15455073

  7. Diversity in gut microflora of Helicoverpa armigera populations from different regions in relation to biological activity of Bacillus thuringiensis δ-endotoxin Cry1Ac.

    PubMed

    Paramasiva, Inakarla; Shouche, Yogesh; Kulkarni, Girish Jayant; Krishnayya, Pulipaka Venkata; Akbar, Shaik Mohammed; Sharma, Hari Chand

    2014-12-01

    Transgenic crops expressing toxin proteins from Bacillus thuringiensis (Bt) have been deployed on a large scale for management of Helicoverpa armigera. Resistance to Bt toxins has been documented in several papers, and therefore, we examined the role of midgut microflora of H. armigera in its susceptibility to Bt toxins. The susceptibility of H. armigera to Bt toxin Cry1Ac was assessed using Log-dose-Probit analysis, and the microbial communities were identified by 16S rRNA sequencing. The H. armigera populations from nine locations harbored diverse microbial communities, and had some unique bacteria, suggesting a wide geographical variation in microbial community in the midgut of the pod borer larvae. Phylotypes belonging to 32 genera were identified in the H. armigera midgut in field populations from nine locations. Bacteria belonging to Enterobacteriaceae (Order Bacillales) were present in all the populations, and these may be the common members of the H. armigera larval midgut microflora. Presence and/or absence of certain species were linked to H. armigera susceptibility to Bt toxins, but there were no clear trends across locations. Variation in susceptibility of F1 neonates of H. armigera from different locations to the Bt toxin Cry1Ac was found to be 3.4-fold. These findings support the idea that insect migut microflora may influence the biological activity of Bt toxins.

  8. Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment.

    PubMed Central

    Cooper, M A; Carroll, J; Travis, E R; Williams, D H; Ellar, D J

    1998-01-01

    The Bacillus thuringiensis Cry1Ac delta-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein-protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles. PMID:9677328

  9. Elite Indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas).

    PubMed

    Khanna, H K; Raina, S K

    2002-08-01

    Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic or Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher (approximately 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.

  10. In-Silico Determination of Insecticidal Potential of Vip3Aa-Cry1Ac Fusion Protein Against Lepidopteran Targets Using Molecular Docking

    PubMed Central

    Ahmad, Aftab; Javed, Muhammad R.; Rao, Abdul Q.; Khan, Muhammad A. U.; Ahad, Ammara; Din, Salah ud; Shahid, Ahmad A.; Husnain, Tayyab

    2015-01-01

    Study and research of Bt (Bacillus thuringiensis) transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac) insecticidal protein and vegetative insecticidal protein (Vip3Aa) have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN) and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, and Spodoptera litura) revealed that the Ser290, Ser293, Leu337, Thr340, and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein. PMID:26697037

  11. In-Silico Determination of Insecticidal Potential of Vip3Aa-Cry1Ac Fusion Protein Against Lepidopteran Targets Using Molecular Docking.

    PubMed

    Ahmad, Aftab; Javed, Muhammad R; Rao, Abdul Q; Khan, Muhammad A U; Ahad, Ammara; Din, Salah Ud; Shahid, Ahmad A; Husnain, Tayyab

    2015-01-01

    Study and research of Bt (Bacillus thuringiensis) transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac) insecticidal protein and vegetative insecticidal protein (Vip3Aa) have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN) and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, and Spodoptera litura) revealed that the Ser290, Ser293, Leu337, Thr340, and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

  12. Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase-N receptor is not directly related to toxicity.

    PubMed

    Jenkins, J L; Lee, M K; Sangadala, S; Adang, M J; Dean, D H

    1999-12-03

    Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509-511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV. However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding. Analysis of toxin binding to aminopeptidase-N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.

  13. Common, but complex, mode of resistance of Plutella xylostella to Bacillus thuringiensis toxins Cry1Ab and Cry1Ac.

    PubMed

    Sayyed, Ali H; Gatsi, Roxani; Ibiza-Palacios, M Sales; Escriche, Baltasar; Wright, Denis J; Crickmore, Neil

    2005-11-01

    A field collected population of Plutella xylostella (SERD4) was selected in the laboratory with Bacillus thuringiensis endotoxins Cry1Ac (Cry1Ac-SEL) and Cry1Ab (Cry1Ab-SEL). Both subpopulations showed similar phenotypes: high resistance to the Cry1A toxins and little cross-resistance to Cry1Ca or Cry1D. A previous analysis of the Cry1Ac-SEL showed incompletely dominant resistance to Cry1Ac with more than one factor, at least one of which was sex influenced. In the present study reciprocal mass crosses between Cry1Ab-SEL and a laboratory susceptible population (ROTH) provided evidence that Cry1Ab resistance was also inherited as incompletely dominant trait with more than one factor, and at least one of the factors was sex influenced. Analysis of single pair mating indicated that Cry1Ab-SEL was still heterogeneous for Cry1Ab resistance genes, showing genes with different degrees of dominance. Binding studies showed a large reduction of specific binding of Cry1Ab and Cry1Ac to midgut membrane vesicles of the Cry1Ab-SEL subpopulation. Cry1Ab-SEL was found to be more susceptible to trypsin-activated Cry1Ab toxin than protoxin, although no defect in toxin activation was found. Present and previous results indicate a common basis of resistance to both Cry1Ab and Cry1Ac in selected subpopulations and suggest that a similar set of resistance genes are responsible for resistance to Cry1Ab and Cry1Ac and are selected whichever toxin was used. The possibility of an incompletely dominant trait of resistant to these toxins should be taken into account when considering refuge resistance management strategies.

  14. Interactions of Bacillus thuringiensis Cry1Ac toxin in genetically engineered cotton with predatory heteropterans.

    PubMed

    Torres, Jorge B; Ruberson, John R

    2008-06-01

    A number of cotton varieties have been genetically transformed with genes from Bacillus thuringiensis (Bt) to continuously produce Bt endotoxins, offering whole plant and season-long protection against many lepidopteran larvae. Constant whole-plant toxin expression creates a significant opportunity for non-target herbivores to acquire and bio-accumulate the toxin for higher trophic levels. In the present study we investigated movement of Cry1Ac toxin from the transgenic cotton plant through specific predator-prey pairings, using omnivorous predators with common cotton pests as prey: (1) the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), with the predator Podisus maculiventris (Heteroptera: Pentatomidae); (2) the two-spotted spider mite, Tetranychus urticae (Acarina: Tetranychidae), with the predatory big-eyed bug Geocoris punctipes (Heteroptera: Geocoridae) and (3) with the predatory damsel bug Nabis roseipennis (Heteropera: Nabidae); and (4) the thrips Frankliniella occidentalis (Thysanoptera: Thripidae) with the predatory pirate bug Orius insidiosus (Heteroptera: Anthocoridae). We quantified Cry1Ac toxin in the cotton plants, and in the pests and predators, and the effects of continuous feeding on S. exigua larvae fed either Bt or non-Bt cotton on life history traits of P. maculiventris. All three herbivores were able to convey Cry1Ac toxin to their respective predators. Among the herbivores, T. urticae exhibited 16.8 times more toxin in their bodies than that expressed in Bt-cotton plant, followed by S. exigua (1.05 times), and F. occidentalis immatures and adults (0.63 and 0.73 times, respectively). Of the toxin in the respective herbivorous prey, 4, 40, 17 and 14% of that amount was measured in the predators G. punctipes, P. maculiventris, O. insidiosus, and N. roseipennis, respectively. The predator P. maculiventris exhibited similar life history characteristics (developmental time, survival, longevity, and fecundity) regardless of the prey's food

  15. Development of bollworms, Helicoverpa zea, on two commercial Bollgard® cultivars that differ in overall Cry1Ac levels

    PubMed Central

    Adamczyk, John J.; Gore, Jeffrey

    2004-01-01

    Research was conducted to quantify the development of the corn earworm (= bollworm), Helicoverpa zea (Boddie), on two different transgenic cotton cultivars (DP 50B and NuCOTN 33B) that contained different levels of the Cry1Ac endotoxin from the soil bacterium, Bacillus thuringiensis Berliner. Using a field cage, an inverse relationship between the amount of Cry1Ac among cultivars versus the weight of bollworm larvae was observed. Larvae that were recovered from the DP 50B cultivar expressing lower Cry1Ac weighed significantly more than larvae collected from the higher expressing NuCOTN 33B cultivar. Cotton plants from NuCOTN 33B were measured as expressing 300% more Cry1Ac than DP 50B plants. The distribution of larval weights indicates that more late-instars (> 200 mg) were collected from the lower expressing DP50B cultivar than the higher expressing NuCOTN 33B cultivar. Within a single population, bollworm larvae were highly variable in their development when feeding on Bollgard® cotton. Possible reasons and consequences for this variation are discussed. PMID:15861247

  16. The heliothis virescens 170 kDa aminopeptidase functions as "receptor A" by mediating specific Bacillus thuringiensis Cry1A delta-endotoxin binding and pore formation.

    PubMed

    Luo, K; Sangadala, S; Masson, L; Mazza, A; Brousseau, R; Adang, M J

    1997-01-01

    The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes. Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine. The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein. Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance. Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN. Each Cry1A toxin recognized two binding sites: a high affinity site with KD ranging from 41 to 95 nM and a lower affinity site with KD in the 325 to 623 nM range. N-acetylgalactosamine inhibited Cry1Ac but not Cry1Aa and Cry1Ab binding to 170 kDa APN. When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced 86Rb+ release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused 86Rb+ release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins. The correlation between toxin-binding specificity and 86Rb+ release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes.

  17. Synergistic interactions between Cry1Ac and natural cotton defenses limit survival of Cry1Ac-resistant Helicoverpa zea (Lepidoptera: Noctuidae) on Bt cotton.

    PubMed

    Anilkumar, Konasale J; Sivasupramaniam, Sakuntala; Head, Graham; Orth, Robert; Van Santen, Edzard; Moar, William J

    2009-07-01

    Larvae of the bollworm Helicoverpa zea (Boddie) show some tolerance to Bacillus thuringiensis (Bt) Cry1Ac, and can survive on Cry1Ac-expressing Bt cotton, which should increase resistance development concerns. However, field-evolved resistance has not yet been observed. In a previous study, a population of H. zea was selected for stable resistance to Cry1Ac toxin. In the present study, we determined in laboratory bioassays if larvae of the Cry1Ac toxin-resistant H. zea population show higher survival rates on field-cultivated Bt cotton squares (= flower buds) collected prebloom-bloom than susceptible H. zea. Our results show that Cry1Ac toxin-resistant H. zea cannot complete larval development on Cry1Ac-expressing Bt cotton, despite being more than 150-fold resistant to Cry1Ac toxin and able to survive until pupation on Cry1Ac toxin concentrations greater than present in Bt cotton squares. Since mortality observed for Cry1Ac-resistant H. zea on Bt cotton was higher than expected, we investigated whether Cry1Ac interacts with gossypol and or other compounds offered with cotton powder in artificial diet. Diet incorporation bioassays were conducted with Cry1Ac toxin alone, and with gossypol and 4% cotton powder in the presence and absence of Cry1Ac. Cry1Ac toxin was significantly more lethal to susceptible H. zea than to resistant H. zea, but no difference in susceptibility to gossypol was observed between strains. However, combinations of Cry1Ac with gossypol or cotton powder were synergistic against resistant, but not against susceptible H. zea. Gossypol concentrations in individual larvae showed no significant differences between insect strains, or between larvae fed gossypol alone vs. those fed gossypol plus Cry1Ac. These results may help explain the inability of Cry1Ac-resistant H. zea to complete development on Bt cotton, and the absence of field-evolved resistance to Bt cotton by this pest.

  18. New Resistance Mechanism in Helicoverpa armigera Threatens Transgenic Crops Expressing Bacillus thuringiensis Cry1Ac Toxin

    PubMed Central

    Gunning, Robin V.; Dang, Ho T.; Kemp, Fred C.; Nicholson, Ian C.; Moores, Graham D.

    2005-01-01

    In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed. PMID:15870346

  19. New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin.

    PubMed

    Gunning, Robin V; Dang, Ho T; Kemp, Fred C; Nicholson, Ian C; Moores, Graham D

    2005-05-01

    In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.

  20. Proteomic analysis of novel Cry1Ac binding proteins in Helicoverpa armigera (Hübner)

    USDA-ARS?s Scientific Manuscript database

    Aminopeptidase N (APN) and cadherin-like proteins have been previously identified as Cry1Ac-binding proteins in Helicoverpa armigera (Hübner). In this study, a proteomic approach was used to identify novel Cry1Ac-binding proteins in H. armigera. Brush border membrane vesicles (BBMV) of H. armigera w...

  1. Acquisition of Cry1Ac Protein by Non-Target Arthropods in Bt Soybean Fields

    PubMed Central

    Yu, Huilin; Romeis, Jörg; Li, Yunhe; Li, Xiangju; Wu, Kongming

    2014-01-01

    Soybean tissue and arthropods were collected in Bt soybean fields in China at different times during the growing season to investigate the exposure of arthropods to the plant-produced Cry1Ac toxin and the transmission of the toxin within the food web. Samples from 52 arthropod species/taxa belonging to 42 families in 10 orders were analysed for their Cry1Ac content using enzyme-linked immunosorbent assay (ELISA). Among the 22 species/taxa for which three samples were analysed, toxin concentration was highest in the grasshopper Atractomorpha sinensis and represented about 50% of the concentration in soybean leaves. Other species/taxa did not contain detectable toxin or contained a concentration that was between 1 and 10% of that detected in leaves. These Cry1Ac-positive arthropods included a number of mesophyll-feeding Hemiptera, a cicadellid, a curculionid beetle and, among the predators, a thomisid spider and an unidentified predatory bug belonging to the Anthocoridae. Within an arthropod species/taxon, the Cry1Ac content sometimes varied between life stages (nymphs/larvae vs. adults) and sampling dates (before, during, and after flowering). Our study is the first to provide information on Cry1Ac-expression levels in soybean plants and Cry1Ac concentrations in non-target arthropods in Chinese soybean fields. The data will be useful for assessing the risk of non-target arthropod exposure to Cry1Ac in soybean. PMID:25110881

  2. Influences of Cry1Ac broccoli on larval survival and oviposition of diamondback moth.

    PubMed

    Yi, Dengxia; Cui, Shusong; Yang, Limei; Fang, Zhiyuan; Liu, Yumei; Zhuang, Mu; Zhang, Yangyong

    2015-01-01

    Larval survival and oviposition behavior of three genotypes of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), (homozygous Cry1Ac-susceptibile, Cry1Ac-resistant, and their F1 hybrids), on transgenic Bacillus thuringiensis (Bt) broccoli expressing different levels of Cry1Ac protein were evaluated in laboratory. These Bt broccoli lines were designated as relative low, medium, and high, respectively, according to the Cry1Ac content. Untransformed brocccoli plants were used as control. Larval survival of diamondback moth on non-Bt leaves was not significantly different among the three genotypes. The Cry1Ac-resistant larvae could survive on the low level of Bt broccoli plants, while Cry1Ac-susceptible and F1 larvae could not survive on them. The three genotypes of P. xylostella larvae could not survive on medium and high levels of Bt broccoli. In oviposition choice tests, there was no significant difference in the number of eggs laid by the three P. xylostella genotypes among different Bt broccoli plants. The development of Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella on intact Bt plants was also tested in greenhouse. All susceptible P. xylostella larvae died on all Bt plants, while resistant larvae could survive on broccoli, which expresses low Cry1Ac protein under greenhouse conditions. The results of the greenhouse trials were similar to that of laboratory tests. This study indicated that high dose of Bt toxins in broccoli cultivars or germplasm lines is required for effective resistance management.

  3. Production and characterization of Bacillus thuringiensis Cry1Ac-resistant cotton bollworm Helicoverpa zea (Boddie).

    PubMed

    Anilkumar, Konasale J; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J

    2008-01-01

    Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments.

  4. ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth.

    PubMed

    Ocelotl, Josue; Sánchez, Jorge; Gómez, Isabel; Tabashnik, Bruce E; Bravo, Alejandra; Soberón, Mario

    2017-05-24

    Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.

  5. Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm

    PubMed Central

    Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E.

    2016-01-01

    Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins. PMID:27257885

  6. Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

    PubMed

    Wei, Jizhen; Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E

    2016-01-01

    Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

  7. Quercetin interacts with Cry1Ac protein to affect larval growth and survival of Helicoverpa armigera.

    PubMed

    Li, Zhen; Guan, Xiumin; Michaud, J P; Zhang, Qingwen; Liu, Xiaoxia

    2016-07-01

    Bt cotton has been widely planted in China for over a decade to control H. armigera, but field surveys indicate increasing resistance in the pest. It has been speculated that accumulating plant secondary compounds in mature cotton may interact with Bt toxins and affect the toxicity of Bt to H. armigera. Both quercetin, one of the main flavonoids in cotton, and the Bt toxin Cry1Ac protein had significant negative effects on the growth, development and survival of H. armigera when added singly to artificial diet, but their effects were inhibited when added in combination. Quercetin was antagonistic to Cry1Ac toxicity at all tested concentrations. The accumulation of quercetin might be one factor contributing to the reduced toxicity of mature Bt cotton plants to H. armigera, and could partially explain the reduced efficacy of Cry1Ac in controlling this pest in the field. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  8. Novel pink bollworm resistance to the Bt toxin Cry 1Ac: effects on mating, oviposition, larval development and survival.

    PubMed

    Fabrick, J A; Forlow Jech, L; Henneberry, T J

    2009-01-01

    Bt cotton plants are genetically engineered to produce insecticidal toxins from the Bacillus thuringiensis (Bt) Berliner (Bacillales: Bacillaceae) bacterium and target key lepidopteran pests. In all previous strains of pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) selected in the laboratory for resistance to insecticidal Cry1Ac toxin using an artificial diet containing the toxin, resistance to Cry1Ac and to Bt cotton is linked to three cadherin alleles (r1, r2, and r3). In contrast, the BG(4) pink bollworm strain was selected for resistance to Bt cotton by feeding larvae for four days in each of 42 generations on bolls of 'NuCOTN33B(R)' that expressed Cry1Ac toxin. After additional selection for eleven generations on Cry1Ac-incorporated diet, the susceptibility to Cry1Ac, fecundity, egg viability, and mating of this strain (Bt4R) was compared with the unselected Cry1Ac-susceptible parent strain. Some larvae of the Bt4R strain survived on diet containing >or= 10 microg Cry1Ac per milliliter artificial diet, but none survived on transgenic cotton bolls. In contrast to strains selected exclusively on Cry1Ac diet, some survival of progeny of reciprocal moth crosses of Bt4R resistant and Bt-susceptible strains occurred on Cry1Ac-treated diet, suggesting differences in levels of dominance. The Bt4R resistant strain does not have the r1, r2, or r3 mutant cadherin genes as do all previous strains of pink bollworm selected on Cry1Ac-treated artificial diet. The combined results suggest a mechanism of resistance to Cry1Ac that is different from previously described cadherin mutations.

  9. Bacillus thuringiensis Cry1Ac Resistance Frequency in Tobacco Budworm (Lepidoptera: Noctuidae)

    USDA-ARS?s Scientific Manuscript database

    The tobacco budworm (Heliothis virescens F.) is one of the most important pests of cotton and has become resistant to a wide range of synthetic insecticides. Cry1Ac-expressing cotton has proven its effectiveness against this insect since its introduction in North America in 1996. However, the consta...

  10. Analysis of resistance to Cry1Ac in field-collected pink bollworm, Pectinophora gossypiella (Lepidoptera:Gelechiidae), populations.

    PubMed

    Ojha, Abhishek; Sree, K Sowjanya; Sachdev, Bindiya; Rashmi, M A; Ravi, K C; Suresh, P J; Mohan, Komarlingam S; Bhatnagar, Raj K

    2014-01-01

    High survivorship of pink bollworrm, Pectinophora gossypiella in bolls of Bollgard® cotton hybrids and resistance to Cry1Ac protein, expressed in Bollgard cotton were reported in field-populations collected from the state of Gujarat (western India) in 2010. We have found Cry1Ac-resistance in pink bollworm populations sourced from Bollgard and non-Bt cotton fields in the adjoining states of Maharashtra and Madhya Pradesh in Central India. Further, we observed reduced binding of labeled Cry1Ac protein to receptors localized on the brush-border membrane of pink bollworm larval strains with high tolerance to Cry1Ac. These strains were sourced from Bollgard and conventional cotton fields. A pooled Cry1Ac-resistant strain, further selected on Cry1Ac diet also showed significantly reduced binding to Cry1Ac protein. The reduced binding of Cry1Ac to receptors could be an underlying mechanism for the observed resistance in pink bollworm populations feeding on Bollgard hybrids.

  11. Binding of Bacillus thuringiensis Cry1Ac Toxin to Aminopeptidase in Susceptible and Resistant Diamondback Moths (Plutella xylostella)

    PubMed Central

    Luo, K.; Tabashnik, B. E.; Adang, M. J.

    1997-01-01

    Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase. PMID:16535536

  12. pH-Controlled Bacillus thuringiensis Cry1Ac Protoxin Loading and Release from Polyelectrolyte Microcapsules

    PubMed Central

    Yang, Wenhui; He, Kanglai; Zhang, Jie; Guo, Shuyuan

    2012-01-01

    Crystal proteins synthesized by Bacillus thuringiensis (Bt) have been used as biopesticides because of their toxicity to the insect larval hosts. To protect the proteins from environmental stress to extend their activity, we have developed a new microcapsule formulation. Poly (acrylic acid) (PAH) and poly (styrene sulfonate) (PSS) were fabricated through layer-by-layer self-assembly based on a CaCO3 core. Cry1Ac protoxins were loaded into microcapsules through layer-by-layer self-assembly at low pH, and the encapsulated product was stored in water at 4°C. Scanning electron microscopy (SEM) was used to observe the morphology of the capsules. To confirm the successful encapsulation, the loading results were observed with a confocal laser scattering microscope (CLSM), using fluorescein-labeled Cry1Ac protoxin (FITC-Cry1Ac). The protoxins were released from the capsule under the alkaline condition corresponding to the midgut of certain insects, a condition which seldom exists elsewhere in the environment. The following bioassay experiment demonstrated that the microcapsules with Cry1Ac protoxins displayed approximately equivalent insecticidal activity to the Asian corn borer compared with free Cry1Ac protoxins, and empty capsules proved to have no effect on insects. Further result also indicated that the formulation could keep stable under the condition of heat and desiccation. These results suggest that this formulation provides a promising methodology that protects protoxins from the environment and releases them specifically in the target insects’ midgut, which has shown potential as biopesticide in the field. PMID:23024810

  13. Association of Cry1Ac Toxin Resistance in Helicoverpa zea (Boddie) with Increased Alkaline Phosphatase Levels in the Midgut Lumen

    PubMed Central

    Caccia, Silvia; Moar, William J.; Chandrashekhar, Jayadevi; Oppert, Cris; Anilkumar, Konasale J.; Jurat-Fuentes, Juan Luis

    2012-01-01

    Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae. PMID:22685140

  14. Improved insecticidal toxicity by fusing Cry1Ac of Bacillus thuringiensis with Av3 of Anemonia viridis.

    PubMed

    Yan, Fu; Cheng, Xing; Ding, Xuezhi; Yao, Ting; Chen, Hanna; Li, Wenping; Hu, Shengbiao; Yu, Ziquan; Sun, Yunjun; Zhang, Youming; Xia, Liqiu

    2014-05-01

    Av3, a neurotoxin of Anemonia viridis, is toxic to crustaceans and cockroaches but inactive in mammals. In the present study, Av3 was expressed in Escherichia coli Origami B (DE3) and purified by reversed-phase liquid chromatography. The purified Av3 was injected into the hemocoel of Helicoverpa armigera, rendering the worm paralyzed. Then, Av3 was expressed alone or fusion expressed with the Cry1Ac in acrystalliferous strain Cry(-)B of Bacillus thuringiensis. The shape of Cry1Ac was changed by fusion with Av3. The expressed fusion protein, Cry1AcAv3, formed irregular rhombus- or crescent-shaped crystalline inclusions, which is quite different from the shape of original Cry1Ac crystals. The toxicity of Cry1Ac was improved by fused expression. Compared with original Cry1Ac expressed in Cry(-)B, the oral toxicity of Cry1AcAv3 to H. armigera was elevated about 2.6-fold. No toxicity was detected when Av3 was expressed in Cry(-)B alone. The present study confirmed that marine toxins could be used in bio-control and implied that fused expression with other insecticidal proteins could be an efficient way for their application.

  15. Vip3Aa tolerance response of Helicoverpa armigera populations from a Cry1Ac cotton planting region.

    PubMed

    An, Jingjie; Gao, Yulin; Wu, Kongming; Gould, Fred; Gao, Jianhua; Shen, Zhicheng; Lei, Chaoliang

    2010-12-01

    Transgenic cotton, Gossypium hirsutum L., that expresses the Bacillus thuringiensis (Bt) Cry1Ac toxin, holds great promise in controlling target insect pests. Evolution of resistance by target pests is the primary threat to the continued efficacy of Bt cotton. To thwart pest resistance evolution, a transgenic cotton culitvar that produces two different Bt toxins, cry1Ac and vip3A genes, was proposed as a successor of cry1Ac cotton. This article reports on levels of Vip3Aa tolerance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) populations from the Cry1Ac cotton planting region in China based on bioassays of the F1 generation of isofemale lines. In total, 80 isofemale families of H. armigera from Xiajin county of Shandong Province (an intensive Bt cotton planting area) and 93 families from Anci county of Hebei Province (a multiple-crop system including corn [Zea mays L.] , soybean [Glycine max (L.) Merr.], peanut (Arachis hypogaea L.), and Bt cotton) were screened with a discriminating concentration of both Cry1Ac- and Vip3A-containing diets in 2009. From data on the relative average development rates and percentage of larval weight inhibition of F1 full-sib families tested simultaneously on Cry1Ac and Vip3Aa, results indicate that responses to Cry1Ac and Vip3Aa were not genetically correlated in field population ofH. armigera. This indicates that the threat of cross-resistance between Cry1Ac and Vip3A is low in field populations of H. armigera. Thus, the introduction of Vip3Aa/Cry1Ac-producing lines could delay resistance evolution in H. armigera in Bt cotton planting area of China.

  16. Coadministration of protoxin Cry1Ac from Bacillus thuringiensis with metacestode extract confers protective immunity to murine cysticercosis.

    PubMed

    Ibarra-Moreno, S; García-Hernández, A L; Moreno-Fierros, L

    2014-06-01

    The Bacillus thuringiensis Cry1Ac protoxin (pCry1Ac) is a promising mucosal immunogen and adjuvant that induces protective immunity against Naegleria fowleri and malaria infection models. We determined whether pCry1Ac acted as a protective adjuvant against infection with Taenia crassiceps. BALB/C mice were thrice i.p. immunized with (i) pCry1Ac, (ii) metacestode extract, (iii) extract + pCry1Ac or (iv) vehicle, challenged with metacestodes on day 26 and then sacrificed 35 days later. Cysticerci in the peritoneal cavity were counted, while the serum antibody response and cytokines were analysed after immunization and during infection. Only immunization with pCry1Ac plus extract conferred a significant protection (up to 47%). This group presented fluctuating antibody peaks during infection and the highest IgG1 and IgM titres. Immunization with extract alone elicited high IgG1 and the highest IgG2a responses after 25 days of infection, while nonimmunized mice presented a poor, mixed-Th1/Th2 response during infection. Sharp peaks of TNFα and IFN-γ occurred immediately after the first immunization with extract, especially in the presence of pCry1Ac, but not after the challenge, while in the control and pCry1Ac-alone groups, cytokines were only detected after the challenge. The data support the protective-adjuvant effect of co-administration of pCry1Ac in cysticercosis.

  17. Increased Long-Flight Activity Triggered in Beet Armyworm by Larval Feeding on Diet Containing Cry1Ac Protoxin

    PubMed Central

    Jiang, Xing Fu; Chen, Jian; Zhang, Lei; Sappington, Thomas W.; Luo, Li Zhi

    2013-01-01

    Evaluating ecological safety and conducting pest risk analysis for transgenic crops are vitally important before their commercial planting. The beet armyworm, Spodoptera exigua, a long-distance migratory insect pest, is not a direct target of transgenic Cry1Ac-expressing cotton in China, but nevertheless it has recently become an important pest. Migrants leaving their natal field arrive in other appropriate habitat far away in a short time, often followed by larval outbreaks. S. exigua has low susceptibility to Cry1Ac. However, our results from laboratory experiments identified (i) sublethal effects of Cry1Ac protoxin on larval development rate, larval and pupal weight, and adult lifetime fecundity, and (ii) increased long-flight behavior triggered by Cry1Ac which may contribute to larval outbreaks elsewhere. No significant differences in larval mortality, pupation rate, adult emergence rate, longevity, pre-oviposition period, or oviposition period were observed between controls and larvae fed on artificial diet incorporating a low concentration of Cry1Ac protoxin. The negative sublethal effects on some developmental and reproductive traits and lack of effect on others suggest they do not contribute to the observed severity of S. exigua outbreaks after feeding on Cry1Ac cotton. Interestingly, the percentage of long fliers increased significantly when larvae were reared on diet containing either of two low-dose treatments of Cry1Ac, suggesting a possible increased propensity to disperse long distances triggered by Cry1Ac. We hypothesize that negative effects on development and reproduction caused by Cry1Ac in the diet are offset by increased flight propensity triggered by the poor food conditions, thereby improving the chances of escaping adverse local conditions before oviposition. Increased long-flight propensity in turn may amplify the area damaged by outbreak populations. This phenomenon might be common in other migratory insect pests receiving sublethal doses

  18. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

  19. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

  20. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

  1. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as...

  2. 40 CFR 174.510 - Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1Ac protein... Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of Bacillus thuringiensis Cry1Ac protein in all plants are exempt from the requirement of a tolerance when used as plant...

  3. Molecular and functional characterization of cry1Ac transgenic pea lines.

    PubMed

    Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

    2016-10-01

    Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

  4. An ABC Transporter Mutation Is Correlated with Insect Resistance to Bacillus thuringiensis Cry1Ac Toxin

    PubMed Central

    Gahan, Linda J.; Pauchet, Yannick; Vogel, Heiko; Heckel, David G.

    2010-01-01

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field. PMID:21187898

  5. An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin.

    PubMed

    Gahan, Linda J; Pauchet, Yannick; Vogel, Heiko; Heckel, David G

    2010-12-16

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt-expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field.

  6. Increased Frequency of Pink Bollworm Resistance to Bt Toxin Cry1Ac in China

    PubMed Central

    Wan, Peng; Huang, Yunxin; Wu, Huaiheng; Huang, Minsong; Cong, Shengbo; Tabashnik, Bruce E.; Wu, Kongming

    2012-01-01

    Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as “refuges” to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on “natural” refuges of non-Bt host plants other than cotton. The “natural” refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005–2007 to 56% in 2008–2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton. PMID:22238687

  7. Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China.

    PubMed

    Wan, Peng; Huang, Yunxin; Wu, Huaiheng; Huang, Minsong; Cong, Shengbo; Tabashnik, Bruce E; Wu, Kongming

    2012-01-01

    Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. The "natural" refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005-2007 to 56% in 2008-2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.

  8. Surface plasmon resonance detection of transgenic Cry1Ac cotton ( Gossypium spp.).

    PubMed

    Zhao, Zhuoya; Chen, Yanshan; Xu, Wenzhong; Ma, Mi

    2013-03-27

    The detection and identification of genetically modified (GM) plants are challenging issues that have arisen from the potential negative impacts of extensive cultivation of transgenic plants. The screening process is a long-term focus and needs specific detection strategies. Surface plasmon resonance (SPR) has been used to detect a variety of biomolecules including proteins and nucleic acids due to its ability to monitor specific intermolecular interactions. In the present study, two high-throughput, label-free, and specific methods based on SPR technology were developed to detect transgenic Cry1Ac cotton ( Gossypium spp.) by separately targeting protein and DNA. In the protein-based detection system, monoclonal anti-Cry1Ac antibodies were immobilized on the surface of a CM5 sensor chip. Conventional cotton samples were used to define the detection threshold. Transgenic cotton was easily identified within 5 min per sample. For the DNA-based model, a 25-mer biotinylated oligonucleotide probe was immobilized on an SA sensor chip. PCR products of Cry1Ac (230 bp) were used to investigate the reaction conditions. The sensitivity of the constructed sensor chip was identified at concentrations as low as 0.1 nM based on its complementary base pairing.

  9. Synthetic fusion-protein containing domains of Bt Cry1Ac and Allium sativum lectin (ASAL) conferred enhanced insecticidal activity against major lepidopteran pests.

    PubMed

    Tajne, Sunita; Boddupally, Dayakar; Sadumpati, Vijayakumar; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

    2014-02-10

    Different transgenic crop plants, developed with δ-endotoxins of Bacillus thuringiensis (Bt) and mannose-specific plant lectins, exhibited significant protection against chewing and sucking insects. In the present study, a synthetic gene (cry-asal) encoding the fusion-protein having 488 amino acids, comprising DI and DII domains from Bt Cry1Ac and Allium sativum agglutinin (ASAL), was cloned and expressed in Escherichia coli. Ligand blot analysis disclosed that the fusion-protein could bind to more number of receptors of brush border membrane vesicle (BBMV) proteins of Helicoverpa armigera. Artificial diet bioassays revealed that 0.025 μg/g and 0.50 μg/g of fusion-protein were sufficient to cause 100% mortality in Pectinophora gossypiella and H. armigera insects, respectively. As compared to Cry1Ac, the fusion-protein showed enhanced (8-fold and 30-fold) insecticidal activity against two major lepidopteran pests. Binding of fusion-protein to the additional receptors in the midgut cells of insects is attributable to its enhanced entomotoxic effect. The synthetic gene, first of its kind, appears promising and might serve as a potential candidate for engineering crop plants against major insect pests.

  10. Intranasal coadministration of the Cry1Ac protoxin with amoebal lysates increases protection against Naegleria fowleri meningoencephalitis.

    PubMed

    Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A; López-Revilla, Rubén; Reséndiz-Albor, Aldo A; Moreno-Fierros, Leticia

    2004-08-01

    Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 x 10(4) live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines.

  11. Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie)▿

    PubMed Central

    Anilkumar, Konasale J.; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J.

    2008-01-01

    Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments. PMID:18024681

  12. Intranasal Coadministration of the Cry1Ac Protoxin with Amoebal Lysates Increases Protection against Naegleria fowleri Meningoencephalitis

    PubMed Central

    Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A.; López-Revilla, Rubén; Reséndiz-Albor, Aldo A.; Moreno-Fierros, Leticia

    2004-01-01

    Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 × 104 live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines. PMID:15271892

  13. Cross-resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against the cotton bollworm

    PubMed Central

    Wei, Jizhen; Guo, Yuyuan; Liang, Gemei; Wu, Kongming; Zhang, Jie; Tabashnik, Bruce E.; Li, Xianchun

    2015-01-01

    To delay evolution of pest resistance to transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt), the "pyramid" strategy uses plants that produce two or more toxins that kill the same pest. We conducted laboratory diet experiments with the cotton bollworm, Helicoverpa armigera, to evaluate cross-resistance and interactions between two toxins in pyramided Bt cotton (Cry1Ac and Cry2Ab). Selection with Cry1Ac for 125 generations produced 1000-fold resistance to Cry1Ac and 6.8-fold cross-resistance to Cry2Ab. Selection with Cry2Ab for 29 generations caused 5.6-fold resistance to Cry2Ab and 61-fold cross-resistance to Cry1Ac. Without exposure to Bt toxins, resistance to both toxins decreased. For each of the four resistant strains examined, 67 to 100% of the combinations of Cry1Ac and Cry2Ab tested yielded higher than expected mortality, reflecting synergism between these two toxins. Results showing minor cross-resistance to Cry2Ab caused by selection with Cry1Ac and synergism between these two toxins against resistant insects suggest that plants producing both toxins could prolong the efficacy of Bt cotton against this pest in China. Including toxins against which no cross-resistance occurs and integrating Bt cotton with other control tactics could also increase the sustainability of management strategies. PMID:25586723

  14. Cross-resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against the cotton bollworm.

    PubMed

    Wei, Jizhen; Guo, Yuyuan; Liang, Gemei; Wu, Kongming; Zhang, Jie; Tabashnik, Bruce E; Li, Xianchun

    2015-01-14

    To delay evolution of pest resistance to transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt), the "pyramid" strategy uses plants that produce two or more toxins that kill the same pest. We conducted laboratory diet experiments with the cotton bollworm, Helicoverpa armigera, to evaluate cross-resistance and interactions between two toxins in pyramided Bt cotton (Cry1Ac and Cry2Ab). Selection with Cry1Ac for 125 generations produced 1000-fold resistance to Cry1Ac and 6.8-fold cross-resistance to Cry2Ab. Selection with Cry2Ab for 29 generations caused 5.6-fold resistance to Cry2Ab and 61-fold cross-resistance to Cry1Ac. Without exposure to Bt toxins, resistance to both toxins decreased. For each of the four resistant strains examined, 67 to 100% of the combinations of Cry1Ac and Cry2Ab tested yielded higher than expected mortality, reflecting synergism between these two toxins. Results showing minor cross-resistance to Cry2Ab caused by selection with Cry1Ac and synergism between these two toxins against resistant insects suggest that plants producing both toxins could prolong the efficacy of Bt cotton against this pest in China. Including toxins against which no cross-resistance occurs and integrating Bt cotton with other control tactics could also increase the sustainability of management strategies.

  15. Transgenic cotton coexpressing Vip3A and Cry1Ac has a broad insecticidal spectrum against lepidopteran pests.

    PubMed

    Chen, Wen-Bo; Lu, Guo-Qing; Cheng, Hong-Mei; Liu, Chen-Xi; Xiao, Yu-Tao; Xu, Chao; Shen, Zhi-Cheng; Wu, Kong-Ming

    2017-10-01

    Although farmers in China have grown transgenic Bt-Cry1Ac cotton to resist the major pest Helicoverpa armigera since 1997 with great success, many secondary lepidopteran pests that are tolerant to Cry1Ac are now reported to cause considerable economic damage. Vip3AcAa, a chimeric protein with the N-terminal part of Vip3Ac and the C-terminal part of Vip3Aa, has a broad insecticidal spectrum against lepidopteran pests and has no cross resistance to Cry1Ac. In the present study, we tested insecticidal activities of Vip3AcAa against Spodoptera litura, Spodoptera exigua, and Agrotis ipsilon, which are relatively tolerant to Cry1Ac proteins. The bioassay results showed that insecticidal activities of Vip3AcAa against these three pests are superior to Cry1Ac, and after an activation pretreatment, Vip3AcAa retained insecticidal activity against S. litura, S. exigua and A. ipsilon that was similar to the unprocessed protein. The putative receptor for this chimeric protein in the brush border membrane vesicle (BBMV) in the three pests was also identified using biotinylated Vip3AcAa toxin. To broaden Bt cotton activity against a wider spectrum of pests, we introduced the vip3AcAa and cry1Ac genes into cotton. Larval mortality rates for S. litura, A. ipsilon and S. exigua that had fed on this new cotton increased significantly compared with larvae fed on non-Bt cotton and Bt-Cry1Ac cotton in a laboratory experiment. These results suggested that the Vip3AcAa protein is an excellent option for a "pyramid" strategy for integrated pest management in China. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea

    PubMed Central

    Wei, Jizhen; Zhang, Min; Liang, Gemei; Wu, Kongming; Guo, Yuyuan; Ni, Xinzhi; Li, Xianchun

    2016-01-01

    Lepidopteran midgut aminopeptidases N (APNs) are phylogenetically divided into eight clusters, designated as APN1–8. Although APN1 has been implicated as one of the receptors for Cry1Ac in several species, its potential role in the mode of action of Cry2Ab has not been functionally determined so far. To test whether APN1 also acts as one of the receptors for Cry1Ac in Helicoverpa zea and even for Cry2Ab in this species, we conducted a gain of function analysis by heterologously expressing H. zea APN1 (HzAPN1) in the midgut and fat body cell lines of H. zea and the ovarian cell line of Spodoptera frugiperda (Sf9) and a loss of function analysis by RNAi (RNA interference) silencing of the endogenous APN1 in the three cell lines using the HzAPN1 double strand RNA (dsRNA). Heterologous expression of HzAPN1 significantly increased the susceptibility of the three cell lines to Cry1Ac, but had no effects on their susceptibility to Cry2Ab. Knocking down of the endogenous APN1 made the three cell lines resistant to Cry1Ac, but didn’t change cell lines susceptibility to Cry2Ab. The findings from this study demonstrate that HzAPN1 is a functional receptor of Cry1Ac, but not Cry2Ab. PMID:26755166

  17. Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

    PubMed Central

    Zhou, Dinggang; Guo, Jinlong; Xu, Liping; Gao, Shiwu; Lin, Qingliang; Wu, Qibin; Wu, Luguang; Que, Youxiong

    2014-01-01

    To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg2+, 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn2+ (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane. PMID:24810230

  18. Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

    PubMed

    Gao, Shiwu; Yang, Yingying; Wang, Chunfeng; Guo, Jinlong; Zhou, Dinggang; Wu, Qibin; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines.

  19. Degradation of Cry1Ac protein within transgenic Bacillus thuringiensis rice tissues under field and laboratory conditions.

    PubMed

    Li, Yunhe; Wu, Kongming; Zhang, Yongjun; Yuan, Guohui

    2007-10-01

    To clarify the environmental fate of the Cry1Ac protein from Bacillus thuringiensis subsp. kurstaki (Bt) contained in transgenic rice plant stubble after harvest, degradation was monitored under field conditions using an enzyme-linked immunosorbent assay. In stalks, Cry1Ac protein concentration decreased rapidly to 50% of the initial amount during the first month after harvest; subsequently, the degradation decreased gradually reaching 21.3% when the experiment was terminated after 7 mo. A similar degradation pattern of the Cry1Ac protein was observed in rice roots. However, when the temperature increased in April of the following spring, protein degradation resumed, and no protein could be detected by the end of the experiment. In addition, a laboratory experiment was conducted to study the persistence of Cry1Ac protein released from rice tissue in water and paddy soil. The protein released from leaves degraded rapidly in paddy soil under flooded conditions during the first 20 d and plateaued until the termination of this trial at 135 d, when 15.3% of the initial amount was still detectable. In water, the Cry1Ac protein degraded more slowly than in soil but never entered a relatively stable phase as in soil. The degradation rate of Cry1Ac protein was significantly faster in nonsterile water than in sterile water. These results indicate that the soil environment can increase the degradation of Bt protein contained in plant residues. Therefore, plowing a field immediately after harvest could be an effective method for decreasing the persistence of Bt protein in transgenic rice fields.

  20. Asymmetrical cross-resistance between Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in pink bollworm.

    PubMed

    Tabashnik, Bruce E; Unnithan, Gopalan C; Masson, Luke; Crowder, David W; Li, Xianchun; Carrière, Yves

    2009-07-21

    Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests and can reduce reliance on insecticide sprays. Sustainable use of such crops requires methods for delaying evolution of resistance by pests. To thwart pest resistance, some transgenic crops produce 2 different Bt toxins targeting the same pest. This "pyramid" strategy is expected to work best when selection for resistance to 1 toxin does not cause cross-resistance to the other toxin. The most widely used pyramid is transgenic cotton producing Bt toxins Cry1Ac and Cry2Ab. Cross-resistance between these toxins was presumed unlikely because they bind to different larval midgut target sites. Previous results showed that laboratory selection with Cry1Ac caused little or no cross-resistance to Cry2A toxins in pink bollworm (Pectinophora gossypiella), a major cotton pest. We show here, however, that laboratory selection of pink bollworm with Cry2Ab caused up to 420-fold cross-resistance to Cry1Ac as well as 240-fold resistance to Cry2Ab. Inheritance of resistance to high concentrations of Cry2Ab was recessive. Larvae from a laboratory strain resistant to Cry1Ac and Cry2Ab in diet bioassays survived on cotton bolls producing only Cry1Ac, but not on cotton bolls producing both toxins. Thus, the asymmetrical cross-resistance seen here does not threaten the efficacy of pyramided Bt cotton against pink bollworm. Nonetheless, the results here and previous evidence indicate that cross-resistance occurs between Cry1Ac and Cry2Ab in some key cotton pests. Incorporating the potential effects of such cross-resistance in resistance management plans may help to sustain the efficacy of pyramided Bt crops.

  1. Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer

    PubMed Central

    Gao, Shiwu; Yang, Yingying; Wang, Chunfeng; Guo, Jinlong; Zhou, Dinggang; Wu, Qibin; Su, Yachun; Xu, Liping

    2016-01-01

    We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines. PMID:27093437

  2. Production of transgenic kiwifruit plants harboring the SbtCry1Ac gene.

    PubMed

    Zhang, H Y; Liu, H M; Liu, X Z

    2015-07-28

    The kiwifruit (Actinidia chinensis Planch.) is an economically and nutritionally important fruit crop that has a remarkably high vitamin C content and is popular throughout the world. However, kiwifruit plants are vulnerable to attack from pests, and effective pest control is urgently required. Transgenic kiwifruit plants containing the synthetic chimeric gene SbtCry1Ac that encodes the insecticidal protein btCrylAc were obtained through an Agrobacterium-mediated transformation of kiwifruit leaf discs. The kanamycin resistance of the transgenic plants was then analyzed. Results from polymerase chain reactions and genomic DNA Southern blot analyses indicated that SbtCrylAc had been integrated into the genomes of these plants. The results of insect bioassays revealed that the average Oraesia excavate inhibition rate of plants tested at 10 days post-infestation was 75.2%. To our knowledge, this is the first study that has developed insect-resistant transgenic kiwifruit plants.

  3. Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.).

    PubMed

    Keshavareddy, G; Rohini, S; Ramu, S V; Sundaresha, S; Kumar, A R V; Kumar, P Ananda; Udayakumar, M

    2013-07-01

    Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. The amplification of required size fragment of 750 bp with npt II primers and 901 bp with cry1AcF gene primers confirmed the integration of the gene. The expression of the cry gene was ascertained by ELISA in T2 generation, and the maximum concentration of cry protein in transgenic plants reached approximately 0.82 μg/g FW. Further, Southern blot analysis of ten T2 transgenic plants proved that transgene had been integrated in the genome of all the plants and Northern analysis of the same plants demonstrated the active expression of cry1AcF gene. The highest mean % larval mortalities 80.0 and 85.0 with an average mean % larval mortalities 16.25 (n = 369) and 26.0 (n = 80) were recorded in T1 and T2 generations, respectively. Segregation analysis of the selected lines in the T3 generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T2 generation, the cry1AcF gene was effective against S. litura only to some extent.

  4. APN1 is a functional receptor of Cry1Ac but not Cry2Ab in Helicoverpa zea

    USDA-ARS?s Scientific Manuscript database

    Lepidopteran midgut aminopeptidases N (APNs) are phylogenetically divided into eight clusters, designated as APN1-8. Although APN1 has been implicated as one of the receptors for Cry1Ac in several species, its potential role in the mode of action of Cry2Ab has not been functionally determined so fa...

  5. Cloning & Characterization of the Cry1Ac-binding Alkaline Phosphatase (HvALP) from Heliothis virescens

    USDA-ARS?s Scientific Manuscript database

    Membrane bound alkaline phosphatases (mALPs) in the insect midgut have been reported as functional receptors for Cry toxins from the bacterium Bacillus thuringiensis. We previously reported the identification of HvALP in the midgut of Heliothis virescens larvae as a Cry1Ac binding protein that is d...

  6. Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

    USDA-ARS?s Scientific Manuscript database

    While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (ATP-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baselin...

  7. Response of Heliothis virescens (Lepidoptera: Noctuidae) strains to Bacillus thuringiensis Cry1Ac incorporated into different insect artificial diets

    USDA-ARS?s Scientific Manuscript database

    Susceptibility to the Cry1Ac toxin from Bacillus thuringiensis in Heliothis virescens is usually measured by performing bioassays under laboratory conditions. Currently there is great interest and research devoted to this insect because it is one of the main targets of B. thuringiensis-expressing tr...

  8. Aminopeptidase N1 is involved in Bacillus thuringiensis Cry1Ac toxicity in the beet armyworm, Spodoptera exigua

    PubMed Central

    Qiu, Lin; Cui, Songhe; Liu, Lang; Zhang, Boyao; Ma, Weihua; Wang, Xiaoping; Lei, Chaoliang; Chen, Lizhen

    2017-01-01

    Understanding how insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) interact with their hosts is crucial to fully explain the molecular bases of Bt specificity and insecticidal activity. Previous studies support ATP binding cassette transporters (ABCC2/3) and one cadherin-like protein are Cry1Ac functional receptors in the beet armyworm (Spodoptera exigua). In this study, a combined one-dimensional gel electrophoresis and immunoblotting approach identified aminopeptidase N (APNs) as putative Cry1Ac binding proteins in the midgut brush border membrane of S. exigua larvae. Functional analyses by gene silencing of six different S. exigua APN genes (SeAPN1, SeAPN2, SeAPN3, SeAPN4, SeAPN5 and SeAPN6) showed that only suppression of SeAPN1 resulted in decreased larval susceptibility to Cry1Ac toxin. These results support that SeAPN1 plays important functional role in Cry1Ac toxicity in S. exigua. PMID:28327568

  9. Cry1Ac Transgenic Sugarcane Does Not Affect the Diversity of Microbial Communities and Has No Significant Effect on Enzyme Activities in Rhizosphere Soil within One Crop Season

    PubMed Central

    Zhou, Dinggang; Xu, Liping; Gao, Shiwu; Guo, Jinlong; Luo, Jun; You, Qian; Que, Youxiong

    2016-01-01

    Cry1Ac transgenic sugarcane provides a promising way to control stem-borer pests. Biosafety assessment of soil ecosystem for cry1Ac transgenic sugarcane is urgently needed because of the important role of soil microorganisms in nutrient transformations and element cycling, however little is known. This study aimed to explore the potential impact of cry1Ac transgenic sugarcane on rhizosphere soil enzyme activities and microbial community diversity, and also to investigate whether the gene flow occurs through horizontal gene transfer. We found no horizontal gene flow from cry1Ac sugarcane to soil. No significant difference in the population of culturable microorganisms between the non-GM and cry1Ac transgenic sugarcane was observed, and there were no significant interactions between the sugarcane lines and the growth stages. A relatively consistent trend at community-level, represented by the functional diversity index, was found between the cry1Ac sugarcane and the non-transgenic lines. Most soil samples showed no significant difference in the activities of four soil enzymes: urease, protease, sucrose, and acid phosphate monoester between the non-transgenic and cry1Ac sugarcane lines. We conclude, based on one crop season, that the cry1Ac sugarcane lines may not affect the microbial community structure and functional diversity of the rhizosphere soil and have few negative effects on soil enzymes. PMID:27014291

  10. Cry1Ac Transgenic Sugarcane Does Not Affect the Diversity of Microbial Communities and Has No Significant Effect on Enzyme Activities in Rhizosphere Soil within One Crop Season.

    PubMed

    Zhou, Dinggang; Xu, Liping; Gao, Shiwu; Guo, Jinlong; Luo, Jun; You, Qian; Que, Youxiong

    2016-01-01

    Cry1Ac transgenic sugarcane provides a promising way to control stem-borer pests. Biosafety assessment of soil ecosystem for cry1Ac transgenic sugarcane is urgently needed because of the important role of soil microorganisms in nutrient transformations and element cycling, however little is known. This study aimed to explore the potential impact of cry1Ac transgenic sugarcane on rhizosphere soil enzyme activities and microbial community diversity, and also to investigate whether the gene flow occurs through horizontal gene transfer. We found no horizontal gene flow from cry1Ac sugarcane to soil. No significant difference in the population of culturable microorganisms between the non-GM and cry1Ac transgenic sugarcane was observed, and there were no significant interactions between the sugarcane lines and the growth stages. A relatively consistent trend at community-level, represented by the functional diversity index, was found between the cry1Ac sugarcane and the non-transgenic lines. Most soil samples showed no significant difference in the activities of four soil enzymes: urease, protease, sucrose, and acid phosphate monoester between the non-transgenic and cry1Ac sugarcane lines. We conclude, based on one crop season, that the cry1Ac sugarcane lines may not affect the microbial community structure and functional diversity of the rhizosphere soil and have few negative effects on soil enzymes.

  11. Inheritance of Cry1Ac resistance and associated biological traits in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

    PubMed

    Nair, Rupa; Kalia, Vinay; Aggarwal, K K; Gujar, Govind T

    2010-05-01

    The analysis of reciprocal genetic crosses between resistant Helicoverpa armigera strain (BH-R) (227.9-fold) with susceptible Vadodara (VA-S) strain showed dominance (h) of 0.65-0.89 and degree of dominance (D) of 0.299-0.782 suggesting Cry1Ac resistance as a semi-dominant trait. The D and h values of F(1) hybrids of female resistant parent were higher than female susceptible parent, showing maternally enhanced dominance of Cry1Ac resistance. The progeny of F(2) crosses, backcrosses of F(1) hybrid with resistant BH-R parent did not differ significantly in respect of mortality response with resistant parent except for backcross with female BH-R and male of F(1) (BH-RxVA-S) cross, suggesting dominant inheritance of Cry1Ac resistance. Evaluation of some biological attributes showed that larval and pupal periods of progenies of reciprocal F(1) crosses, backcrosses and F(2) crosses were either at par with resistant parent or lower than susceptible parent on treated diet (0.01 microg/g). The susceptible strain performed better in terms of pupation and adult formation than the resistant strain on untreated diet. In many backcrosses and F(2) crosses, Cry1Ac resistance favored emergence of more females than males on untreated diet. The normal larval period and the body weight (normal larval growth) were the dominant traits associated with susceptible strain as contrast to longer larval period and the lower body weight (slow growth) associated with resistance trait. Further, inheritance of larval period in F(2) and backcross progeny suggested existence of a major resistant gene or a set of tightly linked loci associated with Cry1Ac sensitivity. (c) 2010 Elsevier Inc. All rights reserved.

  12. Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers.

    PubMed

    De Guglielmo-Cróquer, Z; Altosaar, I; Zaidi, M; Menéndez-Yuffá, A

    2010-05-01

    The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

  13. Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India.

    PubMed

    Dhurua, Sanyasi; Gujar, Govind T

    2011-08-01

    The pink bollworm is one of the most destructive pests of cotton. Transgenic cotton producing Bt toxin Cry1Ac or a combination of Cry1Ac and Cry2Ab2 has been used effectively against this pest. However, some other insects have evolved resistance to Bt toxins in the field. During the 2007-2008 and 2008-2009 seasons, pink bollworm populations in India were surveyed to evaluate their responses to Cry1Ac and seed powder containing Cry1Ac and Cry2Ab2. The results provide evidence that resistance to Cry1Ac had evolved by 2008 in a population sampled from non-Bt cotton in the Amreli district of Gujarat in western India. The median lethal concentration of Cry1Ac for five-day-old larvae (LC50 ) was significantly higher for insects derived in 2008 from Amreli than for any of the other field populations tested from four locations in India. For Cry1Ac, the mean LC50 for the strain derived from Amreli in 2008 was 44 times higher than for the most susceptible population. However, for seed powder of Bollgard II containing primarily Cry2Ab2, the 2008 Amreli population was only slightly less susceptible than the most susceptible population. The data reported here constitute the first evidence of field-evolved resistance of pink bollworm to Cry1Ac. This initial evidence spurred more extensive evaluations during the 2009-2010 growing season, which confirmed field-evolved resistance to Cry1Ac in Amreli. The lack of cross-resistance to Cry2Ab2 suggests that plants producing this toxin are likely to be more effective against resistant populations than plants producing only Cry1Ac. Copyright © 2011 Society of Chemical Industry.

  14. Susceptibilities of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) populations to Cry1Ac insecticidal protein.

    PubMed

    Ali, M I; Luttrell, R G; Young, S Y

    2006-02-01

    Susceptibilities of bollworm, Helicoverpa zea (Boddie) and tobacco budworm, Heliothis virescens (F.) to Cry1Ac were measured via a diet-incorporated assay with MPV II at the University of Arkansas during 2002-2004. Lethal concentration-mortality (LC50) estimates of five laboratory, seven laboratory-cross, and 10 field populations of H. virescens varied 12-fold. Pooled susceptibilities of H. virescens across all laboratory and field populations varied five-fold. The LC50 estimates for H. virescens were higher than those reported by previous research before the introduction of transgenic crops. However, the ratio of susceptibility of laboratory and field populations was similar, suggesting no change in overall species susceptibility. Individual LC50 estimates of five laboratory, nine laboratory-cross, and 57 field populations of H. zea varied over 130-fold. Pooled susceptibilities across laboratory and field populations varied widely. Among the field populations, colonies from non-Bacillus thuringiensis (Bt) crops were generally more susceptible than those from Bt crops. Across the Bt crops expressing Cry protein, colonies from Bollgard (Monsanto Company) cotton had lower susceptibility to CrylAc than those from Bt corn and those from non-Bt crops.

  15. Evaluation of genetically modified sugarcane lines carrying Cry 1AC gene using molecular marker techniques.

    PubMed

    Ismail, Roba M

    2013-01-01

    Five genetically modified insect resistant sugarcane lines harboring the Bt Cry 1AC gene to produce insecticidal proteins were compared with non-transgenic control by using three types of molecular marker techniques namely, RAPD, ISSR and AFLP. These techniques were applied on transgenic and non-transgenic plants to investigate the genetic variations, which may appear in sugarcane clones. This variation might demonstrate the genomic changes associated with the transformation process, which could change important molecular basis of various biological phenomena. Genetic variations were screened using 22 different RAPD primers, 10 ISSR primers and 13 AFLP primer combinations. Analysis of RAPD and ISSR banding patterns gave no exclusive evidence for genetic variations. Meanwhile, the percentage of polymorphic bands was 0.45% in each of RAPD and ISSR, while the polymorphism generated by AFLP analysis was 1.8%. The maximum percentage of polymorphic bands was 1.4%, 1.1% and 5.5% in RAPD, ISSR and AFLP, respectively. These results demonstrate that most transgenic lines showed genomic homogeneity and verified minor genomic changes. Dendrograms revealing the relationships among the transgenic and control plants were developed from the data of each of the three marker types.

  16. [Anther culture generated stem borer-resistance DH lines of Minghui 81(Oryza sativa L. subsp. indica) expressing modified cry1Ac gene].

    PubMed

    Zeng, Qian-Chun; Wu, Qian; Feng, De-Jiang; Zhou, Kai-Da; Liu, Xiang; Zhu, Zhen

    2002-07-01

    2600 Anthers from T0 modified cry1 Ac-transgenic rice lines of Minghui 81, an elite restoring line of commercial CMS indica hybrid rice, were cultured on SK3 media. 83 green plantlets were recovered, 43 double haploid (DH) and 40 haploid among them. Results of PCR analyzes indicated that 55 plants of 83 were harbored the cry1Ac gene, and the ratio of cry1Ac-positive against cry1Ac-negative was 2:1 (55/28). 36 putative transgenic DH plants were further confirmed by Southern blot. ELISA detection showed that Cry1Ac level in different transgenic rice plants of the same cry1Ac-DH clone was almost equal and the highest one amount to 0.25% of the total soluble protein. Pest insect-resistant bioassay at field trials demonstrated that some of the homozygous cry1Ac-transgenic rice plants not only showed high-level resistance against striped stem borer (Chilo suppressalis) but also retained elite agronomy characters. These results demonstrated that rice anther culture has a great value in rice molecular breeding.

  17. Similar genetic basis of resistance to Bt toxin Cry1Ac in Boll-selected and diet-selected strains of pink bollworm.

    PubMed

    Fabrick, Jeffrey A; Tabashnik, Bruce E

    2012-01-01

    Genetically engineered cotton and corn plants producing insecticidal Bacillus thuringiensis (Bt) toxins kill some key insect pests. Yet, evolution of resistance by pests threatens long-term insect control by these transgenic Bt crops. We compared the genetic basis of resistance to Bt toxin Cry1Ac in two independently derived, laboratory-selected strains of a major cotton pest, the pink bollworm (Pectinophora gossypiella [Saunders]). The Arizona pooled resistant strain (AZP-R) was started with pink bollworm from 10 field populations and selected with Cry1Ac in diet. The Bt4R resistant strain was started with a long-term susceptible laboratory strain and selected first with Bt cotton bolls and later with Cry1Ac in diet. Previous work showed that AZP-R had three recessive mutations (r1, r2, and r3) in the pink bollworm cadherin gene (PgCad1) linked with resistance to Cry1Ac and Bt cotton producing Cry1Ac. Here we report that inheritance of resistance to a diagnostic concentration of Cry1Ac was recessive in Bt4R. In interstrain complementation tests for allelism, F(1) progeny from crosses between AZP-R and Bt4R were resistant to Cry1Ac, indicating a shared resistance locus in the two strains. Molecular analysis of the Bt4R cadherin gene identified a novel 15-bp deletion (r4) predicted to cause the loss of five amino acids upstream of the Cry1Ac-binding region of the cadherin protein. Four recessive mutations in PgCad1 are now implicated in resistance in five different strains, showing that mutations in cadherin are the primary mechanism of resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona.

  18. Antibiotics influence the toxicity of the delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa armigera.

    PubMed

    Paramasiva, Inakarla; Sharma, Hari C; Krishnayya, Pulipaka Venkata

    2014-07-24

    The cotton bollworm, Helicoverpa armigera is one of the most important crop pests worldwide. It has developed high levels of resistance to synthetic insecticides, and hence, Bacillus thuringiensis (Bt) formulations are used as a safer pesticide and the Bt genes have been deployed in transgenic crops for controlling this pest. There is an apprehension that H. armigera might develop resistance to transgenic crops in future. Therefore, we studied the role of gut microbes by eliminating them with antibiotics in H. armigera larvae on the toxicity of Bt toxins against this pest. Commercial formulation of Bt (Biolep®) and the pure Cry1Ab and Cry1Ac toxin proteins were evaluated at ED50, LC50, and LC90 dosages against the H. armigera larvae with and without antibiotics (which removed the gut microbes). Lowest H. armigera larval mortality due to Bt formulation and the Bt toxins Cry1Ab and Cry1Ac was recorded in insects reared on diets with 250 and 500 μg ml-1 diet of each of the four antibiotics (gentamicin, penicillin, rifampicin, and streptomycin), while the highest larval mortality was recorded in insects reared on diets without the antibiotics. Mortality of H. armigera larvae fed on diets with Bt formulation and the δ-endotoxins Cry1Ab and Cry1Ac was inversely proportional to the concentration of antibiotics in the artificial diet. Nearly 30% reduction in larval mortality was observed in H. armigera larvae from F1 to F3 generation when the larvae were reared on diets without antibiotics (with gut microbes) and fed on 0.15% Bt or 12 μg Cry1Ab or Cry1Ac ml-1 diet, indicating development of resistance to Bt in the presence of gut microflora. However, there were no differences in larval mortality due to Bt, Cry1Ab or Cry1Ac across generations in insects when they were reared on diets with 250 μg of each antibiotic ml-1 diet (without gut microflora). The results suggested that antibiotics which eliminated gut microflora influenced the toxicity of Bt towards H. armigera

  19. Antibiotics influence the toxicity of the delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa armigera

    PubMed Central

    2014-01-01

    Background The cotton bollworm, Helicoverpa armigera is one of the most important crop pests worldwide. It has developed high levels of resistance to synthetic insecticides, and hence, Bacillus thuringiensis (Bt) formulations are used as a safer pesticide and the Bt genes have been deployed in transgenic crops for controlling this pest. There is an apprehension that H. armigera might develop resistance to transgenic crops in future. Therefore, we studied the role of gut microbes by eliminating them with antibiotics in H. armigera larvae on the toxicity of Bt toxins against this pest. Results Commercial formulation of Bt (Biolep®) and the pure Cry1Ab and Cry1Ac toxin proteins were evaluated at ED50, LC50, and LC90 dosages against the H. armigera larvae with and without antibiotics (which removed the gut microbes). Lowest H. armigera larval mortality due to Bt formulation and the Bt toxins Cry1Ab and Cry1Ac was recorded in insects reared on diets with 250 and 500 μg ml−1 diet of each of the four antibiotics (gentamicin, penicillin, rifampicin, and streptomycin), while the highest larval mortality was recorded in insects reared on diets without the antibiotics. Mortality of H. armigera larvae fed on diets with Bt formulation and the δ-endotoxins Cry1Ab and Cry1Ac was inversely proportional to the concentration of antibiotics in the artificial diet. Nearly 30% reduction in larval mortality was observed in H. armigera larvae from F1 to F3 generation when the larvae were reared on diets without antibiotics (with gut microbes) and fed on 0.15% Bt or 12 μg Cry1Ab or Cry1Ac ml−1 diet, indicating development of resistance to Bt in the presence of gut microflora. However, there were no differences in larval mortality due to Bt, Cry1Ab or Cry1Ac across generations in insects when they were reared on diets with 250 μg of each antibiotic ml−1 diet (without gut microflora). Conclusions The results suggested that antibiotics which eliminated gut microflora

  20. Expression of Cry1Ac in transgenic Bt soybean lines and their efficiency in controlling lepidopteran pests.

    PubMed

    Yu, Huilin; Li, Yunhe; Li, Xiangju; Romeis, Jörg; Wu, Kongming

    2013-12-01

    Two transgenic lines of the soybean Glycine max, MON87701 expressing the Cry1Ac protein and MON87701RR2Y expressing Cry1Ac +  EPSPS proteins, were evaluated for their resistance to four lepidopteran pests in the laboratory using detached-leaf bioassays throughout the soybean growth seasons (before anthesis, during anthesis and after anthesis) in China. Enzyme-linked immunosorbent assays (ELISAs) were used to monitor the Cry1Ac expression in soybean leaves. The bioassay results revealed that both transgenic soybean lines exhibited significantly high resistance against Helicoverpa armigera (Hübner) throughout the soybean growing seasons. The survival rates of H. armigera larvae ranged from 5.4 to 24.4% when feeding on the transgenic soybean leaves, significantly lower than the survival rates when feeding on control leaves (71.1-94.9%). Limited resistance was found for both transgenic soybean lines against Spodoptera litura (Fabricius), although the survival rates and weight of S. litura larvae as well as female fecundity were significantly decreased when feeding on Bt soybean leaves compared with feeding on control leaves. In contrast, both transgenic soybean lines provided almost no resistance to Spodoptera exigua (Hübner) and Agrotis ypsilon (Rottemberg). Cry1Ac expression in the leaves of both transgenic soybean lines was relatively stable throughout the soybean growing season, with a peak occurring at V6 -8 and V11 -12 before anthesis. The ELISA results were positively correlated with the results from the insect bioassays. The results show that, while Cry1Ac-expressing Bt soybeans may provide good protection against H. armigera, alternative control measures are required to manage S. exigua, S. litura and A. ypsilon. © 2013 Society of Chemical Industry.

  1. Food safety assessment of Cry8Ka5 mutant protein using Cry1Ac as a control Bt protein.

    PubMed

    Farias, Davi Felipe; Viana, Martônio Ponte; Oliveira, Gustavo Ramos; Santos, Vanessa Olinto; Pinto, Clidia Eduarda Moreira; Viana, Daniel Araújo; Vasconcelos, Ilka Maria; Grossi-de-Sa, Maria Fátima; Carvalho, Ana Fontenele Urano

    2015-07-01

    Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein.

  2. The cultivation of Bt corn producing Cry1Ac toxins does not adversely affect non-target arthropods.

    PubMed

    Guo, Yanyan; Feng, Yanjie; Ge, Yang; Tetreau, Guillaume; Chen, Xiaowen; Dong, Xuehui; Shi, Wangpeng

    2014-01-01

    Transgenic corn producing Cry1Ac toxins from Bacillus thuringiensis (Bt) provides effective control of Asian corn borer, Ostrinia furnacalis (Guenée), and thus reduces insecticide applications. However, whether Bt corn exerts undesirable effects on non-target arthropods (NTAs) is still controversial. We conducted a 2-yr study in Shangzhuang Agricultural Experiment Station to assess the potential impact of Bt corn on field population density, biodiversity, community composition and structure of NTAs. On each sampling date, the total abundance, Shannon's diversity index, Pielou's evenness index and Simpson's diversity index were not significantly affected by Bt corn as compared to non-Bt corn. The "sampling dates" had a significant effect on these indices, but no clear tendencies related to "Bt corn" or "sampling dates X corn variety" interaction were recorded. Principal response curve analysis of variance indicated that Bt corn did not alter the distribution of NTAs communities. Bray-Curtis dissimilarity and distance analysis showed that Cry1Ac toxin exposure did not increase community dissimilarities between Bt and non-Bt corn plots and that the evolution of non-target arthropod community was similar on the two corn varieties. The cultivation of Bt corn failed to show any detrimental evidence on the density of non-target herbivores, predators and parasitoids. The composition of herbivores, predators and parasitoids was identical in Bt and non-Bt corn plots. Taken together, results from the present work support that Bt corn producing Cry1Ac toxins does not adversely affect NTAs.

  3. Enhancement of Bacillus thuringiensis insecticidal activity by combining Cry1Ac and bi-functional toxin HWTX-XI from spider.

    PubMed

    Sun, Yunjun; Fu, Zujiao; He, Xiaohong; Yuan, Chunhua; Ding, Xuezhi; Xia, Liqiu

    2016-03-01

    In order to assess the potency of bi-functional HWTX-XI toxin from spider Ornithoctonus huwena in improving the insecticidal activity of Bacillus thuringiensis, a fusion gene of cry1Ac and hwtx-XI was constructed and expressed in an acrystalliferous B. thuringiensis strain Cry(-)B. Western blot analysis and microscopic observation revealed that the recombinant strain could express 140-kDa Cry1Ac-HWTX-XI fusion protein and produce parasporal inclusions during sporulation. Bioassay using the larvae of Helicoverpa armigera and Spodoptera exigua showed that the Cry1Ac-HWTX-XI fusion was more toxic than the control Cry1Ac protoxin, as revealed by 95% lethal concentration. Our study indicated that the HWTX-XI from spider might be a candidate for enhancing the toxicity of B. thuringiensis products.

  4. Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

    PubMed Central

    Tabashnik, Bruce E.; Fabrick, Jeffrey A.; Unnithan, Gopalan C.; Yelich, Alex J.; Masson, Luke; Zhang, Jie; Bravo, Alejandra; Soberón, Mario

    2013-01-01

    Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both. PMID:24244692

  5. Adult Exposure to Bt Toxin Cry1Ac Reduces Life Span and Reproduction of Resistant and Susceptible Pink Bollworm (Lepidoptera: Gelechiidae).

    PubMed

    Wang, Ling; Wan, Peng; Cong, Shengbo; Wang, Jintao; Huang, Minsong; Tabashnik, Bruce E; Li, Xianchun; Wu, Kongming

    2016-05-01

    Insecticidal proteins from Bacillus thuringiensis (Bt) are used widely in sprays and transgenic plants to control insect pests. Although much research has elucidated the effects of Bt toxins on larvae, relatively little is known about their effects on adults. Here, we evaluated the effects of exposing adults to Bt toxin Cry1Ac on the life span and reproduction of two strains of pink bollworm (Pectinophora gossypiella (Saunders)). In larval diet bioassays, the concentration of Cry1Ac killing 50% of larvae (LC50) was 640 times higher for the laboratory-selected resistant strain (AZP-R) than the susceptible strain (APHIS-S). In experiments with adults, the highest concentrations of Cry1Ac tested (160 and 640 µg Cry1Ac per ml of 5% honey water) reduced life span for both strains. Treatments with 10, 40, and 160 µg Cry1Ac per ml reduced the duration of the oviposition period as well as the number of eggs laid by both strains, but did not affect the percentage of pairs producing eggs, the duration of the preoviposition period, or the percentage of eggs hatching for either strain. Adult life span did not differ between strains at low to moderate concentrations of Cry1Ac, but it was significantly greater for the resistant strain than the susceptible strain at the two highest concentrations of Cry1Ac tested. The reduced susceptibility to high concentrations of Cry1Ac in adults of the AZP-R strain relative to the APHIS-S strain provides the first evidence of expression of resistance to a Bt toxin in adult Lepidoptera. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Carbohydrate analyses of Manduca sexta aminopeptidase N, co-purifying neutral lipids and their functional interactions with Bacillus thuringiensis Cry1Ac toxin.

    PubMed

    Sangadala, S; Azadi, P; Carlson, R; Adang, M J

    2001-12-01

    Bacillus thuringiensis Cry1Ac insecticidal toxin binds specifically to 120kDa aminopeptidase N (APN) (EC 3.4.11.2) in the epithelial brush border membrane of Manduca sexta midguts. The isolated 120-kDa APN is a member of a functional Cry1 toxin receptor complex (FEBS Lett. 412 (1997) 270). The 120-kDa form is glycosyl-phosphatidylinositol (GPI) anchored and converted to a 115-kDa form upon membrane solubilization. The 115-kDa APN also binds Cry1A toxins and Cry1Ac binding is inhibited by N-acetylgalactosamine (GalNAc). Here we determined the monosaccharide composition of APN. APN is 4.2mol% carbohydrate and contains GalNAc, a residue involved in Cry1Ac interaction. APN remained associated with non-covalently bound lipids through anion-exchange column purification. Most associated lipids were separated from APN by hydrophobic interaction chromatography yielding a lipid aggregate. Chemical analyses of the lipid aggregate separated from APN revealed neutral lipids consisting mostly of diacylglycerol and free fatty acids. The fatty acids were long, unsaturated chains ranging from C:14 to C:22. To test the effect of APN-associated lipids on Cry1Ac function, the lipid aggregate and 115-kDa APN were reconstituted into phosphatidylcholine (PC) vesicles. The lipid aggregate increased the amount of Cry1Ac binding, but binding due to the lipid aggregate was not saturable. In contrast the lipid aggregate promoted Cry1Ac-induced release of 86Rb(+) at the lowest Cry1Ac concentration (50nM) tested. The predominant neutral lipid component extracted from the lipid aggregate promoted Cry1Ac-induced 86Rb(+) release from membrane vesicles in the presence of APN.

  7. Impact of water content and temperature on the degradation of Cry1Ac protein in leaves and buds of Bt cotton in the soil.

    PubMed

    Zhang, Mei-jun; Feng, Mei-chen; Xiao, Lu-jie; Song, Xiao-yan; Yang, Wu-de; Ding, Guang-wei

    2015-01-01

    Determining the influence of soil environmental factors on degradation of Cry1Ac protein from Bt cotton residues is vital for assessing the ecological risks of this commercialized transgenic crop. In this study, the degradation of Cry1Ac protein in leaves and in buds of Bt cotton in soil was evaluated under different soil water content and temperature settings in the laboratory. An exponential model and a shift-log model were used to fit the degradation dynamics of Cry1Ac protein and estimate the DT50 and DT90 values. The results showed that Cry1Ac protein in the leaves and buds underwent rapid degradation in the early stage (before day 48), followed by a slow decline in the later stage under different soil water content and temperature. Cry1Ac protein degraded the most rapidly in the early stage at 35°C with 70% soil water holding capacity. The DT50 values were 12.29 d and 10.17 d and the DT90 values were 41.06 d and 33.96 d in the leaves and buds, respectively. Our findings indicated that the soil temperature was a major factor influencing the degradation of Cry1Ac protein from Bt cotton residues. Additionally, the relative higher temperature (25°C and 35°C) was found to be more conducive to degradation of Cry1Ac protein in the soil and the greater water content (100%WHC) retarded the process. These findings suggested that under appropriate soil temperature and water content, Cry1Ac protein from Bt cotton residues will not persist and accumulate in soil.

  8. Efficacy of Cry1Ac:Cry1F proteins in cotton leaf tissue against fall armyworm, beet armyworm, and soybean looper (Lepidoptera: Noctuidae).

    PubMed

    Tindall, K V; Siebert, M Willrich; Leonard, B R; All, J; Haile, F J

    2009-08-01

    Cotton, Gossypium hirsutum L., plants expressing Cry1Ac and Cry1F insecticidal crystal proteins of Bacillus thuringiensis Berliner (Bt) were evaluated against selected lepidopteran pests including fall armyworm, Spodoptera frugiperda (J. E. Smith), beet armyworm, Spodoptera exigua (Hübner), and soybean looper, Pseudoplusia includens (Walker). Studies were conducted in a range of environments, challenging various cotton tissue types from several varieties containing a combination of Cry1Ac and Cry1F proteins. In fresh tissue bioassays of mature leaves and squares (flower buds) and in artificial field infestations of white flowers, plants containing Cry1Ac:Cry1F significantly reduced levels of damage (leaf defoliation, bract feeding, penetrated squares and bolls, and boll abscission) and induced significantly greater mortality (90-100%) of fall armyworm compared with that on non-Bt cotton plants. Plants containing Cry1Ac:Cry1F conferred high levels (100%) of soybean looper mortality and low levels (0.2%) of leaf defoliation compared with non-Bt cotton. Beet armyworm was relatively less sensitive to Cry1Ac:Cry1F cotton plants compared with fall armyworm and soybean looper. However, beet armyworm larval development was delayed 21 d after infestation (DAI), and ingestion of plant tissue was inhibited (14 and 21 DAI) on the Cry1Ac:Cry1F plants compared with that on non-Bt cotton plants. These results show Cry1Ac:Cry1F cotton varieties can be an effective component in a management program for these lepidopteran pest species. Differential susceptibility of fall armyworm, beet armyworm, and soybean looper larvae to Cry1Ac:Cry1F cotton reinforces the need to sample during plant development and respond with a foliar insecticide if local action thresholds are exceeded.

  9. The Cultivation of Bt Corn Producing Cry1Ac Toxins Does Not Adversely Affect Non-Target Arthropods

    PubMed Central

    Guo, Yanyan; Feng, Yanjie; Ge, Yang; Tetreau, Guillaume; Chen, Xiaowen; Dong, Xuehui; Shi, Wangpeng

    2014-01-01

    Transgenic corn producing Cry1Ac toxins from Bacillus thuringiensis (Bt) provides effective control of Asian corn borer, Ostrinia furnacalis (Guenée), and thus reduces insecticide applications. However, whether Bt corn exerts undesirable effects on non-target arthropods (NTAs) is still controversial. We conducted a 2-yr study in Shangzhuang Agricultural Experiment Station to assess the potential impact of Bt corn on field population density, biodiversity, community composition and structure of NTAs. On each sampling date, the total abundance, Shannon's diversity index, Pielou's evenness index and Simpson's diversity index were not significantly affected by Bt corn as compared to non-Bt corn. The “sampling dates” had a significant effect on these indices, but no clear tendencies related to “Bt corn” or “sampling dates X corn variety” interaction were recorded. Principal response curve analysis of variance indicated that Bt corn did not alter the distribution of NTAs communities. Bray-Curtis dissimilarity and distance analysis showed that Cry1Ac toxin exposure did not increase community dissimilarities between Bt and non-Bt corn plots and that the evolution of non-target arthropod community was similar on the two corn varieties. The cultivation of Bt corn failed to show any detrimental evidence on the density of non-target herbivores, predators and parasitoids. The composition of herbivores, predators and parasitoids was identical in Bt and non-Bt corn plots. Taken together, results from the present work support that Bt corn producing Cry1Ac toxins does not adversely affect NTAs. PMID:25437213

  10. Effects of entomopathogenic nematodes on evolution of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac.

    PubMed

    Gassmann, Aaron J; Hannon, Eugene R; Sisterson, Mark S; Stock, S Patricia; Carrière, Yves; Tabashnik, Bruce E

    2012-06-01

    The evolution of resistance by pests can reduce the efficacy of transgenic crops that produce insecticidal toxins from Bacillus thuringiensis (Bt). However, fitness costs may act to delay pest resistance to Bt toxins. Meta-analysis of results from four previous studies revealed that the entomopathogenic nematode Steinernema riobrave (Rhabditida: Steinernematidae) imposed a 20% fitness cost for larvae of pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), that were homozygous for resistance to Bt toxin Cry1Ac, but no significant fitness cost was detected for heterozygotes. We conducted greenhouse and laboratory selection experiments to determine whether S. riobrave would delay the evolution of pink bollworm resistance to Cry1Ac. We mimicked the high dose/refuge scenario in the greenhouse with Bt cotton (Gossypium hirsutum L.) plants and refuges of non-Bt cotton plants, and in the laboratory with diet containing Cry1Ac and refuges of untreated diet. In both experiments, half of the replicates were exposed to S. riobrave and half were not. In the greenhouse, S. riobrave did not delay resistance. In the laboratory, S. riobrave delayed resistance after two generations but not after four generations. Simulation modeling showed that an initial resistance allele frequency > 0.015 and population bottlenecks can diminish or eliminate the resistance-delaying effects of fitness costs. We hypothesize that these factors may have reduced the resistance-delaying effects of S. riobrave in the selection experiments. The experimental and modeling results suggest that entomopathogenic nematodes could slow the evolution of pest resistance to Bt crops, but only under some conditions.

  11. Inheritance patterns, dominance and cross-resistance of Cry1Ab- and Cry1Ac-selected Ostrinia furnacalis (Guenée).

    PubMed

    Zhang, Tiantao; He, Mingxia; Gatehouse, Angharad M R; Wang, Zhenying; Edwards, Martin G; Li, Qing; He, Kanglai

    2014-09-11

    Two colonies of Asian corn borer, Ostrinia furnacalis (Guenée), artificially selected from a Bt-susceptible colony (ACB-BtS) for resistance to Cry1Ab (ACB-AbR) and Cry1Ac (ACB-AcR) toxins, were used to analyze inheritance patterns of resistance to Cry1 toxins. ACB-AbR and ACB-AcR evolved significant levels of resistance, with resistance ratios (RR) of 39-fold and 78.8-fold to Cry1Ab and Cry1Ac, respectively. The susceptibility of ACB-AbR larvae to Cry1Ac and Cry1F toxins, which had not previously been exposed, were significantly reduced, being >113-fold and 48-fold, respectively. Similarly, susceptibility of ACB-AcR larvae to Cry1Ab and Cry1F were also significantly reduced (RR > nine-fold, RR > 18-fold, respectively), indicating cross-resistance among Cry1Ab, Cry1Ac, and Cry1F toxins. However, ACB-AbR and ACB-AcR larvae were equally susceptible to Cry1Ie as were ACB-BtS larvae, indicating no cross-resistance between Cry1Ie and Cry1Ab or Cry1Ac toxins; this may provide considerable benefits in preventing or delaying the evolution of resistance in ACB to Cry1Ab and Cry1Ac toxins. Backcrossing studies indicated that resistance to Cry1Ab toxin was polygenic in ACB-AbR, but monogenic in ACB-AcR, whilst resistance to Cry1Ac toxin was primarily monogenic in both ACB-AbR and ACB-AcR, but polygenic as resistance increased.

  12. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  13. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  14. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  15. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  16. 40 CFR 180.1107 - Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin of...

  17. Efficacy of Bt maize producing the Cry1Ac protein against two important pests of corn in China.

    PubMed

    Chen, Hong-Xing; Yang, Rui; Yang, Wang; Zhang, Liu; Camara, Ibrahima; Dong, Xue-Hui; Liu, Yi -Qing; Shi, Wang-Peng

    2016-11-01

    Ostrinia furnacalis (Guenée) and Helicoverpa armigera (Hübner) are the most important pests of maize in China. A laboratory study and a 2-year field study on the efficacy of transgenic maize expressing the Cry1Ac protein BT38 against O. furnacalis and H. armigera were performed. We found that the husks, kernels, and silks of BT38 showed significant efficacy against larvae of O. furnacalis and H. armigera. In the field, when neonate larvae of O. furnacalis and H. armigera were on plants at different growth stages and when levels of leaf-damage or number of damaged silks were used to score efficacy, we found that BT38 showed significant insecticidal efficacy against O. furnacalis and H. armigera, but the non-Bt maize did not show significant efficacy against either pest. These results suggest that the insecticidal efficacy of Bt maize expressing the Cry1Ac protein could be useful in the integrated pest management of these key maize pests.

  18. Long-term toxicity study on genetically modified corn with cry1Ac gene in a Wuzhishan miniature pig model.

    PubMed

    Chen, Liang; Sun, Zhe; Liu, Quanwei; Zhong, Ruqing; Tan, Shuyi; Yang, Xiaoguang; Zhang, Hongfu

    2016-09-01

    The objective of the present study was to investigate the chronic effect of transgenic maize lines by the insertion of the cry1Ac gene from Bacillus thuringiensis (Bt) on the growth performance, immune response and health using a Wuzhishan miniature pig model through a 196-day feeding study. Based on the gender and weight, 72 Wuzhishan miniature pigs were randomly assigned one of the diets containing 65% non-transgenic isogenic corn or Bt corn at three stages of growth (day 0-69, 70-134 and 135-196). The potential toxicological effects of transgenic corn on pigs were explored. No difference between the diet treatments for growth performance and haematology parameters at any stages of growth. Although subtle differences in serum content of alanine aminotransferase, relative kidney weight and some immune response were observed between the Bt group and isogenic group, they were not considered as diet treatment-related. Long-term feeding Bt corn carrying cry1Ac genes to Wuzhishan miniature pigs did not indicate adverse effects on the growth, immune response and health indicators at any stages of growth. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  19. Monitoring cotton bollworm resistance to Cry1Ac in two counties of northern China during 2009-2013.

    PubMed

    An, Jingjie; Gao, Yulin; Lei, Chaoliang; Gould, Fred; Wu, Kongming

    2015-03-01

    Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis (Bt) has been deployed for combating cotton bollworm in China since 1997. As a follow-up on research started in 2002, the quantitative shifts in larval Cry1Ac resistance of field Helicoverpa armigera populations were monitored from 2009-2013 using bioassays of isofemale lines. A total of 2837 lines from Xiajin and 2055 lines from Anci were screened for growth rate on normal artificial diet and on a diet containing 1.0 µg mL(-1) of Cry1A(c) toxin. In 2009-2013, the mean relative average development rates (RADRs) of H. armigera larvae in the Xiajin population were 0.62, 0.59, 0.59, 0.58 and 0.62 respectively, and in the Anci population 0.54, 0.58, 0.60, 0.53 and 0.62 respectively. Compared with previous results in 2002, there was an increase in the RADR of H. armigera during 2009-2013, with ratios of 1.53-1.63 and 1.77-2.07 in the respective Xiajin and Anci populations, suggesting that resistance to Cry1Ac has increased in H. armigera populations in northern China. © 2014 Society of Chemical Industry.

  20. Frequency of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae).

    PubMed

    Mahon, R J; Olsen, K M; Downes, S; Addison, S

    2007-12-01

    Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an important lepidopteran pest of cotton (Gossypium spp.) in Australia and the Old World. From 2002, F2 screens were used to examine the frequency of resistance alleles in Australian populations of H. armigera to Bacillus thuringiensis (Bt) CrylAc and Cry2Ab, the two insecticidal proteins present in the transgenic cotton Bollgard II. At that time, Ingard (expressing Cry1Ac) cotton had been grown in Australia for seven seasons, and Bollgard II was about to be commercially released. The principal objective of our study was to determine whether sustained exposure caused an elevated frequency of alleles conferring resistance to Cry1Ac in a species with a track record of evolving resistance to conventional insecticides. No major alleles conferring resistance to Cry1Ac were found. The frequency of resistance alleles for Cry1Ac was <0.0003, with a 95% credibility interval between 0 and 0.0009. In contrast, alleles conferring resistance to Cry2Ab were found at a frequency of 0.0033 (0.0017, 0.0055). The first isolation of this allele was found before the widespread deployment of Bollgard II. For both toxins the experiment-wise detection probability was 94.4%. Our results suggest that alleles conferring resistance to Cry1Ac are rare and that a relatively high baseline frequency of alleles conferring resistance to Cry2Ab existed before the introduction of Bt cotton containing this toxin.

  1. Field resistance to the Bacillus thuringiensis protein Cry1Ac expressed in Bollgard(®) hybrid cotton in pink bollworm, Pectinophora gossypiella (Saunders), populations in India.

    PubMed

    Mohan, Komarlingam S; Ravi, Kadanur C; Suresh, Pennadam J; Sumerford, Douglas; Head, Graham P

    2016-04-01

    Bollgard(®) cotton, expressing Cry1Ac insecticidal protein, was approved for commercial planting in India in 2002, and by 2009 constituted 87% of the Indian crop, reducing losses from lepidopteran pests, including pink bollworm (PBW), Pectinophora gossypiella. Inadequate control of PBW in fields of single-gene Bollgard cotton was reported in 2009; surveys revealed heavy infestations of PBW in Bollgard, restricted to Gujarat state, but not elsewhere in India. Bioassays of PBW strains from Bollgard bolls showed that, while susceptible PBW could not complete development to third and later instar at 10.0 µg Cry1Ac mL(-1) , 66.1% of larvae from Gujarat Bollgard strains could. A field-resistant strain, further selected in the laboratory, had susceptibility to Cry1Ac reduced by >2000-fold. Resistance to Cry1Ac did not confer cross-resistance to the Cry2Ab2 protein. In 2010, Bollgard fields in Gujarat continued to be infested with PBW, and many Bollgard fields in the adjoining states of Maharashtra and Madhya Pradesh showed high-level infestation by PBW. Inadequate planting of refuges for PBW is the likely explanation for the field resistance to Bt cotton observed in Gujarat. These findings underscore the higher vulnerability of single-gene Bt products relative to dual-gene products expressing Cry1Ac and Cry2Ab2, and the increased risk of resistance evolution with low refuge compliance. © 2015 Society of Chemical Industry.

  2. Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.).

    PubMed

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-04-01

    Biopesticides or transgenic crops based on Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) effectively control agricultural insect pests. The sustainable use of Bt biopesticides and Bt crops is threatened, however, by the development of Cry resistance in the target pests. The diamondback moth, Plutella xylostella (L.), is the first pest that developed resistance to a Bt biopesticide in the field, and a recent study has shown that the resistance of P. xylostella to Cry1Ac is caused by a mutation in an ATP-binding cassette (ABC) transporter gene (ABCC2). In this study, we report that down-regulation of a novel ABC transporter gene from ABCG subfamily (Pxwhite) is associated with Cry1Ac resistance in P. xylostella. The full-length cDNA sequence of Pxwhite was cloned and analyzed. Spatial-temporal expression detection revealed that Pxwhite was expressed in all tissues and developmental stages, and highest expressed in Malpighian tubule tissue and in egg stage. Sequence variation analysis of Pxwhite indicated the absence of constant non-synonymous mutations between susceptible and resistant strains, whereas midgut transcript analysis showed that Pxwhite was remarkably reduced in all resistant strains and further reduced when larvae of the moderately resistant SZ-R strain were subjected to selection with Cry1Ac toxin. Furthermore, RNA interference (RNAi)-mediated suppression of Pxwhite gene expression significantly reduced larval susceptibility to Cry1Ac toxin, and genetic linkage analysis confirmed that down-regulation of Pxwhite gene is tightly linked to Cry1Ac resistance in P. xylostella. To our knowledge, this is the first report indicating that Pxwhite gene is involved in Cry1Ac resistance in P. xylostella.

  3. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth.

    PubMed

    Guo, Zhaojiang; Kang, Shi; Chen, Defeng; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhu, Xun; Baxter, Simon W; Zhou, Xuguo; Jurat-Fuentes, Juan Luis; Zhang, Youjun

    2015-04-01

    Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

  4. MAPK Signaling Pathway Alters Expression of Midgut ALP and ABCC Genes and Causes Resistance to Bacillus thuringiensis Cry1Ac Toxin in Diamondback Moth

    PubMed Central

    Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhu, Xun; Baxter, Simon W.; Zhou, Xuguo; Jurat-Fuentes, Juan Luis; Zhang, Youjun

    2015-01-01

    Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella. PMID:25875245

  5. The Spodoptera exigua (Lepidoptera: Noctuidae) ABCC2 Mediates Cry1Ac Cytotoxicity and, in Conjunction with Cadherin, Contributes to Enhance Cry1Ca Toxicity in Sf9 Cells.

    PubMed

    Ren, Xiang-Liang; Jiang, Wei-Li; Ma, Ya-Jie; Hu, Hong-Yan; Ma, Xiao-Yan; Ma, Yan; Li, Guo-Qing

    2016-12-01

    In insects, the mode of Cry1A toxins action has been studied in detail and many receptors that participate in the process are known. Recent evidence has revealed that an ABC transporter (ABCC2) is involved in conferring resistance to Cry1A toxins and that ABCC2 could be a receptor of Cry1A. However, it is not known whether Cry1Ca interacts with the same receptor proteins as Cry1A. In this study, we report the cloning of an ABC transporter gene, SeABCC2b, from the midgut of Spodoptera exigua (Hübner) larvae, and its expression in Sf9 cells for a functional analysis. The addition of Cry1Ca and Cry1Ac to Sf9 cell culture caused swelling in 28.5% and 93.9% of the SeABCC2-expressing cells, respectively. In contrast, only 7.4% and 1.3% of the controls cells swelled in the presence of Cry1Ca and Cry1Ac. Thus, SeABCC2b-expressing Sf9 cells had increased susceptibility to Cry1Ca and Cry1Ac. Similarly, S. exigua cadherin (SeCad1b) expressed in Sf9 cells caused 47.1% and 1.8% of the SeCad1b-expressing cells to swell to Cry1Ca and Cry1Ac exposure. Therefore, Sf9 cells expressing SeCad1b were more sensitive to Cry1Ca than Cry1Ac. Together, our data suggest that SeABCC2b from S. exigua mediates Cry1Ac cytotoxicity and, in conjunction with SeCad1b, contributes to enhance Cry1Ca toxicity in Sf9 cells.

  6. Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac Is Independent of Alteration of the Cadherin-Like Receptor for Cry Toxins

    PubMed Central

    Zhang, Xin; Tiewsiri, Kasorn; Kain, Wendy; Huang, Lihua; Wang, Ping

    2012-01-01

    Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin

  7. Resistance of Trichoplusia ni to Bacillus thuringiensis toxin Cry1Ac is independent of alteration of the cadherin-like receptor for Cry toxins.

    PubMed

    Zhang, Xin; Tiewsiri, Kasorn; Kain, Wendy; Huang, Lihua; Wang, Ping

    2012-01-01

    Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin

  8. Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

    PubMed Central

    Puspito, Agung N.; Rao, Abdul Q.; Hafeez, Muhammad N.; Iqbal, Muhammad S.; Bajwa, Kamran S.; Ali, Qurban; Rashid, Bushra; Abbas, Muhammad A.; Latif, Ayesha; Shahid, Ahmad A.; Nasir, Idrees A.; Husnain, Tayyab

    2015-01-01

    More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered “white gold” because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton. PMID:26617613

  9. Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane.

    PubMed

    Lemes, Ana Rita Nunes; Figueiredo, Camila Soares; Sebastião, Isis; Marques da Silva, Liliane; da Costa Alves, Rebeka; de Siqueira, Herbert Álvaro Abreu; Lemos, Manoel Victor Franco; Fernandes, Odair Aparecido; Desidério, Janete Apparecida

    2017-01-01

    The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests.

  10. Efficacy of genetically modified Bt toxins alone and in combinations against pink bollworm resistant to Cry1Ac and Cry2Ab

    USDA-ARS?s Scientific Manuscript database

    Evolution of resistance in pests threatens the long-term success of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and ...

  11. Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane

    PubMed Central

    2017-01-01

    The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests. PMID:28123906

  12. Cadherin is involved in the action of Bacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm, Spodoptera exigua.

    PubMed

    Qiu, Lin; Hou, Leilei; Zhang, Boyao; Liu, Lang; Li, Bo; Deng, Pan; Ma, Weihua; Wang, Xiaoping; Fabrick, Jeffrey A; Chen, Lizhen; Lei, Chaoliang

    2015-05-01

    Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to "pyramid" two or more Bt proteins that bind to distinct receptor proteins within the insect midgut. The most common Bt pyramid in cotton (Gossypium hirsutum L.) employs Cry1Ac with Cry2Ab to target several key lepidopteran pests, including the beet armyworm, Spodoptera exigua (Hübner), which is a serious migratory pest of many vegetable crops and is increasingly important in cotton in China. While cadherin and aminopeptidase-N are key receptors of Cry1 toxins in many lepidopterans including S. exigua, the receptor for Cry2A toxins remains poorly characterized. Here, we show that a heterologous expressed peptide corresponding to cadherin repeat 7 to the membrane proximal extracellular domain (CR7-MPED) in the S. exigua cadherin 1b (SeCad1b) binds Cry1Ac and Cry2Aa. Moreover, SeCad1b transcription was suppressed in S. exigua larvae by oral RNA interference and susceptibility to Cry1Ac and Cry2Aa was significantly reduced. These results indicate that SeCad1b plays important functional roles of both Cry1Ac and Cry2Aa, having major implications for resistance management for S. exigua in Bt crops.

  13. The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model.

    PubMed

    González-González, Edith; García-Hernández, Ana Lilia; Flores-Mejía, Raúl; López-Santiago, Rubén; Moreno-Fierros, Leticia

    2015-02-25

    Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.

  14. High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigers to Bt Soybean in Brazil.

    PubMed

    Dourado, Patrick M; Bacalhau, Fabiana B; Amado, Douglas; Carvalho, Renato A; Martinelli, Samuel; Head, Graham P; Omoto, Celso

    2016-01-01

    The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL-1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil.

  15. Potential impact of differential production of the Cry2Ab and Cry1Ac proteins in transgenic cotton in response to cold stress.

    PubMed

    Addison, Stewart J; Rogers, D John

    2010-08-01

    Transgenic Bollgard II cotton, Gossypium hirsutum L., expresses Cry1Ac and Cry2Ab proteins that provide control of lepidopteran larvae, including Helicoverpa and Heliothis species (Lepidoptera: Noctuidae) worldwide. Experiments conducted at Katherine, Northern Territory, Australia evaluated the impact of night minimum temperatures on Cry1Ac and Cry2Ab protein levels in Bollgard II cotton. In both 2003 and 2004, potted plants were either grown outside continuously or protected from cold in a glasshouse each night. In 2003, bulked samples of leaves were taken after two periods of low minimum temperature and used to determine a cold-stress threshold and critical period. In 2004, replicated samples were taken on 10 dates spanning five periods of low minimum temperature, allowing analysis of seasonal variation in Cry protein levels. The protein level was markedly higher for Cry2Ab than for Cry1Ac. Cry1Ac protein level peaked midseason and was not adversely affected by minimum temperatures down to 2.6 degrees C. The Cry2Ab protein level remained approximately constant but was reduced by low minimum temperatures (threshold, approximately 14 degrees C) for up to 6 d after each chill. The rate of Cry2Ab protein loss was 1.15 and 1.01% per chilling day-degree below threshold in 2003 and 2004, respectively. Impact would seem to be negligible on both the overall efficacy against lepidopteran larvae in-crop and on the current pyramided genes/high-dose/refuge Bt resistance-management strategies because the cold-stress effect is transient, a high level of Cry2Ab protein is still expressed, and there is no impact of chilling on Cry1Ac protein level.

  16. High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigera to Bt Soybean in Brazil

    PubMed Central

    Bacalhau, Fabiana B.; Amado, Douglas; Carvalho, Renato A.; Martinelli, Samuel; Head, Graham P.; Omoto, Celso

    2016-01-01

    The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F2 screen. The mean Cry1Ac lethal concentration (LC50) ranged from 0.11 to 1.82 μg·mL−1 of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F2 family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil. PMID:27532632

  17. A Comprehensive Assessment of the Effects of Transgenic Cry1Ac/Cry1Ab Rice Huahui 1 on Adult Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae)

    PubMed Central

    Zhou, Xia; Guo, Yunling; Kong, Hua; Zuo, Jiao; Huang, Qixing; Jia, Ruizong; Guo, Anping; Xu, Lin

    2016-01-01

    Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae) is a widely distributed coleoptera predator in southern Asia in rice ecosystem, and adult M. discolor feed on both rice pollen and soft-bodied arthropods. Bitrophic bioassay and tritrophic bioassay were conducted to evaluate the potential impact of Cry1Ac/Cry1Ab-expressing rice Huahui 1 and its non-transgenic counterpart Minghui 63 on fitness parameters of adult M. discolor. The results showed that the survival, and fecundity of this beetle’ adults were not different when they fed on Bt rice or non-Bt rice pollen or Nilaparvata lugens (Stål) reared on Bt rice or non-Bt rice. Toxicity assessment to ensure M. discolor adults were not sensitive to Cry1Ab or Cry1Ac protein independent from the pollen background, M. discolor adults were fed with an artificial diet containing Cry1Ac, Cry1Ab or both protein approximately 10 times higher concentration than in Huahui 1 rice pollen. No difference was detected for any of the life-table parameters tested between Cry protein-containing and pure diet. Artificial diet containing E-64 (N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide) was included as a positive control. In contrast, the pre-oviposition and fecundity of M. discolor were significantly adversely affected by feeding on E-64-containing diet. In both bioassays, the uptakes of Cry protein by adult M. discolor were tested by ELISA measurements. These results indicated that adults of M. discolor are not affected by Cry1Ab- or Cry1Ac-expressing rice pollen and are not sensitive to Cry protein at concentrations exceeding the levels in rice pollen in Huahui1. This suggests that M. discolor adults would not be harmed by Cry1Ac/Cry1Ab rice if Bt rice Huahui 1 were commercialized. PMID:26914608

  18. A Comprehensive Assessment of the Effects of Transgenic Cry1Ac/Cry1Ab Rice Huahui 1 on Adult Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae).

    PubMed

    Zhou, Xia; Guo, Yunling; Kong, Hua; Zuo, Jiao; Huang, Qixing; Jia, Ruizong; Guo, Anping; Xu, Lin

    2016-01-01

    Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae) is a widely distributed coleoptera predator in southern Asia in rice ecosystem, and adult M. discolor feed on both rice pollen and soft-bodied arthropods. Bitrophic bioassay and tritrophic bioassay were conducted to evaluate the potential impact of Cry1Ac/Cry1Ab-expressing rice Huahui 1 and its non-transgenic counterpart Minghui 63 on fitness parameters of adult M. discolor. The results showed that the survival, and fecundity of this beetle' adults were not different when they fed on Bt rice or non-Bt rice pollen or Nilaparvata lugens (Stål) reared on Bt rice or non-Bt rice. Toxicity assessment to ensure M. discolor adults were not sensitive to Cry1Ab or Cry1Ac protein independent from the pollen background, M. discolor adults were fed with an artificial diet containing Cry1Ac, Cry1Ab or both protein approximately 10 times higher concentration than in Huahui 1 rice pollen. No difference was detected for any of the life-table parameters tested between Cry protein-containing and pure diet. Artificial diet containing E-64 (N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide) was included as a positive control. In contrast, the pre-oviposition and fecundity of M. discolor were significantly adversely affected by feeding on E-64-containing diet. In both bioassays, the uptakes of Cry protein by adult M. discolor were tested by ELISA measurements. These results indicated that adults of M. discolor are not affected by Cry1Ab- or Cry1Ac-expressing rice pollen and are not sensitive to Cry protein at concentrations exceeding the levels in rice pollen in Huahui1. This suggests that M. discolor adults would not be harmed by Cry1Ac/Cry1Ab rice if Bt rice Huahui 1 were commercialized.

  19. Development of dipsticks for simultaneous detection of vip3A and cry1Ab/cry1Ac transgenic proteins.

    PubMed

    Kumar, Rajesh

    2012-01-01

    The number of genetically modified (GM) crops being cultivated and its produce reaching market is increasing every year. The transgenes (vip3A, cry1Ab, and cry1Ac) from Bacillus thuringiensis are being used by plant breeders, apart from other transgenes for developing insect pest-resistant GM crops. It is therefore necessary to develop an easy, rapid, and reliable detection assay to discriminate GM crops and non-GM crops. Dipstick strips using colloidal gold-labeled polyclonal antibodies were developed for simultaneous detection of Vip3A and Cry1Ab/CrylAc proteins. The assay was essentially based on the sandwich format of immunoassay, which was completed within 10 min, and the results were evaluated visually. The detection limits were 50 ng/mL (50 ppb) for both CrylAc and CrylAb proteins, and 100 ng/mL (100 ppb) for Vip3A protein. The developed dipsticks are suitable for on-site simultaneous screening of GM crops bearing two proteins, which, in turn, reduce cost and time of the assay.

  20. Bacillus thuringiensis HD-73 Spores Have Surface-Localized Cry1Ac Toxin: Physiological and Pathogenic Consequences

    PubMed Central

    Du, C.; Nickerson, K. W.

    1996-01-01

    Spores from Cry(sup+) strains of Bacillus thuringiensis bound fluorescein isothiocyanate-labeled antibodies specific for the 65-kDa activated Cry 1Ac toxin, whereas spores from Bacillus cereus and Cry(sup-) strains of B. thuringiensis did not. The Cry(sup+) spores could be activated for germination by alkaline conditions (pH 10.3), whereas Cry(sup-) spores could not. Once the surrounding exosporia had been removed or permeabilized, Cry(sup+) spores were able to bind the toxin receptor(s) from insect gut brush border membrane vesicle preparations, and their germination rates were increased ca. threefold in the presence of brush border membrane vesicles. A model is presented whereby in the soil the Cry toxins on the spore surface are protected by the exosporium while in the gut they are exposed and available for binding to the insect receptors. This model explains why the disulfide-rich C terminus of the cry genes is so highly conserved even though it is removed during the processing of the protoxin to the activated toxin. It also highlights the trade-off resulting from having Cry toxins located on the spore surface, i.e., decreased spore resistance versus enhanced insect pathogenesis. PMID:16535421

  1. Agrotis segetum midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 differs from that of Cry1Ac toxin.

    PubMed

    Ben Hamadou-Charfi, Dorra; Boukedi, Hanen; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jaoua, Samir

    2013-10-01

    Considering the fact that Agrotis segetum is one of the most pathogenic insects to vegetables and cereals in the world, particularly in Africa, the mode of action of Vip3Aa16 of Bacillus thuringiensis BUPM95 and Cry1Ac of the recombinant strain BNS3Cry-(pHTcry1Ac) has been examined in this crop pest. A. segetum proteases activated the Vip3Aa16 protoxin (90kDa) yielding three bands of about 62, 45, 22kDa and the activated form of the toxin was active against this pest with an LC50 of about 86ng/cm(2). To be active against A. segetum, Cry1Ac protoxin was activated to three close bands of about 60-65kDa. Homologous and heterologous competition binding experiments demonstrated that Vip3Aa16 bound specifically to brush border membrane vesicles (BBMV) prepared from A. segetum midgut and that it does not inhibit the binding of Cry1Ac. Moreover, BBMV protein blotting experiments showed that the receptor of Vip3Aa16 toxin in A. segetum midgut differs from that of Cry1Ac. In fact, the latter binds to a 120kDa protein whereas the Vip3Aa16 binds to a 65kDa putative receptor. The midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane lysis, vesicle formation in the goblet cells and disintegration of the apical membrane. The distinct binding properties and the unique protein sequence of Vip3Aa16 support its use as a novel insecticidal agent to control the crop pest A. segetum.

  2. Downregulation and Mutation of a Cadherin Gene Associated with Cry1Ac Resistance in the Asian Corn Borer, Ostrinia furnacalis (Guenée)

    PubMed Central

    Jin, Tingting; Chang, Xue; Gatehouse, Angharad M. R.; Wang, Zhenying; Edwards, Martin G.; He, Kanglai

    2014-01-01

    Development of resistance in target pests is a major threat to long-term use of transgenic crops expressing Bacillus thuringiensis (Bt) Cry toxins. To manage and/or delay the evolution of resistance in target insects through the implementation of effective strategies, it is essential to understand the basis of resistance. One of the most important mechanisms of insect resistance to Bt crops is the alteration of the interactions between Cry toxins and their receptors in the midgut. A Cry1Ac-selected strain of Asian corn borer (ACB), Ostrinia furnacalis, a key pest of maize in China, evolved three mutant alleles of a cadherin-like protein (OfCAD) (MPR-r1, MPR-r2 and MPR-r3), which mapped within the toxin-binding region (TBR). Each of the three mutant alleles possessed two or three amino acid substitutions in this region, especially Thr1457→Ser. In highly resistant larvae (ACB-Ac200), MPR-r2 had a 26-amino acid residue deletion in the TBR, which resulted in reduced binding of Cry1Ac compared to the MPR from the susceptible strain, suggesting that the number of amino acid deletions influences the level of resistance. Furthermore, downregulation of OfCAD gene (ofcad) transcription was observed in the Cry1Ac resistant strain, ACB-Ac24, suggesting that Cry1Ac resistance in ACB is associated with the downregulation of the transcript levels of the cadherin-like protein gene. The OfCAD identified from ACB exhibited a high degree of similarity to other members of the cadherin super-family in lepidopteran species. PMID:25216082

  3. Structure of the full-length insecticidal protein Cry1Ac reveals intriguing details of toxin packaging into in vivo formed crystals

    PubMed Central

    Evdokimov, Artem G; Moshiri, Farhad; Sturman, Eric J; Rydel, Timothy J; Zheng, Meiying; Seale, Jeffrey W; Franklin, Sonya

    2014-01-01

    For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability. PMID:25139047

  4. Experimental design and Bayesian networks for enhancement of delta-endotoxin production by Bacillus thuringiensis.

    PubMed

    Ennouri, Karim; Ayed, Rayda Ben; Hassen, Hanen Ben; Mazzarello, Maura; Ottaviani, Ennio

    2015-12-01

    Bacillus thuringiensis (Bt) is a Gram-positive bacterium. The entomopathogenic activity of Bt is related to the existence of the crystal consisting of protoxins, also called delta-endotoxins. In order to optimize and explain the production of delta-endotoxins of Bacillus thuringiensis kurstaki, we studied seven medium components: soybean meal, starch, KH₂PO₄, K₂HPO₄, FeSO₄, MnSO₄, and MgSO₄and their relationships with the concentration of delta-endotoxins using an experimental design (Plackett-Burman design) and Bayesian networks modelling. The effects of the ingredients of the culture medium on delta-endotoxins production were estimated. The developed model showed that different medium components are important for the Bacillus thuringiensis fermentation. The most important factors influenced the production of delta-endotoxins are FeSO₄, K2HPO₄, starch and soybean meal. Indeed, it was found that soybean meal, K₂HPO₄, KH₂PO₄and starch also showed positive effect on the delta-endotoxins production. However, FeSO4 and MnSO4 expressed opposite effect. The developed model, based on Bayesian techniques, can automatically learn emerging models in data to serve in the prediction of delta-endotoxins concentrations. The constructed model in the present study implies that experimental design (Plackett-Burman design) joined with Bayesian networks method could be used for identification of effect variables on delta-endotoxins variation.

  5. A 90-day subchronic feeding study of genetically modified maize expressing Cry1Ac-M protein in Sprague-Dawley rats.

    PubMed

    Liu, Pengfei; He, Xiaoyun; Chen, Delong; Luo, Yunbo; Cao, Sishuo; Song, Huan; Liu, Ting; Huang, Kunlun; Xu, Wentao

    2012-09-01

    The cry1Ac-M gene, coding one of Bacillus thuringiensis (Bt) crystal proteins, was introduced into maize H99 × Hi IIB genome to produce insect-resistant GM maize BT-38. The food safety assessment of the BT-38 maize was conducted in Sprague-Dawley rats by a 90-days feeding study. We incorporated maize grains from BT-38 and H99 × Hi IIB into rodent diets at three concentrations (12.5%, 25%, 50%) and administered to Sprague-Dawley rats (n=10/sex/group) for 90 days. A commercialized rodent diet was fed to an additional group as control group. Body weight, feed consumption and toxicological response variables were measured, and gross as well as microscopic pathology were examined. Moreover, detection of residual Cry1Ac-M protein in the serum of rats fed with GM maize was conducted. No death or adverse effects were observed in the current feeding study. No adverse differences in the values of the response variables were observed between rats that consumed diets containing GM maize BT-38 and non-GM maize H99 × Hi IIB. No detectable Cry1Ac-M protein was found in the serum of rats after feeding diets containing GM maize for 3 months. The results demonstrated that BT-38 maize is as safe as conventional non-GM maize.

  6. Protection against Naegleria fowleri infection in mice immunized with Cry1Ac plus amoebic lysates is dependent on the STAT6 Th2 response.

    PubMed

    Carrasco-Yepez, M; Rojas-Hernandez, S; Rodriguez-Monroy, M A; Terrazas, L I; Moreno-Fierros, L

    2010-01-01

    We previously reported that intranasal administration of Cry1Ac protoxin alone or in combination with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice. Those results suggested that both antibody responses and innate immune mechanisms may be participating in the protective effects observed. The present study was aimed to investigate whether the STAT6-induced Th2 immune response is essential for the resistance to N. fowleri infection, conferred by immunization with amoebic lysates plus Cry1Ac. STAT6-deficient (STAT6-/-) and wild-type (STAT6+/+) BALB/c mice were immunized by the intranasal route with a combination of N. fowleri lysates plus Cry1Ac, and subsequently challenged with lethal doses of N. fowleri trophozoites. STAT6+/+ mice displayed 100% protection, while no protection was observed in STAT6-/- mice. Significantly higher titres of Th2-associated IgG1 as well as interleukin-4 (IL-4) were found in STAT6+/+ mice, whereas in STAT6-/- mice significantly more IL-12 and IFN-gamma as well as significantly higher titres of Th1-associated IgG2a were detected. Thus, whereas protected STAT6+/+-immunized mice elicited a Th-2 type inclined immune response that produced predominantly humoral immunity, unprotected STAT6-/- mice exhibited a polarized Th1 type cellular response. These findings suggest that the STAT6-signalling pathway is critical for defence against N. fowleri infection.

  7. Pretreatment with Cry1Ac Protoxin Modulates the Immune Response, and Increases the Survival of Plasmodium-Infected CBA/Ca Mice

    PubMed Central

    Legorreta-Herrera, Martha; Oviedo Meza, Rodrigo; Moreno-Fierros, Leticia

    2010-01-01

    Malaria is a major global health problem that kills 1-2 million people each year. Despite exhaustive research, naturally acquired immunity is poorly understood. Cry1A proteins are potent immunogens with adjuvant properties and are able to induce strong cellular and humoral responses. In fact, it has been shown that administration of Cry1Ac protoxin alone or with amoebic lysates induces protection against the lethal infection caused by the protozoa Naegleria fowleri. In this work, we studied whether Cry1Ac is able to activate the innate immune response to induce protection against Plasmodium berghei ANKA (lethal) and P. chabaudi AS (nonlethal) parasites in CBA/Ca mice. Treatment with Cry1Ac induced protection against both Plasmodium species in terms of reduced parasitaemia, longer survival time, modulation of pro- and anti-inflammatory cytokines, and increased levels of specific antibodies against Plasmodium. Understanding how to boost innate immunity to Plasmodium infection should lead to immunologically based intervention strategies. PMID:20300584

  8. A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae.

    PubMed

    Hara, Hirotaka; Atsumi, Shogo; Yaoi, Katsuro; Nakanishi, Kazuko; Higurashi, Satoshi; Miura, Nami; Tabunoki, Hiroko; Sato, Ryoichi

    2003-03-13

    Aminopeptidase N (APN) and cadherin-like protein (BtR175) from Bombyx mori larvae were examined for their roles in Cry1Aa- and Cry1Ac-induced lysis of B. mori midgut epithelial cells (MECs). APNs and BtR175 were present in all areas of the midgut, were particularly abundant in the posterior region, and were found only on columnar cell microvilli and not on the lateral membrane that makes cell-cell contacts. This distribution was in accordance with the distribution of Cry1A-susceptible MECs in the midgut. The lytic activity of Cry1Aa and Cry1Ac on collagenase-dissociated MECs was linearly dependent on toxin concentration. Although pre-treatment of MECs with anti-BtR175 antibody was observed to partially inhibit the lytic activity exerted by 0.1-1 nM Cry1Aa toxin or 5 nM Cry1Ac toxin, no significant inhibition was observed when MECs were pre-treated with anti-APN antibody. These results suggest that BtR175 functions as a major receptor for Cry1A toxins in the midgut of B. mori larvae.

  9. Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene.

    PubMed

    Narendran, M; Deole, Satish G; Harkude, Satish; Shirale, Dattatray; Nanote, Asaram; Bihani, Pankaj; Parimi, Srinivas; Char, Bharat R; Zehr, Usha B

    2013-08-01

    Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ). Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

  10. Transgenic Bt rice lines producing Cry1Ac, Cry2Aa or Cry1Ca have no detrimental effects on Brown Planthopper and Pond Wolf Spider.

    PubMed

    Niu, Lin; Mannakkara, Amani; Qiu, Lin; Wang, Xiaoping; Hua, Hongxia; Lei, Chaoliang; Jurat-Fuentes, Juan Luis; Ma, Weihua

    2017-05-16

    Transgenic rice expressing cry genes from the bacterium Bacillus thuringiensis (Bt rice) is highly resistant to lepidopteran pests. The brown planthopper (BPH, Nilaparvata lugens) is the main non-target sap-sucking insect pest of Bt transgenic rice. The pond wolf spider (PWS, Pardosa pseudoannulata) is one of the most dominant predators of BPH in rice fields. Consequently, the safety evaluation of Bt rice on BPH and PWS should be conducted before commercialization. In the current study, two experiments were performed to assess the potential ecological effects of Bt rice on BPH and PWS: (1) a tritrophic experiment to evaluate the transmission of Cry1Ac, Cry2Aa and Cry1Ca protein in the food chain; and (2) binding assays of Cry1Ac, Cry2Aa and Cry1Ca to midgut brush border membrane proteins from BPH and PWS. Trace amounts of the three Cry proteins were detected in BPH feeding on Bt rice cultivars, but only Cry1Ac and Cry2Aa proteins could be transferred to PWS through feeding on BPH. In vitro binding of biotinylated Cry proteins and competition assays in midgut protein vesicles showed weak binding, and ligand blot analysis confirmed the binding specificity. Thus, we inferred that the tested Bt rice varieties have negligible effects on BPH and PWS.

  11. The expression of a recombinant cry1Ac gene with subtilisin-like protease CDEP2 gene in acrystalliferous Bacillus thuringiensis by Red/ET homologous recombination.

    PubMed

    Xia, Liqiu; Zeng, Zhi; Ding, Xuezhi; Huang, Fan

    2009-10-01

    A novel cDNA encoding the subtilisin-like serine protease gene CDEP2 was isolated from Beauveria bassiana by reverse transcription polymerase chain reaction (RT-PCR). It contained an 1137 bp ORF that predicted a protein of 379 amino acids with M = 38863 Da and pI = 8.21. In an attempt to improve insecticidal activity, the CDEP2 gene and the cry1Ac gene from Bacillus thuringiensis were co-fused into the vector pHT315 as pHAc-CDEP2 plasmid by Red/ET homologous recombination. The co-fusion gene was attempted under the control of the native cry1Ac promoter. Plasmid pHAc-CDEP2 was electro-transformed into the B. thuringiensis subsp. kurstaki Cry(-)B. Analyzed by SDS-PAGE and Western blotting, the transformant Cry(-)B-pHAc-CDEP2 strain produced a 130 kDa Cry1Ac protein and 39 kDa CDEP2 protein. The 50% lethal concentration values (LC(50)) of Cry(-)B-pHAc-CDEP2 strain (8.5 microl/ml) to Helicoverpa armigera third instars larvae was clearly higher than the Cry(-)B-pHAc strain (16.7 microl/ml) at 72 h.

  12. Effects of defoliating insect resistance QTLs and a cry1Ac transgene in soybean near-isogenic lines.

    PubMed

    Zhu, S; Walker, D R; Boerma, H R; All, J N; Parrott, W A

    2008-02-01

    The crystal proteins coded by transgenes from Bacillus thuringiensis (Bt) have shown considerable value in providing effective insect resistance in a number of crop species, including soybean, Glycine max (L.) Merr. Additional sources of soybean insect resistance would be desirable to manage the development of tolerance/resistance to crystal proteins by defoliating insects and to sustain the deployment of Bt crops. The objective of this study was to evaluate the effects and interactions of three insect resistance quantitative trait loci (QTLs; QTL-M, QTL-H, and QTL-G) originating from Japanese soybean PI 229358 and a cry1Ac gene in a "Benning" genetic background. A set of 16 BC(6)F(2)-derived near isogenic lines (NILs) was developed using marker-assisted backcrosses and evaluated for resistance to soybean looper [SBL, Pseudoplusia includens (Walker)] and corn earworm [CEW, Helicoverpa zea (Boddie)] in field cage, greenhouse, and detached leaf assays. Both Bt and QTL-M had significantly reduced defoliation by both SBL and CEW and reduced larval weight of CEW. The antibiosis QTL-G had a significant effect on reducing CEW larval weight and also a significant effect on reducing defoliation by SBL and CEW in some assays. The antixenosis QTL-H had no main effect, but it appeared to function through interaction with QTL-M and QTL-G. Adding QTL-H and QTL-G further enhanced the resistance of the Bt and QTL-M combination to CEW in the field cage assay. These results should help guide the development of strategies for effective management of insect pests and for sustainable deployment of Bt genes.

  13. Identification of Residues in Domain III of Bacillus thuringiensis Cry1Ac Toxin That Affect Binding and Toxicity

    PubMed Central

    Lee, Mi Kyong; You, Taek H.; Gould, Fred L.; Dean, Donald H.

    1999-01-01

    Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to Lymantria dispar, Manduca sexta, and Heliothis virescens. Five sets of alanine block mutants were generated at the residues 503SS504, 506NNI508, 509QNR511, 522ST523, and 524ST525. Single alanine substitutions were made at the residues 509Q, 510N, 511R, and 513Y. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations, 503SS504-AA, 506NNI508-AAA, 522ST523-AA, 524ST525-AA, and 510N-A affected neither the protein’s toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward L. dispar and M. sexta, the 509QNR511-AAA, 509Q-A, 511R-A, and 513Y-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward H. virescens, the 509QNR511-AAA, 509Q-A, 511R-A, and 513Y-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only 509QNR511-AAA and 511R-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues 509Q, 511R, and 513Y in domain III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced more than fivefold. PMID:10508083

  14. Effects of four nematode species on fitness costs of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac.

    PubMed

    Hannon, Eugene R; Sisterson, Mark S; Stock, S Patricia; Carrière, Yves; Tabashnik, Bruce E; Gassmann, Aaron J

    2010-10-01

    Evolution of resistance by pests can reduce the efficacy oftransgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bt). In conjunction with refuges of non-Bt host plants, fitness costs can delay the evolution of resistance. Furthermore, fitness costs often vary with ecological conditions, suggesting that agricultural landscapes can be manipulated to magnify fitness costs and thereby prolong the efficacy of Bt crops. In the current study, we tested the effects of four species of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) on the magnitude and dominance of fitness costs of resistance to Bt toxin CrylAc in pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae). For more than a decade, field populations of pink bollworm in the United States have remained susceptible to Bt cotton Gossypium hirsutum L. producing CrylAc; however, we used laboratory strains that had a mixture of susceptible and resistant individuals. In laboratory experiments, dominant fitness costs were imposed by the nematode Steinernema riobrave Cabanillas, Poinar, and Raulston but no fitness costs were imposed by Steinernema carpocapsae Weiser, Steinernema sp. (ML18 strain), or Heterorhabditis sonorensis Stock, Rivera-Orduño, and Flores-Lara. In computer simulations, evolution of resistance to Cry1Ac by pink bollworm was substantially delayed by treating some non-Bt cotton refuge fields with nematodes that imposed a dominant fitness cost, similar to the cost observed in laboratory experiments with S. riobrave. Based on the results here and in related studies, we conclude that entomopathogenic nematodes could bolster insect resistance management, but the success of this approach will depend on selecting the appropriate species of nematode and environment, as fitness costs were magnified by only two of five species evaluated and also depended on environmental factors.

  15. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta endotoxin...

  16. Constitutive and herbivore-inducible glucosinolate concentrations in oilseed rape (Brassica napus) leaves are not affected by Bt Cry1Ac insertion but change under elevated atmospheric CO2 and O3.

    PubMed

    Himanen, Sari J; Nissinen, Anne; Auriola, Seppo; Poppy, Guy M; Stewart, C Neal; Holopainen, Jarmo K; Nerg, Anne-Marja

    2008-01-01

    Glucosinolates are plant secondary compounds involved in direct chemical defence by cruciferous plants against herbivores. The glucosinolate profile can be affected by abiotic and biotic environmental stimuli. We studied changes in glucosinolate patterns in leaves of non-transgenic oilseed rape (Brassica napus ssp. oleifera) under elevated atmospheric CO2 or ozone (O3) concentrations and compared them with those from transgenic for herbivore-resistance (Bacillus thuringiensis Cry1Ac endotoxin), to assess herbivory dynamics. Both elevated CO2 and O3 levels decreased indolic glucosinolate concentrations in transgenic and non-transgenic lines, whereas O3 specifically increased the concentration of an aromatic glucosinolate, 2-phenylethylglucosinolate. The herbivore-inducible indolic glucosinolate response was reduced in elevated O3 whereas elevated CO2 altered the induction dynamics of indolic and aliphatic glucosinolates. Herbivore-resistant Bt plants experienced minimal leaf damage after target herbivore Plutella xylostella feeding, but exhibited comparatively similar increase in glucosinolate concentrations after herbivory as non-transgenic plants, indicating that the endogenous glucosinolate defence was not severely compromised by transgenic modifications. The observed differences in constitutive and inducible glucosinolate concentrations of oilseed rape under elevated atmospheric CO2 and O3 might have implications for plant-herbivore interactions in Brassica crop-ecosystems in future climate scenarios.

  17. FOXA transcriptional factor modulates insect susceptibility to Bacillus thuringiensis Cry1Ac toxin by regulating the expression of toxin-receptor ABCC2 and ABCC3 genes.

    PubMed

    Li, Jianghuai; Ma, Yuemin; Yuan, Wanli; Xiao, Yutao; Liu, Chenxi; Wang, Jia; Peng, Jianxin; Peng, Rong; Soberón, Mario; Bravo, Alejandra; Yang, Yongbo; Liu, Kaiyu

    2017-09-01

    Cry toxins produced by Bacillus thuringiensis (Bt) are insecticidal proteins widely used in insect control. Recently, it was shown that ATP-binding cassette transporter proteins (ABC) such as ABCC2, ABCC3, ABCG1 and ABCA2 are implicated in the insecticidal action of Cry toxins as putative receptors. However, the transcriptional regulators involved in the expression of ABC transporter genes remain unknown. Sequence analysis of promoter regions of ABCC2 gene from Helicoverpa armigera and ABCC3 gene from Spodoptera litura Sl-HP cultured cells, revealed the potential participation of Forkhead box protein A (FOXA), a transcription factor that regulates the expression of genes through remodeling chromatin. To determine if FOXA was involved in regulating expression of ABCC2 and ABCC3 genes, the expression of FOXA, ABCC2 and ABCC3 was compared in Sl-HP cells that are sensitive to Cry1Ac toxin with those in S. frugiperda Sf9 cells that are not sensitive to the toxin. Expression levels of those genes were significantly higher in Sl-HP than in Sf9 cells. Transient expression of FOXA in Sf9 cells activated ABCC2 and ABCC3 transcription, which directly correlated with enhanced Cry1Ac-susceptibility in these cells. Silencing of FOXA gene expression by RNAi in H. armigera larvae resulted in a decreased expression of ABCC2 and ABCC3 without affecting expression of other Cry toxin receptor genes such as alkaline phosphatase, aminopeptidase or cadherin. Silencing of FOXA gene expression also resulted in a Cry1Ac-tolerant phenotype since lower mortality and higher pupation rate were observed in diet containing Cry1Ac protoxin in comparison with the control group. These results demonstrate that FOXA up-regulates expression of the Cry1Ac-toxin receptor ABCC2 and ABCC3 genes, and that lower FOXA expression correlates with tolerance to Cry toxin in cell lines and in lepidopteran larvae. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Quantification of toxins in a Cry1Ac + CpTI cotton cultivar and its potential effects on the honey bee Apis mellifera L.

    PubMed Central

    Han, Peng; Lei, Chao-Liang; Cui, Jin-Jie; Desneux, Nicolas

    2010-01-01

    Transgenic Cry1Ac + CpTI cotton (CCRI41) is increasingly planted throughout China. However, negative effects of this cultivar on the honey bee Apis mellifera L., the most important pollinator for cultivated ecosystem, remained poorly investigated. The objective of our study was to evaluate the potential side effects of transgenic Cry1Ac + CpTI pollen from cotton on young adult honey bees A. mellifera L. Two points emphasized the significance of our study: (1) A higher expression level of insecticidal protein Cry1Ac in pollen tissues was detected (when compared with previous reports). In particular, Cry1Ac protein was detected at 300 ± 4.52 ng g−1 [part per billion (ppb)] in pollen collected in July, (2) Effects on chronic mortality and feeding behaviour in honey bees were evaluated using a no-choice dietary feeding protocol with treated pollen, which guarantee the highest exposure level to bees potentially occurring in natural conditions (worst case scenario). Tests were also conducted using imidacloprid-treated pollen at a concentration of 48 ppb as positive control for sublethal effect on feeding behaviour. Our results suggested that Cry1Ac + CpTI pollen carried no lethal risk for honey bees. However, during a 7-day oral exposure to the various treatments (transgenic, imidacloprid-treated and control), honey bee feeding behaviour was disturbed and bees consumed significantly less CCRI41 cotton pollen than in the control group in which bees were exposed to conventional cotton pollen. It may indicate an antifeedant effect of CCRI41 pollen on honey bees and thus bees may be at risk because of large areas are planted with transgenic Bt cotton in China. This is the first report suggesting a potential sublethal effect of CCRI41 cotton pollen on honey bees. The implications of the results are discussed in terms of risk assessment for bees as well as for directions of future work involving risk assessment of CCRI41 cotton. PMID:20700762

  19. Expression of a Bacillus thuringiensis delta-endotoxin gene by Bacillus pumilus.

    PubMed

    Selinger, L B; Khachatourians, G G; Byers, J R; Hynes, M F

    1998-03-01

    The delta-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a delta-endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and delta-endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that < 0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of delta-endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level delta-endotoxin production occurred concomitant with sporulation. Synthesis of functional delta-endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of delta-endotoxins to the root environment for biocontrol purposes.

  20. Relationship between poly-beta-hydroxybutyrate production and delta-endotoxin for Bacillus thuringiensis var. kurstaki.

    PubMed

    Navarro, A Karin; Farrera, Reynold R; López, Ruth; Pérez-Guevara, Fermín

    2006-05-01

    A linear relationship between total solid concentration (TSC), delta-endotoxin production [Cry = 0.2795(TSC)-0.2472, R2 = 0.8644] and poly-beta-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R2 = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between delta-endotoxin and PHB accumulation [Cry = 2.1573(PHB)-1.1248, R2 = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l(-1) was required before the onset of delta-endotoxin production.

  1. A Comprehensive Assessment of the Effects of Bt Cotton on Coleomegilla maculata Demonstrates No Detrimental Effects by Cry1Ac and Cry2Ab

    PubMed Central

    Li, Yunhe; Romeis, Jörg; Wang, Ping; Peng, Yufa; Shelton, Anthony M.

    2011-01-01

    The ladybird beetle, Coleomegilla maculata (DeGeer), is a common and abundant predator in many cropping systems. Its larvae and adults are predaceous, feeding on aphids, thrips, lepidopteran larvae and plant tissues, such as pollen. Therefore, this species is exposed to insecticidal proteins expressed in insect-resistant, genetically engineered cotton expressing Cry proteins derived from Bacillus thuringiensis (Bt). A tritrophic bioassay was conduced to evaluate the potential impact of Cry2Ab- and Cry1Ac-expressing cotton on fitness parameters of C. maculata using Bt-susceptible and -resistant larvae of Trichoplusia ni as prey. Coleomegilla maculata survival, development time, adult weight and fecundity were not different when they were fed with resistant T. ni larvae reared on either Bt or control cotton. To ensure that C. maculata were not sensitive to the tested Cry toxins independent from the plant background and to add certainty to the hazard assessment, C. maculata larvae were fed artificial diet incorporated with Cry2Ab, Cry1Ac or both at >10 times higher concentrations than in cotton tissue. Artificial diet containing E-64 was included as a positive control. No differences were detected in any life-table parameters between Cry protein-containing diet treatments and the control diet. In contrast, larvae of C. maculata fed the E-64 could not develop to the pupal stage and the 7-d larval weight was significantly negatively affected. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources were confirmed by ELISA and sensitive-insect bioassays. Our results show that C. maculata is not affected by Bt cotton and is not sensitive to Cry2Ab and Cry1Ac at concentrations exceeding the levels in Bt cotton, thus demonstrating that Bt cotton will pose a negligible risk to C. maculata. More importantly, this study demonstrates a comprehensive system for assessing the risk of genetically modified plants on non-target organisms. PMID

  2. High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids

    PubMed Central

    Singh, Amarjeet Kumar; Paritosh, Kumar; Kant, Uma; Burma, Pradeep Kumar; Pental, Deepak

    2016-01-01

    Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2–2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4–5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy. PMID:27391960

  3. Feed intake, milk production and composition of crossbred cows fed with insect-protected Bollgard II® cottonseed containing Cry1Ac and Cry2Ab proteins.

    PubMed

    Singhal, K K; Tyagi, A K; Rajput, Y S; Singh, M; Kaur, H; Perez, T; Hartnell, G F

    2011-09-01

    Twenty crossbred lactating multiparous cows were used in a 28-day study to compare dry matter intake (DMI), milk yield, milk composition and Bacillus thuringiensis (Bt) protein concentrations in plasma when fed diets containing Bollgard II(®) cottonseed (BGII) or a control non-genetically modified isogenic cottonseed (CON). Bollgard II cottonseed contains the Cry1Ac and Cry2Ab insecticidal proteins that protect cotton plants from feeding damage caused by certain lepidopteran insects. Cows were assigned randomly to the BGII or CON treatments after a 2-week adjustment period. Cows consumed a concentrate containing 40% crushed cottonseed according to milk yield and green maize forage ad libitum. All cows received the same diet but with different crushed cottonseed sources. Cottonseed was included to provide approximately 2.9 kg per cow daily (dry matter basis). The ingredient composition of the concentrate was 40% crushed cottonseed, 15% groundnut cake, 20% corn, 22% wheat bran, 1% salt and 2% mineral mixture. Milk and blood plasma were analyzed for Cry1Ac and Cry2Ab proteins. DMI, BW, milk yield and milk components did not differ between cows on the BGII and CON treatments. Although milk yield and milk fat percentage were not affected by treatment, 4% fat-corrected milk (FCM) production and FCM/kg DMI for cows on the BGII treatment (14.0 kg/cow per day, 1.12 kg/kg) were significantly improved compared with cows on the CON treatment (12.1 kg/cow per day, 0.97 kg/kg). Gossypol contents in BGII cottonseed and conventional cottonseed were similar. Cry1Ac and Cry2Ab2 proteins in Bollgard II cottonseed were 5.53 and 150.8 μg/g, respectively, and were not detected in the milk or plasma samples. The findings suggested that Bollgard II cottonseed can replace conventional cottonseed in dairy cattle diets with no adverse effects on performance and milk composition.

  4. Effects of transgenic Cry1Ac + CpTI cotton on non-target mealybug pest Ferrisia virgata and its predator Cryptolaemus montrouzieri.

    PubMed

    Wu, Hongsheng; Zhang, Yuhong; Liu, Ping; Xie, Jiaqin; He, Yunyu; Deng, Congshuang; De Clercq, Patrick; Pang, Hong

    2014-01-01

    Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata.

  5. Effects of Transgenic Cry1Ac + CpTI Cotton on Non-Target Mealybug Pest Ferrisia virgata and Its Predator Cryptolaemus montrouzieri

    PubMed Central

    Wu, Hongsheng; Zhang, Yuhong; Liu, Ping; Xie, Jiaqin; He, Yunyu; Deng, Congshuang; De Clercq, Patrick; Pang, Hong

    2014-01-01

    Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata. PMID:24751821

  6. Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

    PubMed Central

    Kain, Wendy; Song, Xiaozhao; Janmaat, Alida F.; Zhao, Jian-Zhou; Myers, Judith; Shelton, Anthony M.

    2014-01-01

    Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops. PMID:25480752

  7. High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

    PubMed

    Singh, Amarjeet Kumar; Paritosh, Kumar; Kant, Uma; Burma, Pradeep Kumar; Pental, Deepak

    2016-01-01

    Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

  8. Cytotoxicity on human cells of Cry1Ab and Cry1Ac Bt insecticidal toxins alone or with a glyphosate-based herbicide.

    PubMed

    Mesnage, R; Clair, E; Gress, S; Then, C; Székács, A; Séralini, G-E

    2013-07-01

    The study of combined effects of pesticides represents a challenge for toxicology. In the case of the new growing generation of genetically modified (GM) plants with stacked traits, glyphosate-based herbicides (like Roundup) residues are present in the Roundup-tolerant edible plants (especially corns) and mixed with modified Bt insecticidal toxins that are produced by the GM plants themselves. The potential side effects of these combined pesticides on human cells are investigated in this work. Here we have tested for the very first time Cry1Ab and Cry1Ac Bt toxins (10 ppb to 100 ppm) on the human embryonic kidney cell line 293, as well as their combined actions with Roundup, within 24 h, on three biomarkers of cell death: measurements of mitochondrial succinate dehydrogenase, adenylate kinase release by membrane alterations and caspase 3/7 inductions. Cry1Ab caused cell death from 100 ppm. For Cry1Ac, under such conditions, no effects were detected. The Roundup tested alone from 1 to 20 000 ppm is necrotic and apoptotic from 50 ppm, far below agricultural dilutions (50% lethal concentration 57.5 ppm). The only measured significant combined effect was that Cry1Ab and Cry1Ac reduced caspases 3/7 activations induced by Roundup; this could delay the activation of apoptosis. There was the same tendency for the other markers. In these results, we argue that modified Bt toxins are not inert on nontarget human cells, and that they can present combined side-effects with other residues of pesticides specific to GM plants. Copyright © 2012 John Wiley & Sons, Ltd.

  9. Bt crops producing Cry1Ac, Cry2Ab and Cry1F do not harm the green lacewing, Chrysoperla rufilabris.

    PubMed

    Tian, Jun-Ce; Wang, Xiang-Ping; Long, Li-Ping; Romeis, Jörg; Naranjo, Steven E; Hellmich, Richard L; Wang, Ping; Earle, Elizabeth D; Shelton, Anthony M

    2013-01-01

    The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.

  10. Transgenic Cabbage Expressing Cry1Ac1 Does Not Affect the Survival and Growth of the Wolf Spider, Pardosa astrigera L. Koch (Araneae: Lycosidae)

    PubMed Central

    Kim, Young-Joong; Lee, Joon-Ho; Harn, Chee Hark; Kim, Chang-Gi

    2016-01-01

    Both herbivores that consume transgenic crops and their predators can be exposed to insecticidal proteins expressed in those crops. We conducted a tritrophic bioassay to evaluate the ecotoxicological impacts that Bt cabbage (Brassica oleracea var. capitata) expressing Cry1Ac1 protein might have on the wolf spider (Pardosa astrigera), a non-target generalist predator. Enzyme-Linked Immunosorbent Assays indicated that protein levels were 4.61 ng g-1 dry weight in fruit flies (Drosophila melanogaster) fed with the transgenic cabbage and 1.86 ng g-1 dry weight in the wolf spiders that preyed upon them. We also compared the life history traits of spiders collected from Bt versus non-Bt cabbage and found no significant differences in their growth, survival, and developmental rates. Because Bt cabbage did not affect the growth of fruit flies, we conclude that any indirect effects that this crop had on the wolf spider were probably not mediated by prey quality. Therefore, exposure to Cry1Ac1 protein when feeding upon prey containing that substance from transgenic cabbage has only a negligible influence on those non-target predatory spiders. PMID:27055120

  11. Intranasal immunization with Naegleria fowleri lysates and Cry1Ac induces metaplasia in the olfactory epithelium and increases IgA secretion.

    PubMed

    Jarillo-Luna, A; Moreno-Fierros, L; Campos-Rodríguez, R; Rodríguez-Monroy, M A; Lara-Padilla, E; Rojas-Hernández, S

    2008-01-01

    According to previous reports, intranasal administration of the Cry1Ac protein alone or with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice, apparently by eliciting IgA responses in the nasal mucosa. In the current study, we performed an immunohistochemical analysis of IgA in the nasal mucosa of mice immunized intranasally with Cry1Ac, and amoebic lysates or a combination of both. The animals were sacrificed 24 h after the last immunization or after an intranasal lethal challenge with N. fowleri. Our results indicate that all of the intranasal immunizations provoked an increase in areas with metaplasia in the olfactory epithelium, allowing for secretion of IgA. As a result, IgA antibodies were found interacting with trophozoites in the nasal lumen, and there was a marked increase of IgA in the metaplasic epithelium. On the other hand in nonimmunized mice trophozoites were observed invading the nasal mucosa, which was not the case for immunized mice. Our results suggest that intranasal immunization provokes cellular changes in the olfactory epithelium, leading to greater protection against N. fowleri that is probably caused by an increased secretion of IgA. The increased IgA response induced in the nasal mucosa by immunization probably impedes both amoebic adhesion and subsequent invasion of the parasite to the nasal epithelium.

  12. Ingestion of Bt corn pollen containing Cry1Ab/2Aj or Cry1Ac does not harm Propylea japonica larvae.

    PubMed

    Liu, Yanmin; Liu, Qingsong; Wang, Yanan; Chen, Xiuping; Song, Xinyuan; Romeis, Jörg; Li, Yunhe; Peng, Yufa

    2016-03-23

    Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) is a prevalent pollen consumer in corn fields and is therefore exposed to insecticidal proteins contained in the pollen of insect-resistant transgenic corn cultivars expressing Cry proteins derived from Bacillus thuringiensis (Bt). In the present study, the potential effect of Cry1Ab/2Aj- or Cry1Ac-containing transgenic Bt corn pollen on the fitness of P. japonica larvae was evaluated. The results show that the larval developmental time was significantly shorter when P. japonica larvae were fed pollen from Bt corn cultivars rather than control pollen but that pupation rate, eclosion rate, and adult fresh weight were not significantly affected. In the feeding experiments, the stability of the Cry proteins in the food sources was confirmed. When Bt corn pollen passed through the gut of P. japonica, 23% of Cry1Ab/2Aj was digested. The results demonstrate that consumption of Bt corn pollen containing Cry1Ab/2Aj or Cry1Ac has no detrimental effect on P. japonica larvae; the shortened developmental time of larvae that consumed these proteins was likely attributable to unknown differences in the nutritional composition between the Bt-transgenic and control corn pollen.

  13. Effect of crop plants on fitness costs associated with resistance to Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in cabbage loopers.

    PubMed

    Wang, Ran; Tetreau, Guillaume; Wang, Ping

    2016-02-12

    Fitness costs associated with resistance to Bacillus thuringiensis (Bt) toxins critically impact the development of resistance in insect populations. In this study, the fitness costs in Trichoplusia ni strains associated with two genetically independent resistance mechanisms to Bt toxins Cry1Ac and Cry2Ab, individually and in combination, on four crop plants (cabbage, cotton, tobacco and tomato) were analyzed, in comparison with their near-isogenic susceptible strain. The net reproductive rate (R0) and intrinsic rate of increase (r) of the T. ni strains, regardless of their resistance traits, were strongly affected by the host plants. The ABCC2 gene-linked mechanism of Cry1Ac resistance was associated with relatively low fitness costs, while the Cry2Ab resistance mechanism was associated with higher fitness costs. The fitness costs in the presence of both resistance mechanisms in T. ni appeared to be non-additive. The relative fitness of Bt-resistant T. ni depended on the specific resistance mechanisms as well as host plants. In addition to difference in survivorship and fecundity, an asynchrony of adult emergence was observed among T. ni with different resistance mechanisms and on different host plants. Therefore, mechanisms of resistance and host plants available in the field are both important factors affecting development of Bt resistance in insects.

  14. Effect of crop plants on fitness costs associated with resistance to Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in cabbage loopers

    PubMed Central

    Wang, Ran; Tetreau, Guillaume; Wang, Ping

    2016-01-01

    Fitness costs associated with resistance to Bacillus thuringiensis (Bt) toxins critically impact the development of resistance in insect populations. In this study, the fitness costs in Trichoplusia ni strains associated with two genetically independent resistance mechanisms to Bt toxins Cry1Ac and Cry2Ab, individually and in combination, on four crop plants (cabbage, cotton, tobacco and tomato) were analyzed, in comparison with their near-isogenic susceptible strain. The net reproductive rate (R0) and intrinsic rate of increase (r) of the T. ni strains, regardless of their resistance traits, were strongly affected by the host plants. The ABCC2 gene-linked mechanism of Cry1Ac resistance was associated with relatively low fitness costs, while the Cry2Ab resistance mechanism was associated with higher fitness costs. The fitness costs in the presence of both resistance mechanisms in T. ni appeared to be non-additive. The relative fitness of Bt-resistant T. ni depended on the specific resistance mechanisms as well as host plants. In addition to difference in survivorship and fecundity, an asynchrony of adult emergence was observed among T. ni with different resistance mechanisms and on different host plants. Therefore, mechanisms of resistance and host plants available in the field are both important factors affecting development of Bt resistance in insects. PMID:26868936

  15. Ingestion of Bt corn pollen containing Cry1Ab/2Aj or Cry1Ac does not harm Propylea japonica larvae

    PubMed Central

    Liu, Yanmin; Liu, Qingsong; Wang, Yanan; Chen, Xiuping; Song, Xinyuan; Romeis, Jörg; Li, Yunhe; Peng, Yufa

    2016-01-01

    Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) is a prevalent pollen consumer in corn fields and is therefore exposed to insecticidal proteins contained in the pollen of insect-resistant transgenic corn cultivars expressing Cry proteins derived from Bacillus thuringiensis (Bt). In the present study, the potential effect of Cry1Ab/2Aj- or Cry1Ac-containing transgenic Bt corn pollen on the fitness of P. japonica larvae was evaluated. The results show that the larval developmental time was significantly shorter when P. japonica larvae were fed pollen from Bt corn cultivars rather than control pollen but that pupation rate, eclosion rate, and adult fresh weight were not significantly affected. In the feeding experiments, the stability of the Cry proteins in the food sources was confirmed. When Bt corn pollen passed through the gut of P. japonica, 23% of Cry1Ab/2Aj was digested. The results demonstrate that consumption of Bt corn pollen containing Cry1Ab/2Aj or Cry1Ac has no detrimental effect on P. japonica larvae; the shortened developmental time of larvae that consumed these proteins was likely attributable to unknown differences in the nutritional composition between the Bt-transgenic and control corn pollen. PMID:27005950

  16. Transgenic Cabbage Expressing Cry1Ac1 Does Not Affect the Survival and Growth of the Wolf Spider, Pardosa astrigera L. Koch (Araneae: Lycosidae).

    PubMed

    Kim, Young-Joong; Lee, Joon-Ho; Harn, Chee Hark; Kim, Chang-Gi

    2016-01-01

    Both herbivores that consume transgenic crops and their predators can be exposed to insecticidal proteins expressed in those crops. We conducted a tritrophic bioassay to evaluate the ecotoxicological impacts that Bt cabbage (Brassica oleracea var. capitata) expressing Cry1Ac1 protein might have on the wolf spider (Pardosa astrigera), a non-target generalist predator. Enzyme-Linked Immunosorbent Assays indicated that protein levels were 4.61 ng g(-1) dry weight in fruit flies (Drosophila melanogaster) fed with the transgenic cabbage and 1.86 ng g(-1) dry weight in the wolf spiders that preyed upon them. We also compared the life history traits of spiders collected from Bt versus non-Bt cabbage and found no significant differences in their growth, survival, and developmental rates. Because Bt cabbage did not affect the growth of fruit flies, we conclude that any indirect effects that this crop had on the wolf spider were probably not mediated by prey quality. Therefore, exposure to Cry1Ac1 protein when feeding upon prey containing that substance from transgenic cabbage has only a negligible influence on those non-target predatory spiders.

  17. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  18. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  19. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  20. 40 CFR 180.1108 - Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Delta endotoxin of Bacillus... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens; exemption from the requirement of a tolerance. The delta...

  1. Bioactive and total endotoxins in atmospheric aerosols in the Pearl River Delta region, China

    NASA Astrophysics Data System (ADS)

    Cheng, Jessica Y. W.; Hui, Esther L. C.; Lau, Arthur P. S.

    2012-02-01

    Endotoxin, a toxic and pyrogenic substance in gram-negative bacteria in atmospheric aerosols was measured over a period of one year at Nansha, Guangzhou and Hong Kong in the Pearl River Delta region, China. Atmospheric aerosols were collected by high-volume samplers. The bioactive endotoxin levels in the samples were determined using the Limulus Amebocyte Lysate (LAL) assay after extraction with pyrogen-free water while the total endotoxin levels were measured by quantifying the biomarker, 3-hydroxy fatty acids (3-OHFAs) with GC-MS. Results showed that there was no significant difference (0.19 < p < 0.81) in the bioactive endotoxin level in PM 10 among sites (average concentrations ranged from 0.34 to 0.39 EU m -3). However, Hong Kong showed a significantly lower ( p < 0.05) total endotoxin level in PM 10 (average of 17.4 ng m -3) compared with Nansha's 29.4 ng m -3 and Guangzhou's 32.7 ng m -3. The bioactive endotoxins were found to be associated with the coarse mode (PM 2.5-10) of the particulates of natural origins while the total endotoxins were associated more with the fine mode (PM 2.5) of the particulates of anthropogenic origins. When normalized with particulate mass, the endotoxin loading is much higher in summer as a result of the increased growth of the bacteria when climatic conditions are favorable. The chemically determined total endotoxins were 3-4 orders of magnitude higher than the bioactive endotoxins quantified using the LAL assay. Correlation analyses between the bioactive endotoxins and 3-OHFAs with different carbon length were analyzed. Results showed that the correlations detected vary among sites and particulate sizes. Although no generalization between the total and bioactive endotoxins can be drawn from the study, the levels reported in this study suggests that the discrepancies between the two measurement approaches, and the bioactive potential of 3-OHFAs with individual carbon chains deserve further investigation.

  2. Effects of water management practices on residue decomposition and degradation of Cry1Ac protein from crop-wild Bt rice hybrids and parental lines during winter fallow season.

    PubMed

    Xiao, Manqiu; Dong, Shanshan; Li, Zhaolei; Tang, Xu; Chen, Yi; Yang, Shengmao; Wu, Chunyan; Ouyang, Dongxin; Fang, Changming; Song, Zhiping

    2015-12-01

    Rice is the staple diet of over half of the world's population and Bacillus thuringiensis (Bt) rice expressing insecticidal Cry proteins is ready for deployment. An assessment of the potential impact of Bt rice on the soil ecosystem under varied field management practices is urgently required. We used litter bags to assess the residue (leaves, stems and roots) decomposition dynamics of two transgenic rice lines (Kefeng6 and Kefeng8) containing stacked genes from Bt and sck (a modified CpTI gene encoding a cowpea trypsin inhibitor) (Bt/CpTI), a non-transgenic rice near-isoline (Minghui86), wild rice (Oryza rufipogon) and crop-wild Bt rice hybrid under contrasting conditions (drainage or continuous flooding) in the field. No significant difference was detected in the remaining mass, total C and total N among cultivars under aerobic conditions, whereas significant differences in the remaining mass and total C were detected between Kefeng6 and Kefeng8 and Minghui86 under the flooded condition. A higher decomposition rate constant (km) was measured under the flooded condition compared with the aerobic condition for leaf residues, whereas the reverse was observed for root residues. The enzyme-linked immunosorbent assay (ELISA), which was used to monitor the changes in the Cry1Ac protein in Bt rice residues, indicated that (1) the degradation of the Cry1Ac protein under both conditions best fit first-order kinetics, and the predicted DT50 (50% degradation time) of the Cry1Ac protein ranged from 3.6 to 32.5 days; (2) the Cry1Ac protein in the residue degraded relatively faster under aerobic conditions; and (3) by the end of the study (~154 days), the protein was present at a low concentration in the remaining residues under both conditions. The degradation rate constant was negatively correlated with the initial carbon content and positively correlated with the initial Cry1Ac protein concentration, but it was only correlated with the mass decomposition rate constants under

  3. [The effects of transgenic Cry1Ac+Cry2Ab cotton on cotton bollworm control and functional response of predators on whitefly].

    PubMed

    Junyu, Luo; Shuai, Zhang; Limin, L V; Chunyi, Wang; Xiangzhen, Zhu; Jinjie, Cuil

    2015-06-01

    In this study, we detected and clarified the roles of transgenic Cry1Ac+Cry2Ab cotton "639020" in controlling cotton bollworm (Helicoverpa armigera) during critical periods of bud stage (second generation of bollworm), flowering stage (third generation of bollworm) and bolling stage (fourth generation of bollworm) as well as the influences of 639020 cotton on functional response of the main predators (Chrysopa sinica larvae, Propylaea japonica, Orius and Erigonidium graminicola ) on whitefly using transgenic Cry1Ac cotton "CCRI41" and conventional cotton "CCRI49" as the control. Our results showed that the 639020 cotton well controlled the second and third generation of bollworm, and the level of insect resistance increased by 52.85% and 16.22% separately compared with that of CCRI41, with a significant effect on the second generation of bollworm. Moreover, the number of bollworm eggs in 639020 cotton field was lower than that in CCRI41 and CCRI49 cotton fields (except the second generation of bollworm) during the cotton bud, flowering and bolling stages. Although the number of bollworm larvae in 639020 cotton field was significantly lower than that in CCRI49 field, and both under the controlling index, it has no significant difference compared with that in CCRI41 cotton field. There were also no obvious changes in predator functions of Chrysopa sinica, Propylaea japonica, Orius and Erigonidium graminicola on bemisia tabaci between 639020, CCRI41 and CCRI49 cotton filed. This study evaluated the safety of new transgenic cotton on environment, anti-insect activity of exogenous gene and the safety of production and application prospect.

  4. Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant—Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation

    PubMed Central

    Mezzomo, Bélin Poletto; Miranda-Vilela, Ana Luisa; Grisolia, Cesar Koppe

    2015-01-01

    In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt) Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o.) or with a single intraperitoneal (i.p.) injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed). This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling. PMID:26690217

  5. Expression of Cry1Ac in transgenic tobacco plants under the control of a wound-inducible promoter (AoPR1) isolated from Asparagus officinalis to control Heliothis virescens and Manduca sexta.

    PubMed

    Gulbitti-Onarici, Selma; Zaidi, Mohsin Abbas; Taga, Ibrahim; Ozcan, Sebahattin; Altosaar, Illimar

    2009-07-01

    Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6-72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.

  6. Field Performance of Bt Eggplants (Solanum melongena L.) in the Philippines: Cry1Ac Expression and Control of the Eggplant Fruit and Shoot Borer (Leucinodes orbonalis Guenée).

    PubMed

    Hautea, Desiree M; Taylo, Lourdes D; Masanga, Anna Pauleen L; Sison, Maria Luz J; Narciso, Josefina O; Quilloy, Reynaldo B; Hautea, Randy A; Shotkoski, Frank A; Shelton, Anthony M

    2016-01-01

    Plants expressing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling insect pests in maize and cotton globally. However, there are few Bt vegetable crops. Eggplant (Solanum melongena) is a popular vegetable grown throughout Asia that is heavily treated with insecticides to control the eggplant fruit and shoot borer, Leucinodes orbonalis (EFSB). Herein we provide the first publicly available data on field performance in Asia of eggplant engineered to produce the Cry1Ac protein. Replicated field trials with five Bt eggplant open-pollinated (OP) lines from transformation event EE-1 and their non-Bt comparators were conducted over three cropping seasons in the Philippines from 2010-2012. Field trials documented levels of Cry1Ac protein expressed in plants and evaluated their efficacy against the primary target pest, EFSB. Cry1Ac concentrations ranged from 0.75-24.7 ppm dry weight with the highest in the terminal leaves (or shoots) and the lowest in the roots. Cry1Ac levels significantly increased from the vegetative to the reproductive stage. Bt eggplant lines demonstrated excellent control of EFSB. Pairwise analysis of means detected highly significant differences between Bt eggplant lines and their non-Bt comparators for all field efficacy parameters tested. Bt eggplant lines demonstrated high levels of control of EFSB shoot damage (98.6-100%) and fruit damage (98.1-99.7%) and reduced EFSB larval infestation (95.8-99.3%) under the most severe pest pressure during trial 2. Moths that emerged from larvae collected from Bt plants in the field and reared in their Bt eggplant hosts did not produce viable eggs or offspring. These results demonstrate that Bt eggplant lines containing Cry1Ac event EE-1 provide outstanding control of EFSB and can dramatically reduce the need for conventional insecticides.

  7. Field Performance of Bt Eggplants (Solanum melongena L.) in the Philippines: Cry1Ac Expression and Control of the Eggplant Fruit and Shoot Borer (Leucinodes orbonalis Guenée)

    PubMed Central

    Hautea, Desiree M.; Taylo, Lourdes D.; Masanga, Anna Pauleen L.; Sison, Maria Luz J.; Narciso, Josefina O.; Quilloy, Reynaldo B.; Hautea, Randy A.; Shotkoski, Frank A.; Shelton, Anthony M.

    2016-01-01

    Plants expressing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling insect pests in maize and cotton globally. However, there are few Bt vegetable crops. Eggplant (Solanum melongena) is a popular vegetable grown throughout Asia that is heavily treated with insecticides to control the eggplant fruit and shoot borer, Leucinodes orbonalis (EFSB). Herein we provide the first publicly available data on field performance in Asia of eggplant engineered to produce the Cry1Ac protein. Replicated field trials with five Bt eggplant open-pollinated (OP) lines from transformation event EE-1 and their non-Bt comparators were conducted over three cropping seasons in the Philippines from 2010–2012. Field trials documented levels of Cry1Ac protein expressed in plants and evaluated their efficacy against the primary target pest, EFSB. Cry1Ac concentrations ranged from 0.75–24.7 ppm dry weight with the highest in the terminal leaves (or shoots) and the lowest in the roots. Cry1Ac levels significantly increased from the vegetative to the reproductive stage. Bt eggplant lines demonstrated excellent control of EFSB. Pairwise analysis of means detected highly significant differences between Bt eggplant lines and their non-Bt comparators for all field efficacy parameters tested. Bt eggplant lines demonstrated high levels of control of EFSB shoot damage (98.6–100%) and fruit damage (98.1–99.7%) and reduced EFSB larval infestation (95.8–99.3%) under the most severe pest pressure during trial 2. Moths that emerged from larvae collected from Bt plants in the field and reared in their Bt eggplant hosts did not produce viable eggs or offspring. These results demonstrate that Bt eggplant lines containing Cry1Ac event EE-1 provide outstanding control of EFSB and can dramatically reduce the need for conventional insecticides. PMID:27322533

  8. Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria

    PubMed Central

    Downing, Katrina J.; Leslie, Graeme; Thomson, Jennifer A.

    2000-01-01

    The cry1Ac7 gene of Bacillus thuringiensis strain 234, showing activity against the sugarcane borer Eldana saccharina, was cloned under the control of the tac promoter. The fusion was introduced into the broad-host-range plasmid pKT240 and the integration vector pJFF350 and without the tac promoter into the broad-host-range plasmids pML122 and pKmM0. These plasmids were introduced into a Pseudomonas fluorescens strain isolated from the phylloplane of sugarcane and the endophytic bacterium Herbaspirillum seropedicae found in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the tac promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nmr promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher mortality of larvae than did P. fluorescens 14::tox. An increased toxic effect was observed when P. fluorescens 14::ptac-tox was combined with P. fluorescens carrying the Serratia marcescens chitinase gene chiA, under the control of the tac promoter, integrated into the chromosome. PMID:10877771

  9. Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

    PubMed

    Bel, Yolanda; Sheets, Joel J; Tan, Sek Yee; Narva, Kenneth E; Escriche, Baltasar

    2017-06-01

    Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa (125)-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated

  10. Creation of Bt rice expressing a fusion protein of Cry1Ac and Cry1I-like using a green tissue-specific promoter.

    PubMed

    Yang, Yong-Yi; Mei, Feng; Zhang, Wei; Shen, Zhicheng; Fang, Jun

    2014-08-01

    The insecticidal genes from Bacillus thuringiensis Berliner (Bt) have long been successfully used for development of insect-resistant rice. However, commercial planting of Bt rice has been delayed by the concern over food safety, although no scientific evidence is ever found to justify the concern. To address this safety concern, we developed a transgenic insect-resistant rice line using a green tissue promoter to minimize the Bt protein expression in the rice seeds. The Bt protein expressed in the rice was a fusion protein of two different Bt toxins, Cry1Ac and Cry1I-like protein. The fusion of the two toxins may be helpful to delay the development of insect resistance to Bt rice. Laboratory and field bioassays demonstrated that the transgenic rice plants created by this study were highly active against the rice leaf folder Cnaphalocrocis medinalis (Guenée) and the striped stem borer Chilo suppressalis (Walker). Western analysis indicated that the fusion protein was specifically expressed in green tissues but not in seeds. Therefore, the transgenic rice created in this study should be useful to mitigate the food safety concern and to delay the development of insect resistance.

  11. Quantitative Analysis of Fitness Costs Associated with the Development of Resistance to the Bt Toxin Cry1Ac in Helicoverpa armigera

    PubMed Central

    Cao, Guangchun; Feng, Hongqiang; Guo, Fang; Wu, Kongming; Li, Xianchun; Liang, Gemei; Desneux, Nicolas

    2014-01-01

    Transgenic Bacillus thuringiensis (Bt) crops play an increasing role in pest control, and resistance management is a major issue in large-scale cultivation of Bt crops. The fitness cost of resistance in targeted pests is considered to be one of the main factors delaying resistance when using the refuge strategy. By comparing 10 resistant Helicoverpa armigera (Hubner) strains, showing various resistance levels to Bt toxin (Cry1Ac), to a susceptible strain, we showed an increasing fitness cost corresponding with increasing levels of resistance. The relationship between overall fitness cost C and the resistance ratio Rr could be described by C = 24.47/(1 + exp([1.57 - Log10Rr]/0.2)). This model predicted that the maximum overall fitness cost would be ~24% (±5.22) in the strains with the highest resistance level. The overall fitness cost was closely linked to egg hatching rate, fecundity, emergence rate, larval survival rate, and developmental duration of adults. Among fitness components measured, fecundity was the most sensitive trait linked to the resistance selection. To integrate the results into simulation models would be valuable in evaluating how variation in fitness cost may influence the development of resistance in pest populations, thus helping to develop enhanced refuge strategies. PMID:25005122

  12. Comparative Proteomic and Nutritional Composition Analysis of Independent Transgenic Pigeon Pea Seeds Harboring cry1AcF and cry2Aa Genes and Their Nontransgenic Counterparts.

    PubMed

    Mishra, Pragya; Singh, Shweta; Rathinam, Maniraj; Nandiganti, Muralimohan; Ram Kumar, Nikhil; Thangaraj, Arulprakash; Thimmegowda, Vinutha; Krishnan, Veda; Mishra, Vagish; Jain, Neha; Rai, Vandna; Pattanayak, Debasis; Sreevathsa, Rohini

    2017-02-22

    Safety assessment of genetically modified plants is an important aspect prior to deregulation. Demonstration of substantial equivalence of the transgenics compared to their nontransgenic counterparts can be performed using different techniques at various molecular levels. The present study is a first-ever comprehensive evaluation of pigeon pea transgenics harboring two independent cry genes, cry2Aa and cry1AcF. The absence of unintended effects in the transgenic seed components was demonstrated by proteome and nutritional composition profiling. Analysis revealed that no significant differences were found in the various nutritional compositional analyses performed. Additionally, 2-DGE-based proteome analysis of the transgenic and nontransgenic seed protein revealed that there were no major changes in the protein profile, although a minor fold change in the expression of a few proteins was observed. Furthermore, the study also demonstrated that neither the integration of T-DNA nor the expression of the cry genes resulted in the production of unintended effects in the form of new toxins or allergens.

  13. Laboratory evaluation of transgenic Populus davidiana×Populus bolleana expressing Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3 genes against gypsy moth and fall webworm.

    PubMed

    Ding, Liping; Chen, Yajuan; Wei, Xiaoli; Ni, Mi; Zhang, Jiewei; Wang, Hongzhi; Zhu, Zhen; Wei, Jianhua

    2017-01-01

    Transgenic poplar lines 'Shanxin' (Populus davidiana×Populus bolleana) were generated via Agrobacterium-mediated transformation. The transgenic lines carried the expression cassettes of Cry1Ac + SCK, Cry1Ah3, and Cry9Aa3, respectively. The expression levels of the exogenous insect resistance genes in the transgenic lines were determined by Q-PCR and Western blot. Leaves of the transgenic lines were used for insect feeding bioassays on first instar larvae of the gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). At 5 d of feeding, the mean mortalities of larvae feeding on Cry1Ac + SCK and Cry1Ah3 transgenic poplars leaves were 97% and 91%, while mortality on Cry9Aa3 transgenic lines was about 49%. All gypsy moth and fall webworm larvae were killed in 7-9 days after feeding on leaves from Cry1Ac + SCK or Cry1Ah3 transgenic poplars, while all the fall webworm larvae were killed in 11 days and about 80% of gypsy moth larvae were dead in 14 days after feeding on those from Cry9Aa3 transgenic lines. It was concluded that the transgenic lines of Cry1Ac + SCK and Cry1Ah3 were highly toxic to larvae of both insect species while lines with Cry9Aa3 had lower toxicity,and H. cunea larvae are more sensitive to the insecticidal proteins compared to L. dispar. Transgenic poplar lines toxic to L. dispar and H. cunea could be used to provide Lepidoptera pest resistance to selected strains of poplar trees.

  14. Frequency of alleles conferring resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa punctigera (Lepidoptera: Noctuidae) from 2002 to 2006.

    PubMed

    Downes, S; Parker, T L; Mahon, R J

    2009-04-01

    Helicoverpa punctigera and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) are important pests of field and horticultural crops in Australia. The former is endemic to the continent, whereas the latter is also distributed in Africa and Asia. Although H. armigera rapidly developed resistance to virtually every group of insecticide used against it, there is only one report of resistance to an insecticide in H. punctigera. In 1996 the Australian cotton industry adopted Ingard, which expresses the Bacillus thuringiensis (Bt) toxin gene cry1Ac. In 2004/2005, Bollgard II (which expresses Cry1Ac and Cry2Ab) replaced Ingard and has subsequently been grown on 80% of the area planted to cotton, Gossypium hirsutum L. From 2002/2003 to 2006/2007, F2 screens were used to detect resistance to Cry1Ac or Cry2Ab. We detected no alleles conferring resistance to Cry1Ac; the frequency was < 0.0005 (n = 2,180 alleles), with a 95% credibility interval between 0 and 0.0014. However, during the same period, we detected alleles that confer resistance to Cry2Ab at a frequency of 0.0018 (n = 2,192 alleles), with a 95% credibility interval between 0.0005 and 0.0040. For both toxins, the experiment-wise detection probability was 94%, i.e., if there actually was a resistance allele in any tested lines, we would have detected it 94% of the time. The first isolation of Cry2Ab resistance in H. punctigera was before the widespread deployment of Bollgard II. This finding supports our published notion for H. armigera that alleles conferring resistance to Cry2Ab may be present at detectable frequencies in populations before selection by transgenic crops.

  15. Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

    PubMed Central

    Mohammed, S I; Johnson, D E; Aronson, A I

    1996-01-01

    Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins. PMID:8900008

  16. Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs.

    PubMed

    Debode, Frederic; Janssen, Eric; Bragard, Claude; Berben, Gilbert

    2017-08-01

    The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

  17. Capturing the interaction types of two Bt toxins Cry1Ac and Cry2Ab on suppressing the cotton bollworm by using multi-exponential equations.

    PubMed

    Shi, Pei-Jian; Wei, Ji-Zhen; Sandhu, Hardev S; Liang, Ge-Mei

    2016-08-01

    Transgenic crops are increasingly promoted for their practical effects on suppressing certain insect pests, but all transgenic crops are not equally successful. The insect pests can easily develop resistance against single Bacillus thuringiensis (Bt) toxin transgenic crops. Therefore, transgenic crops including two or more mixed Bt-toxins can solve this problem by delaying the resistance development and killing the majority of targeted pests before the evolution of resistance. It is important to test the controlling effects of transgenic crops including multiple mixed toxins on a particular insect pest. Previous research has checked the cross-resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against one susceptible and four resistant strains of cotton bollworm. The results showed that independence was the main interaction type between two toxins for the susceptible strain, whereas synergism was the main interaction type for any one resistant strain. However, the optimal combinations of two toxins were not obtained. In the present study, we developed two multi-exponential equations (namely bi- and tri-exponential equations) to describe the combination effects of two Bt toxins. Importantly, the equations can provide predictions of combination effects of different continuous concentrations of two toxins. We compared these two multi-exponential equations with the generalized linear model (GLM) in describing the combination effects, and found that the bi- and tri-exponential equations are better than GLM. Moreover, the bi-exponential equation can also provide the optimal dose combinations for two toxins. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  18. Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton.

    PubMed

    Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

    2017-01-01

    An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

  19. Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton

    PubMed Central

    Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

    2017-01-01

    An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels. PMID:28099508

  20. Mathematical relationships between spore concentrations, delta-endotoxin levels, and entomotoxicity of Bacillus thuringiensis preparations produced in different fermentation media.

    PubMed

    Vu, Khanh Dang; Tyagi, R D; Surampalli, R Y; Valéro, J R

    2012-11-01

    Mathematic relationships between spore concentrations, delta-endotoxin concentrations and entomotoxicity (Tx) of Bacillus thuringiensis var. kurstaki HD-1 (Btk HD-1) preparations produced in six different media were analysed. The relationship between delta-endotoxin and spore concentration and SpTx-spore (specific Tx per 1000 spore) and spore concentration produced in the different media (starch industry wastewater (SIW) with total solids (TS) concentration of 15g/L, SIW with TS of 30g/L, SIW supplemented with 0.2% (w/v) colloidal chitin, SIW supplemented with 1.25% (w/v) cornstarch and 0.2% (v/v) Tween 80, secondary sludge, and semi-synthetic medium) strictly followed the Power law. Tx and delta endotoxin concentration followed the exponential relation whereas a definite relation between Tx and spore concentration could not be established. Spore and delta-endotoxin produced at the early time (12h) during fermentation might be more toxic than those produced during latter period of fermentation irrespective of media used. Tx and delta-endotoxin concentration exhibited a semi-log linear relationship. Based on these findings, delta-endotoxin concentration can be determined rapidly to monitor the progress of the biopesticide production process. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.

    PubMed

    Tanaka, Shiho; Miyamoto, Kazuhisa; Noda, Hiroaki; Endo, Haruka; Kikuta, Shingo; Sato, Ryoichi

    2016-04-01

    In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.

  2. Haematology, blood biochemistry and tissue histopathology of lambs maintained on diets containing an insect controlling protein (Cry1Ac) in Bt-cottonseed.

    PubMed

    Tripathi, M K; Mondal, D; Somvanshi, R; Karim, S A

    2011-10-01

    This experiment assessed the effect of feeding genetically modified cottonseed (Bt) containing an insect controlling protein (Cry1Ac) on haematology, blood biochemistry and histopathology of lambs. Haemato-biochemicals were estimated at periodic intervals, and histopathology at termination of experiment. Thirty three weaner lambs were fed a composite feed mixture (CFM) ad libitum individually, in three groups for 123 days. The isonitrogenous CFM had roughage (Perl Millet Stover) and concentrate ratio of 350:650. Diet fed to control lambs contained groundnut oil meal as protein source while other two groups received diet containing either whole cottonseed (N-Bt) or Bt-cottonseed (Bt-CS). Daily feed intake and average daily gain were similar among lambs of three groups. Lambs fed N-Bt diet had higher (p < 0.05) serum protein and globulin compared to control and Bt diets, while albumin content was higher (p = 0.018) in Bt diet fed lambs. Serum urea and creatinin content, alkaline phosphatase (ALP) and serum glutamate pyruvic transaminase (SGPT) activities were not different among lamb groups, while urea and creatinin content and ALP activities increased linearly (p < 0.001) with increased feeding period. Blood haemoglobin (Hb), haematocrit (Hc), white blood cells (WBC), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) ranged from 11.1% to 11.2%, 31.8% to 32.8%, 7.0 to 8.3 (× 10(3) /μl), 19.1 to 22.5 fl and 33.2% to 35.5%, respectively, were similar among lamb groups. Erythrocyte sedimentation rate (ESR) (p = 0.008) and red blood cell counts (p = 0.006) were higher in Bt diet fed lambs. Control and N-Bt diet fed lambs had mild fatty infiltration in liver and/or micro-calculi in renal cortex, and such lesions were not seen in Bt diet fed lambs. Growth, haemato-biochemical and histopathology did not change by Bt-CS feeding in growing lamb. However, before recommending the use of Bt-CS in routine feed formulations prolonged feeding

  3. Down-regulation of aminopeptidase N and ABC transporter subfamily G transcripts in Cry1Ab and Cry1Ac resistant Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae)

    PubMed Central

    Zhang, Tiantao; Coates, Brad S.; Wang, Yueqin; Wang, Yidong; Bai, Shuxiong; Wang, Zhenying; He, Kanglai

    2017-01-01

    The Asian corn borer (ACB), Ostrinia furnacalis (Lepidoptera: Crambidae), is a highly destructive pest of cultivated maize throughout East Asia. Bacillus thuringiensis (Bt) crystalline protein (Cry) toxins cause mortality by a mechanism involving pore formation or signal transduction following toxin binding to receptors along the midgut lumen of susceptible insects, but this mechanism and mutations therein that lead to resistance are not fully understood. In the current study, quantitative comparisons were made among midgut expressed transcripts from O. furnacalis susceptible (ACB-BtS) and laboratory selected strains resistant to Cry1Ab (ACB-AbR) and Cry1Ac toxins (ACB-AcR) when feeding on non-Bt diet. From a combined de novo transcriptome assembly of 83,370 transcripts, ORFs of ≥ 100 amino acids were predicted and annotated for 28,940 unique isoforms derived from 12,288 transcripts. Transcriptome-wide expression estimated from RNA-seq read depths predicted significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as a putative ATP binding cassette transporter group G (abcg) gene in both ACB-AbR and -AcR (log2[fold-change] ≥ 1.36; P < 0.0001). The transcripts that were most highly differentially regulated in both ACB-AbR and -AcR compared to ACB-BtS (log2[fold-change] ≥ 2.0; P < 0.0001) included up- and down-regulation of serine proteases, storage proteins and cytochrome P450 monooxygenases, as well as up-regulation of genes with predicted transport function. This study predicted the significant down-regulation of transcripts for previously known Bt resistance genes, aminopeptidase N1 (apn1) and apn3, as well as abccg gene in both ACB-AbR and -AcR. These data are important for the understanding of systemic differences between Bt resistant and susceptible genotypes. PMID:28808417

  4. Development of a Triple Gene Cry1Ac-Cry2Ab-EPSPS Construct and Its Expression in Nicotiana benthamiana for Insect Resistance and Herbicide Tolerance in Plants

    PubMed Central

    Naqvi, Rubab Z.; Asif, Muhammad; Saeed, Muhammad; Asad, Shaheen; Khatoon, Asia; Amin, Imran; Mukhtar, Zahid; Bashir, Aftab; Mansoor, Shahid

    2017-01-01

    Insect pest complex, cotton leaf curl disease and weeds pose major threat to crop production worldwide, including Pakistan. To address these problems, in the present study a triple gene construct harboring Cry1Ac, Cry2Ab, and EPSPS cassettes has been developed for plant specifically in cotton transformation against lepidopteron insect-pests and weeds. Nicotiana benthamiana (tobacco) was used as a model system for characterization of this triple gene construct. The construct has been assembled in plant expression vector and transformed in N. benthamiana. In six transgenic tobacco lines the integration of Cry1Ac-Cry2Ab-EPSPS in tobacco genome was checked by PCR, while successful protein expression of all the three genes was confirmed through immunostrip assay. Efficacy of Cry1Ac and Cry2Ab was evaluated through insect bioassay using armyworm (Spodoptera littoralis). These transgenic tobacco plants showed significant insect mortality as compared to control plants during insect bioassay. Three out of six tested transgenic lines L3, L5, and L9 exhibited 100% mortality of armyworm, while three other lines L1, L10, and L7 showed 86, 80, and 40% mortality, respectively. This construct can readily be used with confidence to transform cotton and other crops for the development of insect resistant and herbicide tolerant transgenic plants. The transgenic crop plants developed using this triple gene construct will provide an excellent germplasm resource for the breeders to improve their efficiency in developing stable homozygous lines as all the three genes being in a single T-DNA border will inherit together. PMID:28174591

  5. Development of a Triple Gene Cry1Ac-Cry2Ab-EPSPS Construct and Its Expression in Nicotiana benthamiana for Insect Resistance and Herbicide Tolerance in Plants.

    PubMed

    Naqvi, Rubab Z; Asif, Muhammad; Saeed, Muhammad; Asad, Shaheen; Khatoon, Asia; Amin, Imran; Mukhtar, Zahid; Bashir, Aftab; Mansoor, Shahid

    2017-01-01

    Insect pest complex, cotton leaf curl disease and weeds pose major threat to crop production worldwide, including Pakistan. To address these problems, in the present study a triple gene construct harboring Cry1Ac, Cry2Ab, and EPSPS cassettes has been developed for plant specifically in cotton transformation against lepidopteron insect-pests and weeds. Nicotiana benthamiana (tobacco) was used as a model system for characterization of this triple gene construct. The construct has been assembled in plant expression vector and transformed in N. benthamiana. In six transgenic tobacco lines the integration of Cry1Ac-Cry2Ab-EPSPS in tobacco genome was checked by PCR, while successful protein expression of all the three genes was confirmed through immunostrip assay. Efficacy of Cry1Ac and Cry2Ab was evaluated through insect bioassay using armyworm (Spodoptera littoralis). These transgenic tobacco plants showed significant insect mortality as compared to control plants during insect bioassay. Three out of six tested transgenic lines L3, L5, and L9 exhibited 100% mortality of armyworm, while three other lines L1, L10, and L7 showed 86, 80, and 40% mortality, respectively. This construct can readily be used with confidence to transform cotton and other crops for the development of insect resistant and herbicide tolerant transgenic plants. The transgenic crop plants developed using this triple gene construct will provide an excellent germplasm resource for the breeders to improve their efficiency in developing stable homozygous lines as all the three genes being in a single T-DNA border will inherit together.

  6. A Spodoptera exigua cadherin serves as a putative receptor for Bacillus thuringiensis Cry1Ca toxin and shows differential enhancement of Cry1Ca and Cry1Ac toxicity.

    PubMed

    Ren, Xiang-Liang; Chen, Rui-Rui; Zhang, Ying; Ma, Yan; Cui, Jin-Jie; Han, Zhao-Jun; Mu, Li-Li; Li, Guo-Qing

    2013-09-01

    Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca.

  7. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda.

    PubMed

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with (125)I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

  8. A Spodoptera exigua Cadherin Serves as a Putative Receptor for Bacillus thuringiensis Cry1Ca Toxin and Shows Differential Enhancement of Cry1Ca and Cry1Ac Toxicity

    PubMed Central

    Ren, Xiang-Liang; Chen, Rui-Rui; Zhang, Ying; Ma, Yan; Cui, Jin-Jie; Han, Zhao-Jun; Mu, Li-Li

    2013-01-01

    Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca. PMID:23835184

  9. The construction of Bacillus thuringiensis strains expressing novel entomocidal delta-endotoxin combinations.

    PubMed Central

    Crickmore, N; Nicholls, C; Earp, D J; Hodgman, T C; Ellar, D J

    1990-01-01

    Using our recently reported method of electroporation to transform Bacillus thuringiensis [Bone & Ellar (1989) FEMS Microbiol. Lett. 58, 171-178], cloned B. thuringiensis entomocidal delta-endotoxin genes have been introduced into several native B. thuringiensis strains. In many cases the resulting transformants expressed both their native toxins and the cloned toxin, producing strains with broader toxicity spectra. The introduction of the var. tenebrionis toxin gene into B. thuringiensis var. israelensis resulted in a strain with activity against Pieris brassicae (cabbage white butterfly), an activity which neither parent strain possesses. We discuss further the possibility of synergism and also the problems associated with introducing cloned DNA by this method. PMID:2168699

  10. Shared Midgut Binding Sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests, Ostrinia nubilalis and Spodoptera frugiperda

    PubMed Central

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with 125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case. PMID:23861865

  11. Growth, sporulation, delta-endotoxins synthesis, and toxicity during culture of bacillus thuringiensis H14.

    PubMed

    Sarrafzadeh, Mohammad H; Guiraud, Joseph P; Lagneau, Christophe; Gaven, Bruno; Carron, Alexandre; Navarro, Jean-Marie

    2005-08-01

    Growth, sporulation, synthesis of delta-endotoxins, and toxicity against the larvae of Aedes aegypti and Culex pipiens were studied during fermentation of Bacillus thuringiensis H14 in a 20-L fermentor. Measurements of optical density and dielectric permittivity for biomass determination suggest a highly promising technique for on-line evaluation of sporulation. The synthesis of 65-, 25- and 130-kDa proteins started at 16, 18, and 23 h, respectively. These proteins were enriched in different ways until the end of culture (48 h). Toxicity in the course of sporulation was significantly different for the larvae of both mosquito species. Maximal activity against Ae. aegypti was obtained at the end of culture, whereas for Cx. pipiens, the sample at 38 h was the most active.

  12. RNA interference knockdown of aminopeptidase N genes decrease the susceptibility of Chilo suppressalis larvae to Cry1Ab/Cry1Ac and Cry1Ca-expressing transgenic rice.

    PubMed

    Qiu, Lin; Fan, Jinxing; Zhang, Boyao; Liu, Lang; Wang, Xiaoping; Lei, Chaoliang; Lin, Yongjun; Ma, Weihua

    2017-03-06

    Transgenic rice expressing Bacillus thuringiensis (Bt) Cry toxins are resistant to lepidopteran pests, such as Chilo suppressalis, a major insect pest of rice in Asia. Understanding how these toxins interact with their hosts is crucial to understanding their insecticidal action. In this study, knockdown of two aminopeptidase N genes (APN1 and APN2) by RNA interference resulted in decreased susceptibility of C. suppressalis larvae to the Bt rice varieties TT51 (Cry1Ab and Cry1Ac fusion genes) and T1C-19 (Cry1Ca), but not T2A-1 (Cry2Aa). This suggests that APN1 and APN2 are receptors for Cry1A and Cry1C toxins in C. suppressalis.

  13. Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by u.v. and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis.

    PubMed

    Ghribi, D; Zouari, N; Jaoua, S

    2004-01-01

    The present work aimed to obtain bioinsecticide over-producing mutants through classical mutagenesis of vegetative cells of Bacillus thuringiensis (Bt) by using u.v. and nitrous acid, and to evidence the involvement of cell metabolism in delta-endotoxin synthesis. Vegetative cells of Bt were treated by nitrous acid (0.17 mg ml(-1)) or exposed to u.v. rays (emitted at a wave length of 240 nm). The isolated survivors were screened on the basis of the production of delta-endotoxins and biomass in glucose and/or in gruel-based media at two aeration conditions. Bioinsecticide over-producing mutants were obtained with high frequencies because random mutations were shown to affect cell metabolism at different pathways related to the regulation of delta-endotoxin synthesis. Classical mutagenesis of Bt cells lead to the isolation of a large variety of delta-endotoxin over-producing mutants that could be classified into six groups based on the location of the mutations, particularly in metabolism pathways and delta-endotoxin synthesis. High frequencies of delta-endotoxin over-producing mutants of Bt could be obtained through classical mutagenesis of vegetative cells. This should contribute to a significant reduction of production and utilization costs of Bt bioinsecticides.

  14. Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein.

    PubMed Central

    Ge, A Z; Shivarova, N I; Dean, D H

    1989-01-01

    Bacillus thuringiensis produces different types of insecticidal crystal proteins (ICPs) or delta-endotoxins. In an effort to identify the insect specificity of ICP toxins, two icp genes were cloned into the Escherichia coli expression vector pKK223-3, and bioassays were performed with purified crystals. The type A protein [from an icpA1, or 4.5-kilobase (kb) gene, from B. thuringiensis var. kurstaki HD-1] was found to be 400 times more active against Bombyx mori than type C protein (from an icpC73, or 6.6-kb gene, from B. thuringiensis var. kurstaki HD-244). The type C protein was 9 times more active against Trichoplusia ni than the type A protein, while both have similar activity against Manduca sexta. To locate the specificity domain of the type A protein for B. mori, site-directed mutagenesis was used to introduce or remove restriction enzyme sites, facilitating the exchange of regions of the two genes. The hybrid genes were overexpressed, and purified ICP was used in bioassays. The B. mori specificity domain for the ICP A toxin is located in the amino-terminal portion of the hypervariable region between amino acids 332 and 450. PMID:2542961

  15. Effects of feeding transgenic corn with mCry1Ac or maroACC gene to laying hens for 12 weeks on growth, egg quality and organ health.

    PubMed

    Zhong, R Q; Chen, L; Gao, L X; Zhang, L L; Yao, B; Yang, X G; Zhang, H F

    2016-08-01

    The objective of the present study was to investigate the effect of feeding two transgenic corn lines containing the mCry1Ac gene from Bacillus thuringiensis strain (BT-799) and the maroACC gene from Agrobacterium tumefaciens strain (CC-2), respectively, on growth, egg quality and organ health indicators. Expression of the mCry1Ac gene confers resistance to Pyrausta nubilalis and the maroACC gene confers tolerance to herbicides. Healthy hens (n=96 placed in cages; 3 hens/cage) were randomly assigned to one of four corn-soybean meal dietary treatments (8 cages/treatment) formulated with the following corn: non-transgenic near-isoline control corn (control), BT-799 corn, CC-2 corn and commercially available non-transgenic reference corn (reference). The experiment was divided into three 4-week phases (week 1 to 4, week 5 to 8 and week 9 to 12), during which hens were fed mash diets. Performance (BW, feed intake and egg production) and egg quality were determined. Following slaughter at the end of 12 weeks of feeding (n=8/treatment), carcass yield and organ weights (heart, liver, spleen, lung, kidneys, stomach and ovary) were recorded; organs and intestines were sampled for histological analysis. Analysis of serum biochemistry parameters to assess the liver and kidney function were performed. No differences in BW, egg production and production efficiency were observed between hens consuming the control diet and hens consuming the BT-799 or CC-2 diet. Haugh unit measures and egg component weights were similar between the control and test groups. Carcass yield was not affected by the diet treatment. Similar organosomatic indices and serum parameters did not indicate the characteristics of organ dysfunction. All observed values of the BT-799 and CC-2 groups were within the calculated tolerance intervals. This research indicates that the performance, egg quality, organ health and carcass yield of laying hens fed diets containing the BT-799 or CC-2 corn line were similar

  16. Bollgard II cotton: compositional analysis and feeding studies of cottonseed from insect-protected cotton (Gossypium hirsutum L.) producing the Cry1Ac and Cry2Ab2 proteins.

    PubMed

    Hamilton, Kathryn A; Pyla, Paul D; Breeze, Matthew; Olson, Tammy; Li, Menghe; Robinson, Edwin; Gallagher, Sean P; Sorbet, Roy; Chen, Yin

    2004-11-17

    Bollgard II cotton event 15985 producing the Cry1Ac and Cry2Ab2 proteins has been developed by genetic modification to broaden the spectrum of insects to which the plant is tolerant and to provide an insect resistance management tool to impede the onset of resistance. The purpose of this study was to evaluate the composition and nutrition of Bollgard II cotton, relative to the use for food and animal feed, compared to that of conventional cotton varieties. Compositional analyses were conducted to measure proximate, fiber, amino acid, fatty acid, gossypol, and mineral contents of cottonseed from a total of 14 U.S. field sites over two years. Compositional analysis results showed that the cottonseed and cottonseed oil from Bollgard II cotton were comparable in their composition to those of the conventional control cotton line and other commercial varieties. The composition data are supported by nutritional safety studies conducted with dairy cows, catfish, and quail. Results from these studies showed that Bollgard II performed similarly to the conventional control cotton varieties. These data demonstrate that Bollgard II cotton is compositionally and nutritionally equivalent to conventional cotton varieties. These data support the conclusion that Bollgard II cotton is as safe and nutritious as conventional cotton for food and feed use.

  17. [Effect of delta-endotoxin of Bacillus thuringiensis israëlensis on biochemical functional relationships in Diptera Aedes aegypti].

    PubMed

    Bounias, M; Nizeyimana, B; Vago, C

    1986-01-01

    A study of the action of very low doses of the delta-endotoxin of Bacillus thuringiensis israëlensis on the larvae of Aedes aegypti (Diptera) gave evidence for peculiar properties of the dose/effect relations based on the variations of the regression slopes of functional relationships between the pairs cysteine/serine and fatty acids/histidine, by contrast with the pair fatty acids/glucose which exhibited a classical shaped relation. This indicates "crypto-toxicological" processes, not lethal by themselves and without pathological symptoms, but able to initiate "pathogen-chaining" mechanisms leading, at the end, to a lethal secondary syndrome.

  18. Does transgenic Cry1Ac + CpTI cotton pollen affect hypopharyngeal gland development and midgut proteolytic enzyme activity in the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

    PubMed

    Han, Peng; Niu, Chang-Ying; Biondi, Antonio; Desneux, Nicolas

    2012-11-01

    The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

  19. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... expression plasmid and cloning vector genetic constructs. 180.1154 Section 180.1154 Protection of Environment... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus... cloning vector genetic constructs are exempt from the requirement of a tolerance when used in or on all...

  20. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... expression plasmid and cloning vector genetic constructs. 180.1154 Section 180.1154 Protection of Environment... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus... cloning vector genetic constructs are exempt from the requirement of a tolerance when used in or on all...

  1. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... expression plasmid and cloning vector genetic constructs. 180.1154 Section 180.1154 Protection of Environment... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus... cloning vector genetic constructs are exempt from the requirement of a tolerance when used in or on all...

  2. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... expression plasmid and cloning vector genetic constructs. 180.1154 Section 180.1154 Protection of Environment... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus... cloning vector genetic constructs are exempt from the requirement of a tolerance when used in or on all...

  3. 40 CFR 180.1154 - CryIA(c) and CryIC derived delta-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... expression plasmid and cloning vector genetic constructs. 180.1154 Section 180.1154 Protection of Environment... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of Bacillus... cloning vector genetic constructs are exempt from the requirement of a tolerance when used in or on all...

  4. The alpha-5 segment of Bacillus thuringiensis delta-endotoxin: in vitro activity, ion channel formation and molecular modelling.

    PubMed Central

    Gazit, E; Bach, D; Kerr, I D; Sansom, M S; Chejanovsky, N; Shai, Y

    1994-01-01

    A peptide with a sequence corresponding to the highly conserved alpha-5 segment of the Cry delta-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA [Gazit and Shai (1993) Biochemistry 32, 3429-3436]), was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of alpha-5 at either the N-terminal amino acid or the epsilon-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled alpha-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled alpha-5 peptides were protected from enzymic proteolysis. Functional characterization of alpha-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of alpha-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by alpha-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for alpha-5 in the toxic mechanism of delta-endotoxins, and assign alpha-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB delta-endotoxin in a loop between alpha-5 and alpha-6 is highly important for its larvicidal activity [Angsuthanasombat, Crickmore and Ellar (1993) FEMS

  5. Leaf morphology and ultrastructure responses to elevated O3 in transgenic Bt (cry1Ab/cry1Ac) rice and conventional rice under fully open-air field conditions.

    PubMed

    Li, Chunyan; Liu, Biao; Li, Chunhua; Zeng, Qing; Hao, Mingzhuo; Han, Zhengmin; Zhu, Jianguo; Li, Xiaogang; Shen, Wenjing

    2013-01-01

    Elevated tropospheric ozone severely affects not only yield but also the morphology, structure and physiological functions of plants. Because of concerns regarding the potential environmental risk of transgenic crops, it is important to monitor changes in transgenic insect-resistant rice under the projected high tropospheric ozone before its commercial release. Using a free-air concentration enrichment (FACE) system, we investigated the changes in leaf morphology and leaf ultrastructure of two rice varieties grown in plastic pots, transgenic Bt Shanyou 63 (Bt-SY63, carrying a fusion gene of cry1Ab and cry1Ac) and its non-transgenic counterpart (SY63), in elevated O3 (E-O3) versus ambient O3 (A-O3) after 64-DAS (Days after seeding), 85-DAS and 102-DAS. Our results indicated that E-O3 had no significant effects on leaf length, leaf width, leaf area, stomatal length and stomatal density for both Bt-SY63 and SY63. E-O3 increased the leaf thickness of Bt-SY63, but decreased that of SY63. O3 stress caused early swelling of the thylakoids of chloroplasts, a significant increase in the proportion of total plastoglobule area in the entire cell area (PCAP) and a significant decrease in the proportion of total starch grain area in the entire cell area (SCAP), suggesting that E-O3 accelerated the leaf senescence of the two rice genotypes. Compared with SY63, E-O3 caused early swelling of the thylakoids of chloroplasts and more substantial breakdown of chloroplasts in Bt-SY63. Our results suggest that the incorporation of cry1Ab/Ac into SY63 could induce unintentional changes in some parts of plant morphology and that O3 stress results in greater leaf damage to Bt-SY63 than to SY63, with the former coupled with higher O3 sensitivity in CCAP (the proportions of total chloroplast area in the entire cell area), PCAP and SCAP. This study provides valuable baseline information for the prospective commercial release of transgenic crops under the projected future climate.

  6. A protein complex from Choristoneura fumiferana gut-juice involved in the precipitation of delta-endotoxin from Bacillus thuringiensis subsp. sotto.

    PubMed

    Milne, R E; Pang, A S; Kaplan, H

    1995-12-01

    A 75 kDa protein from spruce budworm (Choristoneura fumiferana) gut-juice has been isolated and shown to cause a specific precipitation of the delta-endotoxin from Bacillus thuringiensis subsp. sotto. This 75 kDa protein, separated by either column chromatography or SDS-PAGE, caused precipitation of the sotto toxin in both agarose diffusion gels and the PAGE gels. The precipitation event leads to limited proteolysis of the toxin and loss of larval toxicity. SDS-PAGE analysis of the precipitated toxin indicates that proteolysis of the toxin is not a prerequisite for precipitation. The protein responsible for precipitation, exhibits elastase-like activity and appears to be a complex which partially dissociates during boiling in SDS-PAGE sample buffer. Gut-juice from gypsy moth, forest tent caterpillar and white mark tussock moth also precipitated delta-endotoxin, but silkworm gut-juice gave a much weaker response. These results provide further evidence that, in the larval gut, differential processing of delta-endotoxin may play a role in the expression of activity towards various insect larvae.

  7. Restriction map of the 125-kilobase plasmid of Bacillus thuringiensis subsp. israelensis carrying the genes that encode delta-endotoxins active against mosquito larvae.

    PubMed Central

    Ben-Dov, E; Einav, M; Peleg, N; Boussiba, S; Zaritsky, A

    1996-01-01

    A large plasmid containing all delta-endotoxin genes was isolated from Bacillus thuringiensis subsp. israelensis; restricted by BamHI, EcoRI, HindIII, KpnI, PstI, SacI, and SalI; and cloned as appropriate libraries in Escherichia coli. The libraries were screened for inserts containing recognition sites for BamHI, SacI, and SalI. Each was labeled with 32P and hybridized to Southern blots of gels with fragments generated by cleaving the plasmid with several restriction endonucleases, to align at least two fragments of the relevant enzymes. All nine BamHI fragments and all eight SacI fragments were mapped in two overlapping linkage groups (with total sizes of about 76 and 56 kb, respectively). The homology observed between some fragments is apparently a consequence of the presence of transposons and repeated insertion sequences. Four delta-endotoxin genes (cryIVB-D and cytA) and two genes for regulatory polypeptides (of 19 and 20 kDa) were localized on a 21-kb stretch of the plasmid; without cytA, they are placed on a single BamHI fragment. This convergence enables subcloning of delta-endotoxin genes (excluding cryIVA, localized on the other linkage group) as an intact natural fragment. PMID:8795201

  8. Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae.

    PubMed Central

    Regev, A; Keller, M; Strizhov, N; Sneh, B; Prudovsky, E; Chet, I; Ginzberg, I; Koncz-Kalman, Z; Koncz, C; Schell, J; Zilberstein, A

    1996-01-01

    In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form. PMID:8837413

  9. Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance.

    PubMed

    Weng, Li-Xing; Deng, Haihua; Xu, Jin-Ling; Li, Qi; Wang, Lian-Hui; Jiang, Zide; Zhang, Hai Bao; Li, Qiwei; Zhang, Lian-Hui

    2006-02-01

    Five elite sugarcane breeding lines were tested for efficiency in embryogenesis and plant regeneration. All of them produced regenerative embryogenic calli but with varied efficiencies. To engineer strongly insect-resistant sugarcanes, the GC content of a truncated cry1Ac gene, which encodes the active region of Cry1Ac insecticidal delta-endotoxin, was increased from the original 37.4 to 47.5% following the sugarcane codon usage pattern. The synthetic cry1Ac gene (s-cry1Ac) was placed under the control of maize ubiquitin promoter and introduced by microprojectile bombardment into the embryogenic calli of sugarcane lines YT79-177 and ROC16. Southern blotting analysis showed that multicopies of s-cry1Ac were integrated into the genomes of transgenic sugarcane lines. Immunoblotting analysis identified 18 transgenic lines expressing detectable levels of s-Cry1Ac, which were estimated in the range of 1.8-10.0 ng mg(-1) total soluble proteins. Four transgenic and two parental lines were assayed for sugarcane stem borer resistance in leaf tissue feeding trials and greenhouse plant assays. The results showed that, while the untransformed control lines were severely damaged in both leaves and stems, the transgenic sugarcane lines expressing high levels of s-Cry1Ac proteins were highly resistant to sugarcane stem borer attack, resulting in complete mortality of the inoculated insects within 1 week after inoculation.

  10. Hematotoxicity and genotoxicity evaluations in Swiss mice intraperitoneally exposed to Bacillus thuringiensis (var kurstaki) spore crystals genetically modified to express individually Cry1Aa, Cry1Ab, Cry1Ac, or Cry2Aa.

    PubMed

    Mezzomo, Bélin Poletto; Miranda-Vilela, Ana Luisa; Barbosa, Lilian Carla Pereira; Albernaz, Vanessa Lima; Grisolia, Cesar Koppe

    2016-08-01

    Bacillus thuringiensis (Bt) has been widely used in foliar sprays as part of integrated pest management strategies against insect pests of agricultural crops. Since the advent of genetically modified plants expressing Bt δ-endotoxins, the bioavailability of Cry proteins has increased, and therefore for biosafety reasons their adverse effects should be studied, mainly for nontarget organisms. We evaluated, in Swiss mice, the hematotoxicity and genotoxicity of the genetically modified strains of Bt spore crystals Cry1Aa, 1Ab, 1Ac, or 2Aa at 27 mg/kg, and Cry1Aa, 1Ab and 2Aa also at 136 and 270 mg/kg, administered with a single intraperitoneal injection 24 h before euthanasia. Controls received filtered water or cyclophosphamide. Blood samples collected by cardiac puncture were used to perform hemogram, and bone marrow was extracted for the micronucleus test. Bt spore crystals presented toxicity for lymphocytes when in higher doses, which varied according to the type of spore crystal studied, besides promoting cytotoxic and genotoxic effects for the erythroid lineage of bone marrow, mainly at highest doses. Although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results indicated that these Bt spore crystals were not harmless to mice. This suggests that a more specific approach should be taken to increase knowledge about their toxicological properties and to establish the toxicological risks to nontarget organisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 970-978, 2016.

  11. Single amino acid changes in domain II of Bacillus thuringiensis CryIAb delta-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles.

    PubMed Central

    Rajamohan, F; Alcantara, E; Lee, M K; Chen, X J; Curtiss, A; Dean, D H

    1995-01-01

    Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta. PMID:7730254

  12. Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba {delta}-endotoxin complex revealed by electron crystallography: Implications for toxin-pore formation

    SciTech Connect

    Ounjai, Puey; Unger, Vinzenz M.; Sigworth, Fred J.; Angsuthanasombat, Chanan

    2007-10-05

    The insecticidal nature of Cry {delta}-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17 A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.

  13. Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins.

    PubMed Central

    Barloy, F; Delécluse, A; Nicolas, L; Lecadet, M M

    1996-01-01

    A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. PMID:8655486

  14. Enhanced toxicity of Bacillus thuringiensis Cry3A delta-endotoxin in coleopterans by mutagenesis in a receptor binding loop.

    PubMed

    Wu, S J; Koller, C N; Miller, D L; Bauer, L S; Dean, D H

    2000-05-12

    We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R(345)A,Y(350)F,Y(351)F) and A2 (R(345)A,DeltaY(350), DeltaY(351)), showed significantly improved toxicity against Tenebrio molitor (yellow mealworm). The mutant toxin A1 was also more potent against both Leptinotarsa decemlineata (Colorado potato beetle) and Chrysomela scripta (cottonwood leaf beetle), while A2 displayed enhanced toxicity only in L. decemlineata. Competitive binding assays of L. decemlineata brush border membrane vesicles (BBMV) revealed that binding affinities for the A1 and A2 mutant toxins were ca. 2.5-fold higher than for the wild-type Cry3 toxin. Similar binding assays with C. scripta BBMV revealed a ca. 5-fold lower dissociation rate for the A1 mutant as compared to that of Cry3A.

  15. Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

    PubMed Central

    de Maagd, R A; Kwa, M S; van der Klei, H; Yamamoto, T; Schipper, B; Vlak, J M; Stiekema, W J; Bosch, D

    1996-01-01

    To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua. PMID:8633853

  16. Response of larval Ephestia kuehniella (Lepidoptera: Pyralidae) to individual Bacillus thuringiensis kurstaki toxins mixed with Xenorhabdus nematophila.

    PubMed

    BenFarhat, Dalel; Dammak, Mariam; Khedher, Saoussen Ben; Mahfoudh, Salima; Kammoun, Schema; Tounsi, Slim

    2013-09-01

    Bacillus thuringiensis kurstaki strain BNS3 produces parasporal crystals formed by Cry1Aa, Cry1Ac and Cry2Aa delta-endotoxins. In a previous work, we showed that the latter exhibited individually, a weak insecticidal activity against Ephestia kuehniella. In order to improve their toxicities, we studied the combined effect of each delta-endotoxin with X. nematophila cells on E. kuehniella larvae growth. Xenorhabdus cells were used in combination with spore crystal mixture of the wild strain BNS3, known to be active against E. kuehniella, but no improvement in toxicity was observed. This could be due to the high efficiency of BNS3 crystals against this insect. However, when X. nematophila was combined with each of Cry1Aa, Cry1Ac and Cry2Aa, improvement of toxicity was noticed. The best improvements were obtained with Cry1Ac and Cry2Aa, which are more toxic to E. kuehniella than Cry1Aa. The difference in toxicity improvement was attributed to the low affinity of Cry1Aa to BBMV receptors, compared to those of Cry1Ac and Cry2Aa. This synergism between Cry toxins and Xenorhabdus cells could be exploited on control target insect, particularly in case of resistance to Cry toxins.

  17. DELTAE

    SciTech Connect

    Ward, W.C.; Swift, G.W. )

    1993-11-01

    In thermoacoustic engines and refrigerators, and in many simple acoustic systems, a one dimensional wave equation determines the spatial dependence of the acoustic pressure and velocity. DELTAE numerically integrates such wave equations in the acoustic approximation, in gases or liquids, in user-defined geometries. Boundary conditions can include conventional acoustic boundary conditions of geometry and impedance, as well as temperature and thermal power in thermoacoustic systems. DELTAE can be used easily for apparatus ranging from simple duct networks and resonators to thermoacoustic engines refrigerators and combinations thereof. It can predict how a given apparatus will perform, or can allow the user to design an apparatus to achieve desired performance. DELTAE views systems as a series of segments; twenty segment types are supported. The purely acoustic segments include ducts and cones, and lumped impedances including compliances, series impedances, and endcaps. Electroacoustics tranducer segments can be defined using either frequency-independent coefficients or the conventional parameters of loudspeaker-style drivers: mass, spring constant, magnetic field strength, etc. Tranducers can be current driven, voltage driven, or connected to an electrical load impedance. Thermoacoustic segment geometries include parallel plates, circular and rectangular pores, and pin arrays. Side branches can be defined with fixed impedances, frequency-dependent radiation impedances, or as an auxiliary series of segments of any types. The user can select working fluids from among air, helium, neon, argon, hydrogen, deuterium, carbon dioxide, nitrogen, helium-argon mixtures, helium-xenon mixtures, liquid sodium, and eutectic sodium-potassium. Additional fluids and solids can be defined by the user.

  18. DELTAE

    SciTech Connect

    Ward, W.C. ); Swift, G.W. )

    1993-11-01

    In thermoacoustic engines and refrigerators, and in many simple acoustic systems, a one dimensional wave equation determines the spatial dependence of the acoustic pressure and velocity. DELTAE numerically integrates such wave equations in the acoustic approximation, in gases or liquids, in user-defined geometries. Boundary conditions can include conventional acoustic boundary conditions of geometry and impedance, as well as temperature and thermal power in thermoacoustic systems. DELTAE can be used easily for apparatus ranging from simple duct networks and resonators to thermoacoustic engines refrigerators and combinations thereof. It can predict how a given apparatus will perform, or can allow the user to design an apparatus to achieve desired performance. DELTAE views systems as a series of segments; twenty segment types are supported. The purely acoustic segments include ducts and cones, and lumped impedances including compliances, series impedances, and endcaps. Electroacoustics tranducer segments can be defined using either frequency-independent coefficients or the conventional parameters of loudspeaker-style drivers: mass, spring constant, magnetic field strength, etc. Tranducers can be current driven, voltage driven, or connected to an electrical load impedance. Thermoacoustic segment geometries include parallel plates, circular and rectangular pores, and pin arrays. Side branches can be defined with fixed impedances, frequency-dependent radiation impedances, or as an auxiliary series of segments of any types. The user can select working fluids from among air, helium, neon, argon, hydrogen, deuterium, carbon dioxide, nitrogen, helium-argon mixtures, helium-xenon mixtures, liquid sodium, and eutectic sodium-potassium. Additional fluids and solids can be defined by the user.

  19. Lipopolysaccharide Endotoxins

    PubMed Central

    Raetz, Christian R. H.; Whitfield, Chris

    2008-01-01

    Summary Since lipopolysaccharide endotoxins of Gram-negative bacteria were last reviewed in this series in 1990, much has been learned about the assembly and signaling functions of these remarkable glycoconjugates. Lipopolysaccharides typically consist of a hydrophobic domain known as lipid A (or endotoxin), a non-repeating “core” oligosaccharide, and a distal polysaccharide (or O-antigen). The flood of recent genomic data has made it possible to study lipopolysaccharide assembly in diverse Gram-negative bacteria, many of which are human or plant pathogens, and to create mutants or hybrid constructs with novel properties. Unexpectedly, key genes for lipid A biosynthesis have also been found in higher plants, indicating that eucaryotic lipid A-like molecules may exist. The carbohydrate diversity of lipopolysaccharides is better appreciated now than ten years ago, but much remains to be learned about function. Sequence comparisons suggest that extensive lateral transfer of genes for the assembly of O-antigens has occurred among bacteria. The most significant finding in the field of endotoxin biology since 1990 has been the identification of the plasma membrane protein TLR4 as the lipid A signaling receptor of animal cells. The latter belongs to a family of innate immunity receptors, all of which possess a large extracellular domain of leucine-rich repeats, a single trans-membrane segment and a smaller cytoplasmic signaling region that engages the adaptor protein MyD88. The expanding knowledge of TLR4 specificity and its downstream signaling pathways should provide new opportunities for blocking the inflammatory side effects of sepsis. Future progress will require insights into lipopolysaccharide-protein recognition at the atomic level, greater understanding of intra- and inter-cellular lipopolysaccharide trafficking, and incisive biological approaches that combine the tools of bacterial and animal genetics. PMID:12045108

  20. Methods of Endotoxin Detection.

    PubMed

    Su, Wenqiong; Ding, Xianting

    2015-08-01

    Endotoxin, present in the outer membrane of all gram-negative bacteria, can pose serious risks to human health, from irreversible shock to death. Therefore, it is essential to develop sensitive, accurate, and rapid methods for its detection. The rabbit pyrogen test is the first standard technique for endotoxin detection and, nowadays, has been replaced by the Limulus Amoebocyte Lysate test, which is the most popular detection technique for endotoxin. With in-depth understanding of endotoxin, biosensors based on endotoxin-sensing components are promising alternatives to pursue in developing low-cost, easy-operation, and fast-response endotoxin detection techniques. This article summarizes the recent advances of endotoxin detection methods with a particular emphasis on optical and electrochemical biosensors based on various sensing elements ranging from nature biomolecules to artificial materials. As the research and technological revolution continues, the highly integrated and miniaturized commercial devices for sensitively and reliably detecting endotoxin will provide a wide range of applications in people's daily life.

  1. MECHANISMS OF ENDOTOXIN TOLERANCE

    PubMed Central

    Greisman, Sheldon E.; Woodwards, William E.

    1965-01-01

    Bacterial endotoxins were administered by continuous intravenous infusions at constant rates to normal man and rabbits. An initial progressive febrile reaction was followed by progressive defervescence to baseline. The resulting pyrogenic refractory state was characterized as follows: (a) reticuloendothelial blockade with thorotrast neither prevented nor reversed its course; (b) passive transfer was unsuccessful with refractory phase plasma; (c) infusions of normal plasma or fresh whole blood failed to restore responsiveness; (d) a minimum of 4 hours of continuous endotoxin infusion was required for full development of unresponsiveness; (e) circulating antibody titers to endotoxin remained unaltered; (f) peripheral leukocytosis appeared; (g) infusion of febrile phase plasma reevoked an immediate, monophasic fever; (h) endotoxinemia could be demonstrated by pyrogen bioassay; (i) 10-fold increases in endotoxin infusion rates reevoked fever; (j) impaired responsiveness extended to heterologous endotoxins; (k) dermal inflammatory responses to endotoxin were suppressed in man while tuberculin reactivity remained unimpaired; dermal inflammatory responses to endotoxin were enhanced in rabbits; and (l) pyrogenic reactivity to endotoxin reappeared within 24 hours in man; refractoriness persisted in rabbits. It is concluded that the pyrogenic refractory state reflects an inability of the host to continue to mobilize endogenous pyrogen during sustained endotoxinemia. Such observations, together with previous studies, are consistent with two distinct immunologic mechanisms of resistance to endotoxin pyrogenicity: (a) desensitization at the cellular level; and (b) elaboration of circulating antibodies which assist reticuloendothelial clearance and destruction of endotoxin. Whereas both such mechanisms may contribute to pyrogenic tolerance, the characteristics of the pyrogenic refractory state suggest the participation only of the former. PMID:14319407

  2. Intra- and extracellular domains of the Helicoverpa armigera cadherin mediate Cry1Ac cytotoxicity

    USDA-ARS?s Scientific Manuscript database

    Diverse midgut cadherin mutations confer resistance to Cry1A toxins in at least three lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Most of these cadherin mutations are inherited as recessive alleles and result in changes within the cadherin repeat (CR) regions of the extr...

  3. Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication

    USDA-ARS?s Scientific Manuscript database

    Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well ch...

  4. Endotoxin contamination of engineered nanomaterials.

    PubMed

    Esch, R Keith; Han, Li; Foarde, Karin K; Ensor, David S

    2010-03-01

    Endotoxin has established health impacts and may be a potential confounding factor in toxicity studies of engineered nanomaterials (ENM). We aimed to characterize endotoxin contamination for a representative set of carbon-based ENM. The established method for quantifying endotoxin relies on its activity in a complex biochemical assay system. Because of their physical and chemical properties, measurement of endotoxin associated with many ENM presents non-trivial technical challenges. We have made progress in identifying and implementing methods for ENM analysis with respect to endotoxin content, revealing varying levels of endotoxin contamination in the ENM examined here. The physical association of ENM and endotoxin and their shared physiological effects suggest the possibility that contaminating endotoxin may contribute to the toxicity that is ascribed to ENM. We found in this small number of samples that endotoxin levels were not related to type of ENM or surface area but may be introduced randomly during manufacture.

  5. Endotoxins in commercial vaccines.

    PubMed Central

    Geier, M R; Stanbro, H; Merril, C R

    1978-01-01

    Twenty samples of commercial vaccines intended for administration to humans were assayed for the presence of bacterial endotoxins by using the Limulus amebocyte lysate test. Sixteen of the vaccines contained more than 0.1 ng of endotoxin per ml (which corresponds to 103 bacterial cell wall equivalents per ml in the undiluted vaccines). These results suggest that at some stage of preparation, the vaccines have contained varying amounts of gram-negative bacteria and may indicate the presence of other bacterial products as well. It might be useful to list the level of endotoxins, phage, and other contaminants on each vaccine lot to facilitate studies on any side effects of these contaminants. Selection of vaccine lots with the least endotoxin might reduce some of the adverse effects of vaccinations. PMID:727776

  6. Neurotoxic Properties on Endotoxins.

    DTIC Science & Technology

    1980-06-01

    REPRODUCE LEGIBLY. 4 t’ ABS TRACT Some endotoxins produce a biphasic effect on spontaneous transmitter secretion, with an early facilitation of release...spontaneous and evoked trans- mitter secretion under physiological conditions. However, glycolipid produces a bipitasic effect on both spontaneous and...the abolition of transmitter release might be effected by a more general mechanism. Endotoxin may isolate the presynaptic terminal from the

  7. Endotoxin Interactions with Platelets

    DTIC Science & Technology

    1985-01-01

    irreversible aggregation of human platelets (Hamberg and Sainuelsson 1974; Hlamberg et al 1975). Acetylsalicylic acid , an inhibitor of cyclooxygenase aud...exposure to endotoxin (100 ttg/nil). To simulate the lipopolysac- charide of endotoxin, several different fatty acids were added individually to platelet...platelet lytic capability. Similarity, iflegaradt doses of ganima radiation 6wCo destroy fatty acid groups on lipid A (L. Bertok, personal communication

  8. Endotoxin Elimination in Patients with Septic Shock: An Observation Study.

    PubMed

    Adamik, Barbara; Zielinski, Stanislaw; Smiechowicz, Jakub; Kübler, Andrzej

    2015-12-01

    To evaluate the effectiveness of endotoxin elimination with an adsorption column in patients with septic shock and endotoxemia. The elimination therapy was guided by a new bedside method of measuring endotoxin activity (EA). Intensive care unit (ICU) patients with septic shock and suspected Gram-negative infection were consecutively added to the study group within the first 24 h. Endotoxin elimination was performed using hemoperfusion with the Alteco LPS Adsorber. The primary endpoint was improvement in organ function within the first 24 h of treatment. A secondary objective was to assess the usefulness of a new method of measuring EA to help guide endotoxin elimination therapy. Out of 64 patients 18 had a high baseline EA [0.70 EA units (0.66-0.77)]. Those patients had endotoxin elimination treatment in addition to conventional medical therapy. At 24 h after endotoxin elimination, the EA had decreased to 0.56 EA units (0.43-0.77), (p = 0.005); MAP increased from 69 (62-80) to 80 mm Hg (68-88), (p = 0.002), and noradrenaline use decreased from 0.28 (0.15-0.80) to 0.1 μg/kg/min (0.00-0.70) at the same time (p = 0.04). The SOFA score had decreased from 11 (9-15) to 9 (7-14) points 24 h after endotoxin elimination (p = 0.01) with a median delta SOFA -2 points. Endotoxin elimination did not have a significant effect on the ICU length of stay or ICU mortality. Effective endotoxin elimination resulted in a significant improvement in hemodynamic parameters and of organ function. The application of the EA assay was useful for the bedside monitoring of endotoxemia in critically ill ICU patients.

  9. Biosensor of endotoxin and sepsis

    NASA Astrophysics Data System (ADS)

    Shao, Yang; Wang, Xiang; Wu, Xi; Gao, Wei; He, Qing-hua; Cai, Shaoxi

    2001-09-01

    To investigate the relation between biosensor of endotoxin and endotoxin of plasma in sepsis. Method: biosensor of endotoxin was designed with technology of quartz crystal microbalance bioaffinity sensor ligand of endotoxin were immobilized by protein A conjugate. When a sample soliton of plasma containing endotoxin 0.01, 0.03, 0.06, 0.1, 0.5, 1.0Eu, treated with perchloric acid and injected into slot of quartz crystal surface respectively, the ligand was released from the surface of quartz crystal to form a more stable complex with endotoxin in solution. The endotoxin concentration corresponded to the weight change on the crystal surface, and caused change of frequency that occurred when desorbed. The result was biosensor of endotoxin might detect endotoxin of plasma in sepsis, measurements range between 0.05Eu and 0.5Eu in the stop flow mode, measurement range between 0.1Eu and 1Eu in the flow mode. The sensor of endotoxin could detect the endotoxin of plasm rapidly, and use for detection sepsis in clinically.

  10. Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus).

    PubMed

    Girijashankar, V; Sharma, H C; Sharma, Kiran K; Swathisree, V; Prasad, L Sivarama; Bhat, B V; Royer, Monique; Secundo, Blanca San; Narasu, M Lakshmi; Altosaar, I; Seetharama, N

    2005-11-01

    Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T(1) plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1-8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt delta-endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.

  11. Interaction between Cry9Ca and two Cry1A delta-endotoxins from Bacillus thuringiensis in larval toxicity and binding to brush border membrane vesicles of the spruce budworm, Choristoneura fumiferana Clemens.

    PubMed

    Pang, Anthony S D; Gringorten, J Lawrence; van Frankenhuyzen, Kees

    2002-09-24

    A genetically altered variant of Cry9Ca from Bacillus thuringiensis shows high potency against the spruce budworm, Choristoneura fumiferana Clemens. Its activity, as measured by feeding inhibition in frass-failure assays, is estimated to be four to seven times greater than B. thuringiensis subsp. kurstaki HD-1, the strain currently used in commercial products to control this insect. Bioassays against budworm of mixtures of the modified Cry9Ca and two of the Cry1A endotoxin proteins produced by HD-1 show neither synergism nor antagonism. Experiments with brush border membrane vesicles from budworm midgut revealed that Cry9Ca and the Cry1A toxins share a common binding site and that bound Cry9Ca can be displaced from the membrane to some extent by the Cry1A toxins. However, it is uncertain whether the binding site is actually the receptor molecule or a membrane protein associated with pore formation.

  12. Ibuprofen in canine endotoxin shock.

    PubMed Central

    Jacobs, E R; Soulsby, M E; Bone, R C; Wilson, F J; Hiller, F C

    1982-01-01

    The participation of prostaglandins in the physiologic alterations of endotoxin shock has been well established with the aid of prostaglandin synthetase inhibitors. Our study was designed to investigate the potential of ibuprofen, a highly specific cyclooxygenase inhibitor, to reverse the hemodynamic and acid base abnormalities of canine endotoxin shock. Mean blood pressure fell to 49.8 +/- 6.6 mm Hg in dogs given endotoxin by 5 min after injection, and remained below 83 mm Hg for the duration of the 120-min observation period. In animals given endotoxin followed by ibuprofen, a similar initial drop of systemic blood pressure was seen, but it subsequently recovered to 150.2 +/- 4.1 mm Hg by 120 min (P less than 0.001). Cardiac index increased in animals given ibuprofen (2.3 +/- 0.28 liter/m2 per min) compared with animals given endotoxin alone (1.0 +/- 0.09 liter/m2 per min) by termination of the experiment. The arterial pH dropped in endotoxin treated animals to 7.18 +/- 0.03 by 120 min. Ibuprofen prevented the acidosis, the final pH in ibuprofen and endotoxin treated animals measuring 7.36 +/- 0.01. We conclude that ibuprofen protects against the hypotension, acidosis, and depression of cardiac index of canine endotoxin shock. PMID:7107893

  13. Endotoxin hitchhiking on polymer nanoparticles

    NASA Astrophysics Data System (ADS)

    Donnell, Mason L.; Lyon, Andrew J.; Mormile, Melanie R.; Barua, Sutapa

    2016-07-01

    The control of microbial infections is critical for the preparation of biological media including water to prevent lethal septic shock. Sepsis is one of the leading causes of death in the United States. More than half a million patients suffer from sepsis every year. Both gram-positive and gram-negative bacteria are responsible for septic infection by the most common organisms i.e., Escherichia coli and Pseuodomonas aeruginosa. The bacterial cell membrane releases negatively charged endotoxins upon death and enzymatic destruction, which stimulate antigenic response in humans to gram-negative infections. Several methods including distillation, ethylene oxide treatment, filtration and irradiation have been employed to remove endotoxins from contaminated samples, however, the reduction efficiency remains low, and presents a challenge. Polymer nanoparticles can be used to overcome the current inability to effectively sequester endotoxins from water. This process is termed endotoxin hitchhiking. The binding of endotoxin on polymer nanoparticles via electrostatic and hydrophobic interactions offers efficient removal from water. However, the effect of polymer nanoparticles and its surface areas has not been investigated for removal of endotoxins. Poly(ε-caprolactone) (PCL) polymer was tested for its ability to effectively bind and remove endotoxins from water. By employing a simple one-step phase separation technique, we were able to synthesize PCL nanoparticles of 398.3 ± 95.13 nm size and a polydispersity index of 0.2. PCL nanoparticles showed ∼78.8% endotoxin removal efficiency, the equivalent of 3.9 × 105 endotoxin units (EU) per ml. This is 8.34-fold more effective than that reported for commercially available membranes. Transmission electron microscopic images confirmed binding of multiple endotoxins to the nanoparticle surface. The concept of using nanoparticles may be applicable not only to eliminate gram-negative bacteria, but also for any gram

  14. SUBCHRONIC ENDOTOXIN INHALATION CAUSES CHRONIC AIRWAY DISEASE IN ENDOTOXIN-SENSITIVE BUT NOT ENDOTOXIN-RESISTANT MICE

    EPA Science Inventory

    SUBCHRONIC ENDOTOXIN INHALATION CAUSES CHRONIC AIRWAY DISEASE IN ENDOTOXIN-SENSITIVE BUT NOT ENDOTOXIN-RESISTANT MICE. D. M. Brass, J. D. Savov, *S. H. Gavett, ?C. George, D. A. Schwartz. Duke Univ Medical Center Durham, NC, *U.S. E.P.A. Research Triangle Park, NC, ?Univ of Iowa,...

  15. SUBCHRONIC ENDOTOXIN INHALATION CAUSES CHRONIC AIRWAY DISEASE IN ENDOTOXIN-SENSITIVE BUT NOT ENDOTOXIN-RESISTANT MICE

    EPA Science Inventory

    SUBCHRONIC ENDOTOXIN INHALATION CAUSES CHRONIC AIRWAY DISEASE IN ENDOTOXIN-SENSITIVE BUT NOT ENDOTOXIN-RESISTANT MICE. D. M. Brass, J. D. Savov, *S. H. Gavett, ?C. George, D. A. Schwartz. Duke Univ Medical Center Durham, NC, *U.S. E.P.A. Research Triangle Park, NC, ?Univ of Iowa,...

  16. THE MECHANISM OF ACTION OF ENDOTOXIN

    DTIC Science & Technology

    Evidence from this laboratory and others indicates that endotoxin shock is a form of anaphylactic shock mediated through natural antibody and...bacteria may influence susceptibility to endotoxin by their effect on the production of both natural and acquired antibody. The immunologic factors...in the production of endotoxin shock were investigated further by the following studies: ( 1 ) A Systematic Analysis of the Reaction Between Endotoxin

  17. Effects of four entomopathogenic nematode species on fitness costs of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac

    USDA-ARS?s Scientific Manuscript database

    Evolution of resistance by pests can reduce the efficacy of transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). However, fitness costs can slow the evolution of resistance. We tested whether four species of entomopathogenic nematodes (Steinernematidae ...

  18. Increased long-flight activity triggered in beet armyworm by larval feeding on diet containing Cry1Ac protoxin

    USDA-ARS?s Scientific Manuscript database

    Evaluating ecological safety and conducting pest risk analysis for transgenic crops are vitally important before their commercial planting. The beet armyworm, Spodoptera exigua, is not a direct target of transgenic cotton in China but nevertheless recently has become an important pest. In laboratory...

  19. Effects of entomopathogenic nematodes on the evolution of pink bollworm resistance to Bt toxin Cry1Ac.

    USDA-ARS?s Scientific Manuscript database

    The evolution of resistance by pests can reduce the efficacy of transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). Conversely, fitness costs may act to delay pest resistance to Bt. The entomopathogenic nematode Steinernema riobrave (Rhabditida: Steinern...

  20. Effects of four nematodes species on fitness costs of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac

    USDA-ARS?s Scientific Manuscript database

    Evolution of resistance by pests can reduce efficacy of transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). In conjunction with refuges of non-Bt host plants, fitness costs can delay the evolution of resistance. Furthermore, fitness costs often vary wit...

  1. Biological Activity of Masked Endotoxin

    PubMed Central

    Schwarz, Harald; Gornicec, Jan; Neuper, Theresa; Parigiani, Maria Alejandra; Wallner, Michael; Duschl, Albert; Horejs-Hoeck, Jutta

    2017-01-01

    Low endotoxin recovery (LER) is a recently discovered phenomenon describing the inability of limulus amebocyte lysate (LAL)-based assays to detect lipopolysaccharide (LPS) because of a “masking effect” caused by chelators or detergents commonly used in buffer formulations for medical products and recombinant proteins. This study investigates the masking capacities of different buffer formulations and whether masked endotoxin is biologically active. We show that both naturally occurring endotoxin as well as control standard endotoxin can be affected by LER. Furthermore, whereas masked endotoxin cannot be detected in Factor C based assays, it is still detectable in a cell-based TLR4-NF-κB-luciferase reporter gene assay. Moreover, in primary human monocytes, masked LPS induces the expression of pro-inflammatory cytokines and surface activation markers even at very low concentrations. We therefore conclude that masked LPS is a potent trigger of immune responses, which emphasizes the potential danger of masked LPS, as it may pose a health threat in pharmaceutical products or compromise experimental results. PMID:28317862

  2. Endotoxin Studies And Biosolids Stabilization Research

    EPA Science Inventory

    This presentation has three parts; a review of bench-scale endotoxin research, a review of observations from a field scale endotoxin release study, and discussion of biosolids stabilization and characterization by PLFA/FAME microbial community analysis. Endotoxins are part of th...

  3. SUBCHRONIC ENDOTOXIN INHALATION CAUSES PERSISTENT AIRWAY DISEASE

    EPA Science Inventory

    ABSTRACT

    The endotoxin component of organic dusts causes acute reversible airflow obstruction and airway inflammation. To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin ...

  4. Endotoxin Studies And Biosolids Stabilization Research

    EPA Science Inventory

    This presentation has three parts; a review of bench-scale endotoxin research, a review of observations from a field scale endotoxin release study, and discussion of biosolids stabilization and characterization by PLFA/FAME microbial community analysis. Endotoxins are part of th...

  5. Cotton dust and endotoxin exposure-response relationships in cotton textile workers

    SciTech Connect

    Kennedy, S.M.; Christiani, D.C.; Eisen, E.A.; Wegman, D.H.; Greaves, I.A.; Olenchock, S.A.; Ye, T.T.; Lu, P.L.

    1987-01-01

    Endotoxin exposure has been implicated in the etiology of lung disease in cotton workers. We investigated this potential relationship in 443 cotton workers from 2 factories in Shanghai and 439 control subjects from a nearby silk mill. A respiratory questionnaire was administered and pre- and postshift forced expiratory volume (FVC) and flow in one second (FEV1) were determined for each worker. Multiple area air samples were analyzed for total elutriated dust concentration (range: 0.15 to 2.5 mg/m3) and endotoxin (range: 0.002 to 0.55 microgram U.S. Reference Endotoxin/m3). The cotton worker population was stratified by current and cumulative dust or endotoxin exposure. Groups were compared for FEV1, FVC, FEV1/FVC%, % change in FEV1 over the shift (delta FEV1%), and prevalences of chronic bronchitis and byssinosis, and linear and logistic regression models were constructed. No dose-response relationships were demonstrated comparing dust concentration to any pulmonary function or symptom variable. A dose-response trend was seen with the current endotoxin level and FEV1, delta FEV1%, and the prevalence of byssinosis and chronic bronchitis, except for the highest exposure level group in which a reversal of the trend was seen. The regression coefficients for current endotoxin exposure were significant (p less than 0.05) in the models for FEV1 and chronic bronchitis but not in the models for delta FEV1% (i.e., acute change in FEV1) or byssinosis prevalence. The coefficient for dust level was never significant in the models.

  6. Role of Tryptophan Pyrrolase in Endotoxin Poisoning

    PubMed Central

    Moon, Robert J.; Berry, L. Joe

    1968-01-01

    Using substrate induction as a tool, we attempted to determine the role of tryptophan pyrrolase in the response to endotoxin in mice. Previous results have shown that the administration of the ld50 of endotoxin lowers tryptophan pyrrolase activity. α-Methyltryptophan was found to maintain tryptophan pyrrolase activity above control levels in endotoxin-poisoned mice without increasing survival. 5-Hydroxytryptophan, by contrast, lowered tryptophan pyrrolase activity but did not sensitize mice to endotoxin. These results suggest that tryptophan pyrrolase per se does not play a unique role in survival of mice poisoned with endotoxin. This enzyme, however, may reflect the fate of other liver enzymes inducible by adrenocorticoids. In mice given concurrent injections of tryptophan and endotoxin, tryptophan pyrrolase activity was elevated to a level intermediate between that of normal mice and that of mice given tryptophan alone. The mice injected with tryptophan and endotoxin also had about the same mortality as mice given endotoxin alone. Mice treated with tryptophan 4 hr after endotoxin, at a time when the substrate did not fully elevate tryptophan pyrrolase activity, died convulsively and in larger numbers than those given endotoxin alone. This effect was reversed by prior treatment with cyproheptadine, an antiserotonin drug. These results indicate that the depression of tryptophan pyrrolase activity previously observed in vitro after injection of endotoxin reflects an actual decrease in the in vivo activity of the enzyme. PMID:4869211

  7. Endotoxin emissions from commercial composting activities

    PubMed Central

    2009-01-01

    This paper describes an exploratory study of endotoxin emissions and dispersal from a commercial composting facility. Replicated samples of air were taken by filtration at different locations around the facility on 10 occasions. Measurements were made of endotoxin and associated culturable microorganisms. The inflammatory response of cell cultures exposed to extracts from the filters was measured. Endotoxin was detected in elevated concentrations close to composting activities. A secondary peak, of lesser magnitude than the peak at source was detected at 100-150 m downwind of the site boundary. Unexpectedly high concentrations of endotoxin were measured at the most distant downwind sampling point. Extracted endotoxin was found to stimulate human monocytes and a human lung epithelial cell line to produce significant amounts of pro-inflammatory cytokines. On a weight basis, endotoxin extracted from the composting source has a greater inflammatory cytokine inducing effect than commercial E. coli endotoxin. PMID:20102594

  8. Endotoxin emissions from commercial composting activities.

    PubMed

    Deacon, Lewis; Pankhurst, Louise; Liu, Jian; Drew, Gillian H; Hayes, Enda T; Jackson, Simon; Longhurst, James; Longhurst, Philip; Pollard, Simon; Tyrrel, Sean

    2009-12-21

    This paper describes an exploratory study of endotoxin emissions and dispersal from a commercial composting facility. Replicated samples of air were taken by filtration at different locations around the facility on 10 occasions. Measurements were made of endotoxin and associated culturable microorganisms. The inflammatory response of cell cultures exposed to extracts from the filters was measured. Endotoxin was detected in elevated concentrations close to composting activities. A secondary peak, of lesser magnitude than the peak at source was detected at 100-150 m downwind of the site boundary. Unexpectedly high concentrations of endotoxin were measured at the most distant downwind sampling point. Extracted endotoxin was found to stimulate human monocytes and a human lung epithelial cell line to produce significant amounts of pro-inflammatory cytokines. On a weight basis, endotoxin extracted from the composting source has a greater inflammatory cytokine inducing effect than commercial E. coli endotoxin.

  9. Recent advances in biosensor based endotoxin detection.

    PubMed

    Das, A P; Kumar, P S; Swain, S

    2014-01-15

    Endotoxins also referred to as pyrogens are chemically lipopolysaccharides habitually found in food, environment and clinical products of bacterial origin and are unavoidable ubiquitous microbiological contaminants. Pernicious issues of its contamination result in high mortality and severe morbidities. Standard traditional techniques are slow and cumbersome, highlighting the pressing need for evoking agile endotoxin detection system. The early and prompt detection of endotoxin assumes prime importance in health care, pharmacological and biomedical sectors. The unparalleled recognition abilities of LAL biosensors perched with remarkable sensitivity, high stability and reproducibility have bestowed it with persistent reliability and their possible fabrication for commercial applicability. This review paper entails an overview of various trends in current techniques available and other possible alternatives in biosensor based endotoxin detection together with its classification, epidemiological aspects, thrust areas demanding endotoxin control, commercially available detection sensors and a revolutionary unprecedented approach narrating the influence of omics for endotoxin detection.

  10. Lipopolysaccaride Detoxification by Endotoxin Neutralizing Protein

    DTIC Science & Technology

    1993-08-01

    than 3 mg/kg). ENP also reduces or eliminates $ - a FLETCHER ET AL.: ENDOTOXIN NEUTRALIZING PROTEIN 153 TABLE 4 Microscopic Pathology of Organs Liver...bUBTITLE S. FUNDING NUMBERS Lipopolysaccharide detoxification by endotoxin neutralizing protein PE- 61153N PR- MR04120 6. AUTHOR(S) Fletcher MA...Lipopolysaccharide Detoxification by Endotoxin Neutralizing Protein MARK A. FLETCHER, M.D., THOMAS M. MCKENNA, PH.D., JERRY L. QCANCE, D.V.M., NORMAN R. WAINWRIGHT

  11. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  12. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  13. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  14. Movement of Endotoxin Through Soil Columns

    PubMed Central

    Goyal, Sagar M.; Gerba, Charles P.; Lance, J. Clarence

    1980-01-01

    Land treatment of wastewater is an attractive alternative to conventional sewage treatment systems and is gaining widespread acceptance. Although land application systems prevent surface water pollution and augment the available water supplies, the potential dangers to human health should be evaluated. Since sewage may contain high amounts of bacterial endotoxin, the removal of endotoxin from sewage by percolation through soil was investigated. It was found that 90 to 99% of the endotoxin was removed after travel of sewage through 100 to 250 cm of loamy sand soil. When distilled water was allowed to infiltrate into the soil to simulate rainfall, the endotoxin was mobilized and moved in a concentrated band through the soil column. On testing samples from actual land treatment sites, as much as 480 ng of endotoxin per milliliter was found in some groundwater samples. The presence of endotoxin in potable water is known to be a potential problem under some circumstances, but the importance of endotoxin in water supplies has not been fully assessed. Therefore, the design, operation, and management of land application systems should take into account the fate of endotoxin in groundwater beneath the sites. PMID:7387154

  15. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  16. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  17. Current trends in endotoxin detection and analysis of endotoxin-protein interactions.

    PubMed

    Dullah, Elvina Clarie; Ongkudon, Clarence M

    2017-03-01

    Endotoxin is a type of pyrogen that can be found in Gram-negative bacteria. Endotoxin can form a stable interaction with other biomolecules thus making its removal difficult especially during the production of biopharmaceutical drugs. The prevention of endotoxins from contaminating biopharmaceutical products is paramount as endotoxin contamination, even in small quantities, can result in fever, inflammation, sepsis, tissue damage and even lead to death. Highly sensitive and accurate detection of endotoxins are keys in the development of biopharmaceutical products derived from Gram-negative bacteria. It will facilitate the study of the intermolecular interaction of an endotoxin with other biomolecules, hence the selection of appropriate endotoxin removal strategies. Currently, most researchers rely on the conventional LAL-based endotoxin detection method. However, new methods have been and are being developed to overcome the problems associated with the LAL-based method. This review paper highlights the current research trends in endotoxin detection from conventional methods to newly developed biosensors. Additionally, it also provides an overview of the use of electron microscopy, dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET) and docking programs in the endotoxin-protein analysis.

  18. EFFECTS OF BACTERIAL ENDOTOXINS ON METABOLISM

    PubMed Central

    Berry, L. Joe; Smythe, Dorothy S.

    1961-01-01

    In vitro secretion of glycocorticoids by adrenal glands pooled from several control mice was compared with that of glands removed from animals following injections of either ACTH or endotoxin. Both substances prevent glycocorticoid synthesis stimulated in vitro with ACTH. Cholesterol content of adrenal glands under these conditions was nearly depleted, indicating maximal response to ACTH or endotoxin prior to their removal for the in vitro tests. In an effort to account physiologically for the manner in which endotoxin suppresses or prevents the rise in urinary nitrogen excreted in response to ACTH, blood non-protein nitrogen levels (NPN) were determined. The following experimental conditions resulted in increased urinary nitrogen excretion but did not alter blood NPN: cortisone given alone or at the same time as endotoxin; ACTH alone; dichloroisoproterenol (DCI) given concurrently with endotoxin; and lactalbumin digest injected intraperitoneally. Increases (2- to 3-fold) in blood NPN were observed when endotoxin was given alone, concurrently with ACTH, or 3 hours prior to cortisone, DCI, or lactalbumin digest. Urinary nitrogen excretion showed no change under these conditions. The elevation in blood NPN in endotoxin-poisoned mice was found to be due almost entirely to urea nitrogen and not to amino acid nitrogen or to other nitrogenous wastes. Blood clearance of mulin, phenol red excretion, and urea elimination were each determined in control and in endotoxin-poisoned mice. The latter mice showed impaired renal function. Treatment with diuretics (diuril and aminophylline) failed to alter oliguria or elevated blood NPN. Hydergine treatment was also without effect. Total carcass NPN and urinary nitrogen excretion data were combined to give a picture of total protein catabolized by mice under different experimental conditions. Cortisone injected at the same time as endotoxin or 3 hours later resulted in the same increase in total NPN. However, in the former case all

  19. Nile Delta

    Atmospheric Science Data Center

    2013-04-15

    article title:  The Nile River Delta     View Larger Image ... of eastern Africa. At the apex of the fertile Nile River Delta is the Egyptian capital city of Cairo. To the west are the Great Pyramids ...

  20. Field Studies Measuring the aerosolization of Endotoxin ...

    EPA Pesticide Factsheets

    Endotoxin is a component of the cell walls of Gram-negative bacteria and is known to be present in biosolids. Endotoxins have been shown to be a potent stimulator of the innate immune response causing airway irritation and shortness of breath. Class B biosolids are routinely applied to agricultural lands in the US to enhance soil properties and can be used as an alternative to chemical fertilizers. This study investigated the aerosolized endotoxin produced during the land application of Class B biosolids from various wastewater treatment plants on agricultural land and a concrete surface at two sites in Colorado, USA. Aerosolized endotoxin was captured using HiVol sampler fitted with glass fiber filter, polycarbonate filter cassette (both open and closed), and BioSampler impinger air samplers. Endotoxins were also measured in the bulk biosolids to allow for correlating bulk biosolids concentrations with aerosol emission rates. Endotoxin concentrations in biosolids, impinger solutions, and filter extracts were determined using the kinetic Limulus amebocyte lysate assay. Aerosolized endotoxin concentration was detected from all sites with levels ranging from 0.5 to 642 EU/m3. The four types of sampling apparatus were compared and the HiVol and open-faced cassette samplers used produced higher TWA measurements (EU/m3) than the impinger and closed cassette samplers. Ambient wind speed at the sites was found to be the variable best describing the results wit

  1. Volga Delta

    Atmospheric Science Data Center

    2013-04-17

    article title:  Volga Delta and the Caspian Sea     View ... appear reddish. A small cloud near the center of the delta separates into red, green, and blue components due to geometric parallax ... include several linear features located near the Volga Delta shoreline. These long, thin lines are artificially maintained shipping ...

  2. Endotoxins in urban air in Stockholm, Sweden

    NASA Astrophysics Data System (ADS)

    Nilsson, S.; Merritt, A. S.; Bellander, T.

    2011-01-01

    Endotoxins, i.e. components originating from the outer membrane in the cell wall of Gram-negative bacteria, activate the human immune system, which may result in airway symptoms such as shortness of breath and airway inflammation. Endotoxins are present in the environment, both outdoors and indoors, and stay airborne for a long time. In order to investigate the levels of endotoxins in urban air and the influence of traffic and meteorological factors, particles (PM 10 and PM 2.5) were collected at five sites in Stockholm, Sweden on four occasions per site between May and September 2009. Endotoxins were extracted from the filters and analysis was conducted with the Limulus Amebocyte Lysate (LAL)-assay. Endotoxins were present in urban air in Stockholm, albeit in low levels, and were similar to levels found in urban areas outside Sweden. To our knowledge, this is the northernmost location where endotoxins have been measured. The endotoxin levels found in PM 10 ranged from 0.020 to 0.107 EU m -3 with a geometric mean of 0.050 EU m -3 and the levels found in PM 2.5 ranged from 0.005 to 0.064 EU m -3 with a geometric mean of 0.015 EU m -3. No obvious effects of traffic or meteorological factors on endotoxin levels were observed, although a moderate correlation could be seen with soot. The small number of sampling sites is however a shortcoming of the present study. In future studies, more sites and sampling during all seasons would be preferable in order to get a better picture of the influence of different sources on endotoxin levels.

  3. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

    PubMed

    Badran, Ahmed H; Guzov, Victor M; Huai, Qing; Kemp, Melissa M; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H; Wang, Ping; Malvar, Thomas; Liu, David R

    2016-05-05

    The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

  4. Continuous evolution of B. thuringiensis toxins overcomes insect resistance

    PubMed Central

    Badran, Ahmed H.; Guzov, Victor M.; Huai, Qing; Kemp, Melissa M.; Vishwanath, Prashanth; Kain, Wendy; Nance, Autumn M.; Evdokimov, Artem; Moshiri, Farhad; Turner, Keith H.; Wang, Ping; Malvar, Thomas; Liu, David R.

    2016-01-01

    The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. We developed a phage-assisted continuous evolution (PACE) selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. PMID:27120167

  5. Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins.

    PubMed

    Bird, Lisa J; Akhurst, Raymond J

    2007-02-01

    Intra-specific variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) in Australia to the Cry1Ac and Cry2Ab delta-endotoxins from Bacillus thuringiensis (Berliner) (Bt) was determined to establish a baseline for monitoring changes that might occur with the use of Bt cotton. Strains of H. armigera and H. punctigera were established from populations collected primarily from commercial farms throughout the Australian cotton belts. Strains were evaluated for susceptibility using two bioassay methods (surface treatment and diet incorporation) by measuring the dose response for mortality (LC50) and growth inhibition (IC50). The variation in LC50 among H. armigera (n=17 strains) and H. punctigera (n=12 strains) in response to Cry1Ac was 4.6- and 3.2-fold, respectively. The variation in LC50 among H. armigera (n=19 strains) and H. punctigera (n=12 strains) to Cry2Ab was 6.6- and 3.5-fold, respectively. The range of Cry1Ac induced growth inhibition from the 3rd to 4th instar in H. armigera (n=15 strains) was 3.6-fold and in H. punctigera (n=13 strains) was 2.6-fold, while the range of Cry2Ab induced growth inhibition from neonate to 3rd instar in H. armigera (n=13 strains) was 4.3-fold and in H. punctigera (n=12 strains) was 6.1-fold. Variation in susceptibility was also evaluated for two age classes (neonates and 3rd instars) in laboratory strains of H. armigera and H. punctigera. Neonates of H. punctigera had the same or higher sensitivity to Bt than 3rd instars. Neonates of H. armigera were more sensitive to Cry2Ab than 3rd instars, while being less sensitive to Cry1Ac than 3rd instars. Differences in the two methods of bioassay used affected relative sensitivity of species to Bt toxins, highlighting the need to standardize bioassay protocols.

  6. Influence of oxalic and malic acids in chickpea leaf exudates on the biological activity of CryIAc towards Helicoverpa armigera.

    PubMed

    Devi, V Surekha; Sharma, Hari C; Rao, P Arjuna

    2013-04-01

    Efforts are being made to express toxin genes from the bacterium, Bacillus thuringiensis (Bt) in chickpea for minimizing the losses due to the pod borer, Helicoverpa armigera. However, there is an apprehension that acidic exudates in chickpea leaves may influence the protoxin-toxin conversion in the insect midgut, and thus, reduce the efficacy of Bt toxins. Therefore, we studied the influence of organic acids (oxalic acid and malic acid) present in the trichome exudates of chickpea on the biological activity and binding of Bt δ-endotoxin Cry1Ac to brush border membrane vesicles (BBMV) of the pod borer, H. armigera. Oxalic and malic acids in combination at concentrations present in chickpea leaves did not influence the biological activity of Bt toxin Cry1Ac towards H. armigera larvae. Amounts of Cry1Ac protein in the midgut of insects reared on diets with organic acids were similar to those reared on artificial diet without the organic acids. However, very high concentrations of the organic acids reduced the amounts of Cry1Ac in the midgut of H. armigera larvae. Organic acids in the artificial diet also increased the excretion of Cry1Ac in the fecal matter. Organic acids reduced the amount of protein in the BBMV of insects reared on diets with Cry1Ac, possibly because of reduced size of the larvae. Oxalic and malic acids at concentrations present in chickpea leaves did not affect the biological activity of Cry1Ac, but it will be desirable to have high levels of expression of Cry1Ac toxin proteins in chickpea for effective control of the pod borer, H. armigera. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Endotoxin levels and contribution factors of endotoxins in resident, school, and office environments - A review

    NASA Astrophysics Data System (ADS)

    Salonen, Heidi; Duchaine, Caroline; Létourneau, Valérie; Mazaheri, Mandana; Laitinen, Sirpa; Clifford, Sam; Mikkola, Raimo; Lappalainen, Sanna; Reijula, Kari; Morawska, Lidia

    2016-10-01

    As endotoxin exposure has known effects on human health, it is important to know the generally existing levels of endotoxins as well as their contributing factors. This work reviews current knowledge on the endotoxin loads in settled floor dust, concentrations of endotoxins in indoor air, and different environmental factors potentially affecting endotoxin levels. The literature review consists of peer-reviewed manuscripts located using Google and PubMed, with search terms based on individual words and combinations. References from relevant articles have also been searched. Analysis of the data showed that in residential, school, and office environments, the mean endotoxin loads in settled floor dust varied between 660 and 107,000 EU/m2, 2180 and 48,000 EU/m2, and 2700 and 12,890 EU/m2, respectively. Correspondingly, the mean endotoxin concentrations in indoor air varied between 0.04 and 1610 EU/m3 in residences, and 0.07 and 9.30 EU/m3 in schools and offices. There is strong scientific evidence indicating that age of houses (or housing unit year category), cleaning, farm or rural living, flooring materials (the presence of carpets), number of occupants, the presence of dogs or cats indoors, and relative humidity affect endotoxin loads in settled floor dust. The presence of pets (especially dogs) was extremely strongly associated with endotoxin concentrations in indoor air. However, as reviewed articles show inconsistency, additional studies on these and other possible predicting factors are needed.

  8. INTERACTION OF IONIZING RADIATION AND E. COLI ENDOTOXIN. I. EFFECT OF RADIATION ON ENDOTOXIN SHOCK.

    DTIC Science & Technology

    E . coli endotoxin and total body irradiation has been studied in 38 adult mongrel dogs. Results indicate that prior irradiation (1000 r) abolishes the endotoxin shock syndrome, and the dogs die of radiation sickness on an average of 62 hr after exposure. Unirradiated dogs given E . coli endotoxin die on an average of 5 hr after injection of the toxin. It is postulated that irradiation interferes with, or actually prevents, the ’trigger mechanism’ of endotoxin shock. This interaction may involve the immune mechanism, complement, liberation

  9. Staghorn calculus endotoxin expression in sepsis.

    PubMed

    McAleer, Irene M; Kaplan, George W; Bradley, John S; Carroll, Stephen F

    2002-04-01

    Staghorn calculi are infrequent and generally are infected stones. Struvite or apatite calculi are embedded with gram-negative bacteria, which can produce endotoxin. Sepsis syndrome may occur after surgical therapy or endoscopic manipulation of infected or staghorn calculi. Sepsis, which can occur despite perioperative antibiotic use, may be due to bacteremia or endotoxemia. We present a child with an infected staghorn calculus who developed overwhelming sepsis and died after percutaneous stone manipulation. Endotoxin assay of stone fragments demonstrated an extremely high level of endotoxin despite low colony bacterial culture growth. This is the first reported case in which endotoxin was demonstrated in stone fragments from a child who died of severe sepsis syndrome after percutaneous staghorn stone manipulation.

  10. Endotoxins in cotton: washing effects and size distribution

    SciTech Connect

    Olenchock, S.A.; Mull, J.C.; Jones, W.G.

    1983-01-01

    Endotoxin contamination was measured in washed and unwashed cottons from three distinct growing areas, California, Mississippi, and Texas. The data show differences in endotoxin contamination based upon the geographic source of the cotton. It is also shown that washing bulk cotton before the carding process results in lower endotoxin in the cotton dust. Washing conditions can affect the endotoxin levels, and all size fractions of the airborne dust contain quantifiable endotoxin contamination. Endotoxin analyses provide a simple and reliable method for monitoring the cleanliness of cotton or airborne cotton dusts.

  11. Role of platelets in the pathogenesis of canine endotoxin shock.

    PubMed Central

    From, A H; Fong, J S; Chiu, T; Good, R A

    1976-01-01

    Endotoxin-platelet interactions are thought to be of major importance in the response of dogs and other species to bacterial endotoxin; the mechanisms postulated are: (i) the release of vasoactive substances, (ii) the formation of occlusive platelet aggregates, and (iii) induction of intravascular coagulation. The role of platelets in canine endotoxin shock was examined in animals with thrombocytopenia induced by estrogen pretreatment (less than 10,000 platelets/mm3) and in controls. After intravenously administered endotoxin, the hemodynamic responses, mortality, and gross necropsy findings were similar in both groups. These data indicate that endotoxin-platelet interactions are not determinative in the pathogenesis of canine endotoxin shock. PMID:786877

  12. Isolation of Endotoxin Eliminating Lactic Acid Bacteria and a Property of Endotoxin Eliminating Protein.

    PubMed

    Kondo, Ayaka; Asami, Kyoko; Suda, Yoshihito; Shimoyamada, Makoto; Kanauchi, Makoto

    2016-06-01

    Recently, many scholars have reported lactic acid bacteria (LAB) functions, such as anticancer activity and anti-inflammatory activity for intestines. To decrease inflammatory substances such as endotoxins, LAB consumed safely with meals were isolated from food and food ingredients. First, LAB were isolated as 168 strains of bacillus LAB (49 strain) and coccus LAB (119 strains) from food ingredients and fermented foods such as rice, rice bran, malt, grains, miso soy paste, and some pickles. Their LAB (168 strains) were cultivated in medium containing endotoxin from Escherichia coli O18 LPS at 15 and 30 °C for 64 h to identify endotoxin-eliminating LAB. Consequently, the AK-23 strain was screened as an endotoxin-eliminating LAB strain. The strain decreased endotoxin in YP medium without sugar at 30 °C for 64 h until 9% of endotoxin. The strain was identified as Pediococcus pentosaceus according to morphological characteristics such as its cell shape, physiological characteristics related to its fermentation type, assimilation of sugars, pH tolerance, optimum growth temperature, and molecular biological characteristics as its homology to 16S rRNA. To investigate the location of the endotoxin-eliminating substance, 4 fractions were separated from AK-23 cells as extracellular, cell wall digestion, cytoplasm, and cell membrane fractions. The endotoxin-decreasing substance, located on a cell wall, was identified as a 217 kDa protein. © 2016 Institute of Food Technologists®

  13. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    SciTech Connect

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H. )

    1988-11-01

    Binding studies were performed with two {sup 125}I-labeled Bacillus thuringiensis {delta}-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One {delta}-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other {delta}-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis {delta}-endotoxins active against M. sexta compete for binding of {sup 125}I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.

  14. Phospholipid imprinted polymers as selective endotoxin scavengers

    NASA Astrophysics Data System (ADS)

    Sulc, Robert; Szekely, Gyorgy; Shinde, Sudhirkumar; Wierzbicka, Celina; Vilela, Filipe; Bauer, David; Sellergren, Börje

    2017-03-01

    Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of polymerizable bis-imidazolium and urea hosts were evaluated as cationic and neutral hosts for phosphates and phosphonates, the latter used as mimics of the phospholipid head groups. The bis-imidazolium hosts interacted with the guests in a cooperative manner leading to the presence of tight and well defined 1:2 ternary complexes. Optimized monomer combinations were subsequently used for imprinting of phosphatidic acid as an endotoxin dummy template. Presence of the aforementioned ternary complexes during polymerization resulted in imprinting of lipid dimers – the latter believed to crudely mimic the endotoxin Lipid A motif. The polymers were characterized with respect to template rebinding, binding affinity, capacity and common structural properties, leading to the identification of polymers which were thereafter subjected to an industrially validated endotoxin removal test. Two of the polymers were capable of removing endotoxin down to levels well below the accepted threshold (0.005 EU/mg API) in pharmaceutical production.

  15. Phospholipid imprinted polymers as selective endotoxin scavengers

    PubMed Central

    Sulc, Robert; Szekely, Gyorgy; Shinde, Sudhirkumar; Wierzbicka, Celina; Vilela, Filipe; Bauer, David; Sellergren, Börje

    2017-01-01

    Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of polymerizable bis-imidazolium and urea hosts were evaluated as cationic and neutral hosts for phosphates and phosphonates, the latter used as mimics of the phospholipid head groups. The bis-imidazolium hosts interacted with the guests in a cooperative manner leading to the presence of tight and well defined 1:2 ternary complexes. Optimized monomer combinations were subsequently used for imprinting of phosphatidic acid as an endotoxin dummy template. Presence of the aforementioned ternary complexes during polymerization resulted in imprinting of lipid dimers – the latter believed to crudely mimic the endotoxin Lipid A motif. The polymers were characterized with respect to template rebinding, binding affinity, capacity and common structural properties, leading to the identification of polymers which were thereafter subjected to an industrially validated endotoxin removal test. Two of the polymers were capable of removing endotoxin down to levels well below the accepted threshold (0.005 EU/mg API) in pharmaceutical production. PMID:28303896

  16. EFFECTS OF BACTERIAL ENDOTOXIN ON RABBIT PLATELETS

    PubMed Central

    Des Prez, Roger M.; Bryant, Richard E.

    1966-01-01

    The divalent ion requirements of rabbit platelet injury by endotoxin have been defined by the use of various anticoagulant solutions and have been compared to the divalent ion requirements of platelet injury produced by addition of antigen to immune platelet-rich plasma. The endotoxin-platelet interaction takes place in citrated blood. Platelet damage by antigen is inhibited by citrate, but preincubation of antigen and immune platelet-poor plasma in the absence of citrate results in a substance, presumably antigen-antibody complement complex, which then does injure platelets in the presence of citrate. Neither endotoxin nor preincubated antigen injures platelets in the presence of sodium EDTA in concentrations sufficient to interact with all divalent cations present in plasma. These observations have been interpreted by viewing the platelet-endotoxin interaction as a consequence of platelet phagocytosis of endotoxin, a reaction not requiring complement but requiring definite small concentrations of divalent cations. The interaction of antigen and platelets is regarded as a two phase reaction, the first requiring the participation of complement and concentrations of divalent cation larger than those provided in citrated plasma, the second requiring smaller concentrations of divalent cation, no further participation of complement, and active in citrated plasma. This second phase is regarded as representing platelet phagocytosis of immune complexes. PMID:5951281

  17. Effects of endotoxin on the lactating mouse

    SciTech Connect

    Carr, J.K.

    1985-01-01

    The regulation of endogenous mouse mammary tumor virus (MMTV) sequences in trans by a host gene, the Lps locus on mouse chromosome 4, was suspected from a genetic linkage analysis. The Lps locus mediates the mouse's response to the injection of lipopolysaccharide (LPS) in the responder mouse while mice with the deficient allele are incapable of responding. Others have found that endotoxin exposure reduces milk production in lactating animals. This observation was confirmed in mice and extended by examining /sup 125/I-prolactin binding to liver membranes of lactating mice. Endotoxin treatment of responder mice increases liver prolactin binding within 15 minutes, followed by a decline over 6 hours. Scatchard analysis shows that the immediate increase comes from both increased affinity and abundance of the prolactin receptor. No such change in prolactin binding is seen in the non-responder following endotoxin treatment nor in /sup 125/I-insulin binding in responders.

  18. Delta II

    NASA Technical Reports Server (NTRS)

    1990-01-01

    The Delta II expendable launch vehicle with the ROSAT (Roentgen Satellite), cooperative space X-ray astronomy mission between NASA, Germany and United Kingdom, was launched from the Cape Canaveral Air Force Station on June 1, 1990.

  19. Combinatorial effect of Bacillus thuringiensis kurstaki and Photorhabdus luminescens against Spodoptera littoralis (Lepidoptera: Noctuidae).

    PubMed

    Benfarhat-Touzri, Dalel; Ben Amira, Amal; Ben khedher, Saoussen; Givaudan, Alain; Jaoua, Samir; Tounsi, Slim

    2014-11-01

    Spodoptera littoralis, one of the major pests of many important crop plants, is more susceptible to Bacillus thuringiensis aizawai delta-endotoxins than to those of Bacillus thuringiensis kurstaki. Within the framework of the development of efficient bioinsecticides and the prevention against insect resistance, we tested the effect of mixing B. thuringiensis kurstaki delta-endotoxins and Photorhabdus luminescens cells on S. littoralis growth. The obtained results showed that the growth inhibition of this insect was more effective when B. thuringiensis kurstaki spore-crystal mixture and Photorhabdus luminescens cells were used in combination. Furthermore, this synergism is mainly due to the presence of Cry1Ac, which is one of the three delta-endotoxins that form the crystal of B. thuringiensis kurstaki strain BNS3 in addition to Cry1Aa and Cry2Aa. This work shows a possibility to use B. thuringiensis as a delivery means for Photorhabdus bacteria in order to infect the insect hemocoel and to reduce the risk of developing resistance in the target organism. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Allantoin as a solid phase adsorbent for removing endotoxins.

    PubMed

    Vagenende, Vincent; Ching, Tim-Jang; Chua, Rui-Jing; Gagnon, Pete

    2013-10-04

    In this study we present a simple and robust method for removing endotoxins from protein solutions by using crystals of the small-molecule compound 2,5-dioxo-4-imidazolidinyl urea (allantoin) as a solid phase adsorbent. Allantoin crystalline powder is added to a protein solution at supersaturated concentrations, endotoxins bind and undissolved allantoin crystals with bound endotoxins are removed by filtration or centrifugation. This method removes an average of 99.98% endotoxin for 20 test proteins. The average protein recovery is ∼80%. Endotoxin binding is largely independent of pH, conductivity, reducing agent and various organic solvents. This is consistent with a hydrogen-bond based binding mechanism. Allantoin does not affect protein activity and stability, and the use of allantoin as a solid phase adsorbent provides better endotoxin removal than anion exchange, polymixin affinity and biological affinity methods for endotoxin clearance.

  1. Ambient endotoxin concentrations in PM10 from Southern California.

    PubMed Central

    Mueller-Anneling, Linda; Avol, Ed; Peters, John M; Thorne, Peter S

    2004-01-01

    Concentrations of endotoxin in urban air pollution have not previously been extensively characterized. We measured 24-hr levels of PM10 (particulate matter < 10 microm in aerodynamic diameter) and the associated endotoxin component once every 6 weeks for 1 year in 13 communities in Southern California. All the samples collected had detectable PM10 and endotoxin levels. The geometric mean PM10 was 34.6 microg/m3 [geometric SD (GSD), 2.1; range, 3.0-135]. By volume, the endotoxin geometric mean was 0.44 endotoxin units (EU)/m3 (GSD, 3.1; range, 0.03-5.44). Per unit material collected, the geometric mean of endotoxin collected was 13.6 EU/mg (GSD, 3.2; range, 0.7-96.8). No correlation was found between endotoxin concentrations and other ambient pollutants concurrently measured [ozone, nitrogen dioxide, total acids, or PM2.5 (particulate matter < 2.5 micro m in aerodynamic diameter]. PM10 and endotoxin concentrations were significantly correlated, most strongly in summer. Samples collected in more rural and agricultural areas had lower PM10 and mid-range endotoxin levels. The high desert and mountain communities had lower PM10 levels but endotoxin levels comparable with or higher than the rural agricultural sites. By volume, endotoxin levels were highest at sites downwind of Los Angeles, California, which were also the locations of highest PM10. Endotoxin concentrations measured in this study were all < 5.5 EU/m3, which is lower than recognized thresholds for acute adverse health effects for occupational exposures but in the same range as indoor household concentrations. This study provides the first extensive characterization of endotoxin concentration across a large metropolitan area in relation to PM10 and other pollutant monitoring, and supports the need for studies of the role of endotoxin in childhood asthma in urban settings. PMID:15064165

  2. Seasonal variability of endotoxin in ambient fine particulate matter.

    PubMed

    Carty, Cara L; Gehring, Ulrike; Cyrys, Josef; Bischof, Wolfgang; Heinrich, Joachim

    2003-12-01

    Endotoxin is a toxic, pro-inflammatory compound that has been detected in indoor air and dust in homes and occupational settings, and also in outdoor air. Data on the outdoor sampling of endotoxin are limited. Currently, little is known about the seasonal variation and influence of temperature on outdoor endotoxin levels. In the present study, we report endotoxin levels in fine fraction particulate matter with a 50% aerodynamic cutoff diameter of 2.5 microm (PM2.5) and describe the seasonal variation of endotoxin in Munich, Germany. In 1999-2000, PM2.5 was collected at forty outdoor monitoring sites across Munich. Approximately four samples were collected at each site for a total of 158 samples. Endotoxin concentrations in the PM2.5 samples were determined using the kinetic chromogenic Limulus Amebocyte Lysate (LAL) assay. The geometric mean endotoxin concentration was 1.07 EU mg PM2.5(-1) (95% C.I.: 0.915-1.251) or 0.015 EU m(-3) of sampled air (95% C.I.: 0.013-0.018). Munich endotoxin levels were significantly related to ambient temperature (p < 0.0001) and percent relative humidity (p < 0.0001). Sampling periods with higher average temperatures yielded higher levels of endotoxin in PM2.5 (r = 0.641), whereas decreases in percent relative humidity were associated with increased endotoxin levels in PM2.5 (r = -0.388). Endotoxin levels were significantly higher during the warmer seasons of spring [means ratio (MR): 2.5-2.7] and summer (MR: 2.1-3.0) than during winter. Although temperature and relative humidity do not explain all of the variability in endotoxin levels, their effects were significant in our data set. Temperature effects and seasonal variation of endotoxin should be considered in future studies of outdoor endotoxin.

  3. Removal of endotoxin from dairy wastewater

    USDA-ARS?s Scientific Manuscript database

    The efficacy of various treatments on removing endotoxin (ET) from wastewater was tested by using the treated water to induce a systemic reaction via intratracheal inoculation (20 ml/goat, 6 goats/group). Treatments (T1-T7) of wastewater were as follows: 1) autoclaved 15 min, centrifuged and contain...

  4. Enteric vascular endothelial response to bacterial endotoxin.

    PubMed Central

    Koshi, R.; Mathan, V. I.; David, S.; Mathan, M. M.

    1993-01-01

    The response of enteric vasculature to endotoxin was examined at the ultrastructural level using a murine model of endotoxin-induced acute diarrhoea. Morphological changes indicative of endothelial damage were evident as early as 15 minutes following endotoxin challenge. These changes, characterized by widening of intercellular spaces, increased microvillous projections and the appearance of stress fibres, preceded the leucocytic response. Endothelial damage increased with time, being associated with progressive degenerative changes in the plasma membrane, cytoplasm and organelles, ultimately leading to desquamation. These latter changes were temporally associated with margination of neutrophils and platelet adhesion to the denuded subendothelium. The venules were the primary site of these changes while the capillaries were the least affected. The arterioles were markedly constricted with minimal endothelial damage. These changes suggest that the enteric vascular endothelium may be an important target organ, and the resultant endothelial injury may have implications in host responses to endotoxin. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8292557

  5. Endotoxin contamination delays the foreign body reaction.

    PubMed

    van Putten, Sander M; Wübben, Maike; Plantinga, Josée A; Hennink, Wim E; van Luyn, Marja J A; Harmsen, Martin C

    2011-09-15

    Biomaterials are at continuous risk of bacterial contamination during production and application. In vivo, bacterial contamination of biomaterials delays the foreign body reaction (FBR). Endotoxins such as lipopolysaccharides (LPS), major constituents of the bacterial cell wall, are potent stimulators of the immune system in vitro and in vivo. In vitro, biomaterials contaminated with LPS induce the production of proinflammatory cytokines by adherent macrophages. This suggests that the presence of endotoxins on biomaterials will intensify the FBR. The effects of LPS on the course of the FBR have never been studied in vivo. In this study, the influence of LPS contamination on the FBR to subcutaneously implanted Puramatrix-loaded hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) disks was studied in rats. During the onset phase of the FBR, a massive influx of granulocytes was detected in LPS-contaminated disks, while their presence was prolonged. IL-10 production inside LPS-contaminated disks was increased at days 10 and 21. Macrophage densities were not affected by the presence of LPS. However, macrophage functionality was altered: giant cell formation and biomaterial degradation were delayed by LPS-contamination up to 21 days. On the basis of these results, we conclude that LPS delays the FBR. This finding indicates that endotoxin contamination has significant implications for the in vivo function of biomaterials and medical devices and emphasizes the importance of endotoxin testing. Copyright © 2011 Wiley Periodicals, Inc.

  6. Kinetics of Hydrothermal Inactivation of Endotoxins

    PubMed Central

    Li, Lixiong; Wilbur, Chris L.; Mintz, Kathryn L.

    2011-01-01

    A kinetic model was established for the inactivation of endotoxins in water at temperatures ranging from 210°C to 270°C and a pressure of 6.2 × 106 Pa. Data were generated using a bench scale continuous-flow reactor system to process feed water spiked with endotoxin standard (Escherichia coli O113:H10). Product water samples were collected and quantified by the Limulus amebocyte lysate assay. At 250°C, 5-log endotoxin inactivation was achieved in about 1 s of exposure, followed by a lower inactivation rate. This non-log-linear pattern is similar to reported trends in microbial survival curves. Predictions and parameters of several non-log-linear models are presented. In the fast-reaction zone (3- to 5-log reduction), the Arrhenius rate constant fits well at temperatures ranging from 120°C to 250°C on the basis of data from this work and the literature. Both biphasic and modified Weibull models are comparable to account for both the high and low rates of inactivation in terms of prediction accuracy and the number of parameters used. A unified representation of thermal resistance curves for a 3-log reduction and a 3 D value associated with endotoxin inactivation and microbial survival, respectively, is presented. PMID:21193667

  7. Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae)

    USDA-ARS?s Scientific Manuscript database

    Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. Proactive action to examine trypsin gene profiles and proteinase inhibitors for interaction with Bt toxin is necessary ...

  8. Bacillus thuringiensis plants expressing Cry1Ac, Cry2Ab and Cry1F are not toxic to the assassin bug, Zelus renardii

    USDA-ARS?s Scientific Manuscript database

    Cotton and maize delivering insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non-target organisms, especially natural enemies that suppress pest populations. In th...

  9. Bt crops producing Cry1Ac, Cry2Ab and Cry1F do not harm the green lacewing, chrysoperla rufilabris

    USDA-ARS?s Scientific Manuscript database

    The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Le...

  10. Using resistant prey demonstrates that Bt plants producing Cry1Ac, Cry2Ab and Cry1F have no negative effects on Geocoris punctipes and Orius insidiosus

    USDA-ARS?s Scientific Manuscript database

    Geocoris punctipes (Say) and Orius insidiosus (Say) are generalist predators found in a wide range of crops, including cotton (Gossypium hirsutum L.) and maize (Zea mays L.), where they provide important biological control services by feeding on an array of pests including eggs and small larvae of l...

  11. Interaction of proteinase inhibitors with Cry1Ac toxicity and presence of fifteen chymotrypsin cDNAs in midgut of the tobacco budworm Heliothis virescens (F.) (Lepidoptera: noctuidae)

    USDA-ARS?s Scientific Manuscript database

    Extensive adoption of Bt cotton places heavy selection pressure on lepidopteran insects. The potential development of Bt resistance is a major risk in the durability of Bt plant technology. Another concern is the surging of non-lepidopteran pests not controlled by Bt toxins. Intensive cloning and se...

  12. Down-regulation of aminopeptidase N and ABC transporter subfamily G transcripts in Cry1Ab and Cry1Ac resistant Asian corn borer, Ostrina furnacalis (Lepidoptera: Crambidae)

    USDA-ARS?s Scientific Manuscript database

    Bacillus thuringiensis (Bt) crystalline protein (Cry) toxins cause mortality by a mechanism involving pore formation or signal transduction following toxin binding to receptors along the midgut lumen of susceptible insects, but this mechanism and mutations therein that lead to resistance remain poor...

  13. Effects of different Brush Border Membrane Vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

    USDA-ARS?s Scientific Manuscript database

    The brush border membrane vesicles (BBMV) isolated from midgut cells of insect have been widely used for studying of the binding receptors and action mode of Cry proteins produced by Bacillus thuringiensis (Bt). There are several methods for isolating insect BBMV used in one-dimension electrophoresi...

  14. Airborne endotoxin in fine particulate matter in Beijing

    NASA Astrophysics Data System (ADS)

    Guan, Tianjia; Yao, Maosheng; Wang, Junxia; Fang, Yanhua; Hu, Songhe; Wang, Yan; Dutta, Anindita; Yang, Junnan; Wu, Yusheng; Hu, Min; Zhu, Tong

    2014-11-01

    Endotoxin is an important biological component of particulate matter (PM) which, upon inhalation, can induce adverse health effects, and also possibly complicate the diseases in combination with other pollutants. From 1 March 2012 to 27 February 2013 we collected air samples using quartz filters daily for the quantification of airborne endotoxin and also fine PM (PM2.5) in Beijing, China. The geometric means for endotoxin concentration and the fraction of endotoxin in PM were 0.65 EU/m3 (range: 0.10-75.02) and 10.25 EU/mg PM2.5 (range: 0.38-1627.29), respectively. The endotoxin concentrations were shown to vary greatly with seasons, typically with high values in the spring and winter seasons. Temperature and relative humidity, as well as concentrations of sulfur dioxide and nitrogen oxides were found to be significantly correlated with airborne endotoxin concentrations (p < 0.05). Additionally, positive correlations were also detected between endotoxin concentrations and natural sources of Na+, K+, Mg2+, and F-, while negative correlations were observed between endotoxin concentrations and anthropogenic sources of P, Co, Zn, As, and Tl. Oxidative potential analysis revealed that endotoxin concentrations were positively correlated with reactive oxygen species (ROS), but not dithiothreitol (DTT) of PM. This study provided the first continuous time series of airborne endotoxin concentrations in Beijing, and identifies its potential associations with atmospheric factors. The information developed here can assist in the assessment of health effects of air pollution in Beijing.

  15. Detecting endotoxin with a flow cytometry-based magnetic aptasensor.

    PubMed

    Zuo, Ming-Yan; Chen, Li-Juan; Jiang, Hao; Tan, Lin; Luo, Zhao-Feng; Wang, Yan-Mei

    2014-12-01

    Endotoxin, which is also known as lipopolysaccharide (LPS), is a marker for intruding gram-negative pathogens. It is essential to detect endotoxin quickly and sensitively in a complex milieu. A new flow cytometry (FCM)-based magnetic aptasensor assay that employs two endotoxin-binding aptamers and magnetic beads has been developed to detect endotoxin. The endotoxin-conjugated sandwich complex on magnetic beads was observed by scanning confocal laser microscopy. The resulting magnetic aptasensor rapidly detected (<1 min) endotoxin within a broad dynamic detection range of 10(-8) to 10(0)mg/ml in the presence of bovine serum albumin (BSA), RNA, sucrose, and glucose, which are most likely to coexist with endotoxin in the majority of biological liquids. Only 2 μl of magnetic aptasensor was required to quantify the endotoxin solution. Furthermore, the magnetic aptasensor could be regenerated seven times and still presented an outstanding response to the endotoxin solution. Therefore, the magnetic aptasensor exhibited high sensitivity, selectivity, and reproducibility, thereby serving as a powerful tool for the quality control and high-throughput detection of endotoxin in the food and pharmaceutical industries.

  16. Mediated effect of endotoxin and lead upon hepatic metabolism

    SciTech Connect

    Kuttner, R.E.; Ebata, T.; Schumer, W.

    1984-10-01

    A test was made of the possibility that gram-negative bacterial cell wall lipopolysaccharides acted directly on key glucoregulatory enzymes in rat liver cytosol to cause the characteristic hypoglycemia of severe endotoxemia. Fasted male rats were sensitized to endotoxin by the simultaneous intravenous injection of lead acetate. The minimum systemic dosage of endotoxin necessary to perturb the normal pattern of hepatic glycolytic intermediates was determined by serial testing with diminishing dosages of endotoxin. The hepatocyte concentration of endotoxin was then calculated from this minimum dosage by use of literature data on the fraction of endotoxin delivered to liver cells after a systemic intravenous injection of radiochromium labeled lipopolysaccharides. Accepting a molecular weight of 118,000 daltons for the smallest endotoxin monomer capable of evoking a physiologic response, the molar amount of endotoxin present in 1 gram of hepatocytes was readily calculated. The concentration of glucoregulatory enzymes in parenchymal cells was then estimated from other literature sources. It was found that the amount of endotoxin in the hepatocytes was insufficient to combine directly with even 1 per cent of the quantity of a single key glucoregulatory enzyme in liver parenchyma. Since a one to one stoichiometric reaction between endotoxin and enzyme could not occur in the liver cytosol, a direct interaction mechanism between agonist and biocatalyst can be ruled out. It is concluded that bacterial endotoxin must act on hepatic glucoregulation by an indirect mechanism presumably based upon the release and operation of mediators.

  17. Chorioamnionitis induced by subchorionic endotoxin infusion in sheep.

    PubMed

    Moss, Timothy J M; Nitsos, Ilias; Newnham, John P; Ikegami, Machiko; Jobe, Alan H

    2003-12-01

    This study was undertaken to determine whether subchorionic endotoxin infusion causes chorioamnionitis and preterm lung maturation, as occurs after intra-amniotic endotoxin. From day 118 of pregnancy, sheep received infusions of endotoxin (subchorionic 7.5 mg/d, n=11; intra-amniotic 2.5 mg/d, n=9) until delivery of lambs at 120 or 124 days. Other sheep received a single intra-amniotic injection of endotoxin (10 mg, n=7) at 118 days before delivery at 124 days. Controls (n=9) received equivalent saline solution treatments. Chorioamnionitis accompanied all endotoxin treatments. Lung inflammation occurred after intra-amniotic endotoxin infusion or injection but not after subchorionic endotoxin. Umbilical arterial pH was lower and Pco(2) was higher than control after subchorionic endotoxin. Lung compliance and surfactant were increased after intra-amniotic endotoxin infusion or injection but not after subchorionic endotoxin. Chorioamnionitis may result from inflammatory stimuli at various intrauterine sites, with different sites causing different fetal effects and not all cases of chorioamnionitis being accompanied by enhanced lung maturation.

  18. Genetic variants in endotoxin signalling pathway, domestic endotoxin exposure and asthma exacerbations.

    PubMed

    Kljaic-Bukvic, Blazenka; Blekic, Mario; Aberle, Neda; Curtin, John A; Hankinson, Jenny; Semic-Jusufagic, Aida; Belgrave, Danielle; Simpson, Angela; Custovic, Adnan

    2014-10-01

    We investigated the interaction between genetic variants in endotoxin signalling pathway and domestic endotoxin exposure in relation to asthma presence, and amongst children with asthma, we explored the association of these genetic variants and endotoxin exposure with hospital admissions due to asthma exacerbations. In a case-control study, we analysed data from 824 children (417 asthmatics, 407 controls; age 5-18 yr). Amongst asthmatics, we extracted data on hospitalization for asthma exacerbation from medical records. Endotoxin exposure was measured in dust samples collected from homes. We included 26 single-nucleotide polymorphisms (SNPs) in the final analysis (5 CD14, 7LY96 and 14 TLR4). Two variants remained significantly associated with hospital admissions with asthma exacerbations after correction for multiple testing: for CD14 SNP rs5744455, carriers of T allele had decreased risk of repeated hospital admissions compared with homozygotes for C allele [OR (95% CI), 0.42 (0.25-0.88), p = 0.01, False Discovery Rate (FDR) p = 0.02]; for LY96 SNP rs17226566, C-allele carriers were at a lower risk of hospital admissions compared with T-allele homozygotes [0.59 (0.38-0.90), p = 0.01, FDR p = 0.04]. We observed two interactions between SNPs in CD14 and LY96 with environmental endotoxin exposure in relation to hospital admissions due to asthma exacerbation which remained significant after correction for multiple testing (CD14 SNPs rs2915863 and LY96 SNP rs17226566). Amongst children with asthma, genetic variants in CD14 and LY96 may increase the risk of hospital admissions with acute exacerbations. Polymorphisms in endotoxin pathway interact with domestic endotoxin exposure in further modification of the risk of hospitalization. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates.

    PubMed

    Ochiai, Masaki; Yamamoto, Akihiko; Naito, Seishiro; Maeyama, Jun-Ichi; Masumi, Atsuko; Hamaguchi, Isao; Horiuchi, Yoshinobu; Yamaguchi, Kazunari

    2010-11-01

    Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1-3)-β-D-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.

  20. Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences

    PubMed Central

    2013-01-01

    Endotoxin tolerance was first described in a study that exposed animals to a sublethal dose of bacterial endotoxin. The animals subsequently survived a lethal injection of endotoxin. This refractory state is associated with the innate immune system and, in particular, with monocytes and macrophages, which act as the main participants. Several mechanisms are involved in the control of endotoxin tolerance; however, a full understanding of this phenomenon remains elusive. A number of recent reports indicate that clinical examples of endotoxin tolerance include not only sepsis but also diseases such as cystic fibrosis and acute coronary syndrome. In these pathologies, the risk of new infections correlates with a refractory state. This review integrates the molecular basis and clinical implications of endotoxin tolerance in various pathologies. PMID:24229432

  1. Endotoxin Inhalation Alters Lung Development in Neonatal Mice

    PubMed Central

    Kulhankova, Katarina; George, Caroline L.S.; Kline, Joel N.; Darling, Melissa; Thorne, Peter S.

    2012-01-01

    Background Childhood asthma is a significant public health problem. Epidemiologic evidence suggests an association between childhood asthma exacerbations and early life exposure to environmental endotoxin. Although the pathogenesis of endotoxin-induced adult asthma is well studied, questions remain about the impact of environmental endotoxin on pulmonary responsiveness in early life. Methods We developed a murine model of neonatal/juvenile endotoxin exposures approximating those in young children and evaluated the lungs inflammatory and remodeling responses. Results Persistent lung inflammation induced by the inhalation of endotoxin in early life was demonstrated by the influx of inflammatory cells and pro-inflammatory mediators to the airways and resulted in abnormal alveolarization. Conclusions Results of this study advance the understanding of the impact early life endotoxin inhalation has on the lower airways, and demonstrates the importance of an experimental design that approximates environmental exposures as they occur in young children. PMID:22576659

  2. Effects of endotoxin on monoamine metabolism in the rat.

    NASA Technical Reports Server (NTRS)

    Pohorecky, L. A.; Wurtman, R. J.; Taam, D.; Fine, J.

    1972-01-01

    Examination of effects of administered endotoxin on catecholamine metabolism in the rat brain, sympathetic neurons, and adrenal medulla. It is found that endotoxin, administered intraperitoneally, lowers the norepinephrine content in peripheral sympathetic neurons and the brain, and the catecholamine content in the adrenal medulla. It also accelerates the disappearance of H3-norepinephrine from all these tissues. It is therefore suggested that the effects of endotoxin on body temperature may be mediated in part by central non-adrenergic neurons.

  3. Predictors of Endotoxin Levels in U.S. Housing

    PubMed Central

    Thorne, Peter S.; Cohn, Richard D.; Mav, Deepak; Arbes, Samuel J.; Zeldin, Darryl C.

    2009-01-01

    Background The relationship of domestic endotoxin exposure to allergy and asthma has been widely investigated. However, few studies have evaluated predictors of household endotoxin, and none have done so for multiple locations within homes and on a national scale. Objectives We assayed 2,552 house dust samples in a nationwide study to understand the predictors of household endotoxin in bedroom floors, family room floors, beds, kitchen floors, and family room sofas. Methods Reservoir house dust from five locations within homes was assayed for endotoxin and demographic and housing information was assessed through questionnaire and onsite evaluation of 2,456 residents of 831 homes selected to represent national demographics. We performed repeated-measures analysis of variance (rANOVA) for 37 candidate variables to identify independent predictors of endotoxin. Meteorologic data were obtained for each primary sampling unit and tested as predictors of indoor endotoxin to determine if wetter or warmer microclimates were associated with higher endotoxin levels. Results Weighted geometric mean endotoxin concentration ranged from 18.7 to 80.5 endotoxin units (EU)/mg for the five sampling locations, and endotoxin load ranged from 4,160 to 19,500 EU/m2. Bivariate analyses and rANOVA demonstrated that major predictors of endotoxin concentration were sampling location in the home, census division, educational attainment, presence of children, current dog ownership, resident-described problems with cockroaches, food debris, cockroach stains, and evidence of smoking observed by field staff. Low household income entered the model if educational attainment was removed. Conclusion Increased endotoxin in household reservoir dust is principally associated with poverty, people, pets, household cleanliness, and geography. PMID:19479019

  4. Silica enhancement of murine endotoxin sensitivity.

    PubMed

    Vogel, S N; English, K E; O'Brien, A D

    1982-11-01

    Silica has been used for many years as an agent which selectively alters macrophage functions and, as such, has been used to assess the role of macrophages in the immune response to a variety of microbial and chemically defined agents. Silica treatment of C3H/HeN mice 1 day before challenge with protein-free Escherichia coli endotoxin (lipopolysaccharide [LPS]) resulted in a marked increase in LPS sensitivity, as evidenced by accelerated signs of endotoxemia as well as a fourfold decrease in the LPS 50% lethal dose. The silica-mediated increase in responsiveness to LPS was associated with increased production of macrophage-derived soluble factors both in vivo (interferon) and in vitro (Interleukin 1; previously referred to as lymphocyte activating factor or LAF) upon endotoxin stimulation. These findings support the central role of the macrophage and its products in mediating endotoxic reactions.

  5. Endotoxin-induced uveitis in rodents.

    PubMed

    Yadav, Umesh C S; Ramana, Kota V

    2013-01-01

    Uveitis is a common cause of vision loss, accounting for 10-15 % of all cases of blindness worldwide and affects individuals of all ages, genders, and races. Uveitis represents a broad range of intraocular inflammatory conditions due to complications of autoimmune diseases, bacterial infections, viral infections, and chemical and metabolic injuries. Endotoxin-induced uveitis (EIU) in rodents is an efficient experimental model to investigate the pathological mechanism and pharmacological efficacy of potential drug agents. EIU is characterized by clinically relevant classical signs of inflammation, including inflammatory exudates and cells in the anterior and vitreous chambers. EIU in small animal models such as rats, mice, and rabbits is a short-lived uveal inflammation that can be developed subsequent to administration of bacterial endotoxin, such as lipopolysaccharide. Here, we present a reproducible, reliable, and simplified protocol to induce EIU in mice. This method could be used with similar efficacy for EIU induction in other small animals as well.

  6. Are Cockroaches an Important Source of Indoor Endotoxins?

    PubMed Central

    Lai, Ka Man

    2017-01-01

    Endotoxins are common indoor biocontaminants. Their levels have been shown to link to many sources and factors. One of them is cockroach infestation but the role of cockroaches and contamination mechanisms are unclear. We hypothesized that not only is cockroach infestation a sign of poor hygiene, but it also contributes to indoor endotoxins via fecal contamination. In this study, different cockroach species were caught in homes. The endotoxin and allergen levels and their ratios in cockroach feces were determined. To estimate the amount of indoor endotoxins that originated from cockroaches, a new approach of using these new cockroach endotoxin and allergen ratios to compare with environmental data was employed. We found that Supella (S.) longipalpa, Periplaneta (P.) australasiae, and Blattella (B.) germanica were dominant in homes. On average, P. australasiae feces had a higher level but greater variation of endotoxins. B. germanica feces had the highest levels of allergens measured. Depending on environmental bacterial load and the type of cockroaches present, cockroach endotoxins in the environment may vary greatly. Cockroaches directly contribute to indoor endotoxins rather than just being a sign of poor hygiene. The type and extent of cockroach infestation should be taken into consideration when assessing and remediating indoor endotoxin contamination. PMID:28106812

  7. Fluorescent nanodiamonds as highly stable biomarker for endotoxin verification

    NASA Astrophysics Data System (ADS)

    Bergmann, Thorsten; Burg, Jan Michael; Lilholt, Maria; Maeder, Ulf; Beer, Sebastian; Salzig, Denise; Ebrahimi, Mehrdad; Czermak, Peter; Fiebich, Martin

    2012-03-01

    Fluorescent nanodiamonds (ND) provide advantageous properties as a fluorescent biomarker for in vitro and in vivo studies. The maximum fluorescence occurs around 700 nm, they do not show photobleaching or blinking and seem to be nontoxic. After a pretreatment with strong acid fluorescent ND can be functionalized and coupled to endotoxin. Endotoxin is a decay product of bacteria and causes strong immune reactions. Therefore endotoxin has to be removed for most applications. An effective removal procedure is membrane filtration. The endotoxin, coupled to fluorescent ND can be visualized by using confocal microscopy which allows the investigation of the separation mechanisms of the filtration process within the membranes.

  8. Risks associated with endotoxins in feed additives produced by fermentation.

    PubMed

    Wallace, R John; Gropp, Jürgen; Dierick, Noël; Costa, Lucio G; Martelli, Giovanna; Brantom, Paul G; Bampidis, Vasileios; Renshaw, Derek W; Leng, Lubomir

    2016-01-15

    Increasingly, feed additives for livestock, such as amino acids and vitamins, are being produced by Gram-negative bacteria, particularly Escherichia coli. The potential therefore exists for animals, consumers and workers to be exposed to possibly harmful amounts of endotoxin from these products. The aim of this review was to assess the extent of the risk from endotoxins in feed additives and to calculate how such risk can be assessed from the properties of the additive. Livestock are frequently exposed to a relatively high content of endotoxin in the diet: no additional hazard to livestock would be anticipated if the endotoxin concentration of the feed additive falls in the same range as feedstuffs. Consumer exposure will be unaffected by the consumption of food derived from animals receiving endotoxin-containing feed, because the small concentrations of endotoxin absorbed do not accumulate in edible tissues. In contrast, workers processing a dusty additive may be exposed to hazardous amounts of endotoxin even if the endotoxin concentration of the product is low. A calculation method is proposed to compare the potential risk to the worker, based on the dusting potential, the endotoxin concentration and technical guidance of the European Food Safety Authority, with national exposure limits.

  9. Are Cockroaches an Important Source of Indoor Endotoxins?

    PubMed

    Lai, Ka Man

    2017-01-18

    Endotoxins are common indoor biocontaminants. Their levels have been shown to link to many sources and factors. One of them is cockroach infestation but the role of cockroaches and contamination mechanisms are unclear. We hypothesized that not only is cockroach infestation a sign of poor hygiene, but it also contributes to indoor endotoxins via fecal contamination. In this study, different cockroach species were caught in homes. The endotoxin and allergen levels and their ratios in cockroach feces were determined. To estimate the amount of indoor endotoxins that originated from cockroaches, a new approach of using these new cockroach endotoxin and allergen ratios to compare with environmental data was employed. We found that Supella (S.) longipalpa, Periplaneta (P.) australasiae, and Blattella (B.) germanica were dominant in homes. On average, P. australasiae feces had a higher level but greater variation of endotoxins. B. germanica feces had the highest levels of allergens measured. Depending on environmental bacterial load and the type of cockroaches present, cockroach endotoxins in the environment may vary greatly. Cockroaches directly contribute to indoor endotoxins rather than just being a sign of poor hygiene. The type and extent of cockroach infestation should be taken into consideration when assessing and remediating indoor endotoxin contamination.

  10. Bacterial endotoxins: biological properties and mechanisms of action

    PubMed Central

    Freudenberg, M. A.

    1993-01-01

    Endotoxins (lipopolysaccharides, LPS) are agents of pathogenicity of Gram-negative bacteria, implicated in the development of Gram-negative shock. Endotoxin reacts with lipopolysaccharide-sensitive cells producing endogenous mediators such as tumour necrosis factor alpha (TNFα). Macrophages are cells mediating the toxic activities of LPS and TNFα is the primary mediator of the lethal action of endotoxin. This review article discusses the various mechanisms by which endotoxin hypersensitivity in bacteria-sensitized animals develops. The paper concludes with a discussion on the possible protective effect of carnitine congeners against the lethal action of LPS. PMID:18475562

  11. Hormone and Cytokine Responses to Repeated Endotoxin Exposures-No Evidence of Endotoxin Tolerance After 5 Weeks in Humans.

    PubMed

    Rittig, Nikolaj; Thomsen, Henrik H; Bach, Ermina; Jørgensen, Jens Otto L; Møller, Niels

    2015-07-01

    Endotoxin administrations are used in experimental models of inflammatory disease. Short-term endotoxin tolerance in response to repeated endotoxin exposure is well known, but the duration of endotoxin tolerance in humans remains unknown. The main purpose of this study was to test whether endotoxin tolerance is present in vivo when separating endotoxin exposures with more than 5 weeks, a time span often used between individual investigations in clinical experimental studies. Seventeen healthy young men were exposed twice to Escherichia coli endotoxin. The inflammatory response was calculated as area under the curve between the first and second endotoxin exposures for heart rate, mean arterial blood pressure, temperature, cortisol, tumor necrosis factor α, and interleukin (IL)-1β, IL-6, and IL-10. The median interval between exposures was 90 days (range, 37-244). The ratio between the inflammatory responses during the second and the first endotoxin exposures was 0.89 ± 0.09 (P = 0.28) for tumor necrosis factor α, 0.96 ± 0.07 (P = 0.53) for IL-1β, 0.97 ± 0.11 (P = 0.78) for IL-6, 1.30 ± 0.18 (P = 0.12) for IL-10, and 0.92 ± 0.04 (P = 0.10) for cortisol. Our data do not show evidence of in vivo tolerance to repeated endotoxin exposure when administrations are separated with at least 5 weeks. This observation is important in the planning and interpretation of future experimental endotoxin studies.

  12. Mississippi Delta

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The streamers of clouds draped over the Gulf of Mexico in this true-color MODIS image from February 27, 2002, suggest that a cold, dry wind was blowing southward over the United States and began to pick up moisture over the Gulf, causing these strips of clouds. That the clouds didn't pick up until some distance from the coastline allowed MODIS to get a perfect view of the dynamic Gulf Coast environment spanning (left to right) Texas, Louisiana, Mississippi, Alabama, and Florida's Western Panhandle. The Mississippi River runs roughly down the center of the image, and is joined in Louisiana by the Red River coming in from the northwest. Over the past 7000 years, the actual delta, where the main river channel empties into the Gulf, has wandered around what we now think of as the Louisiana coast. Considering all the sediment visible in this image, it's not hard to imagine that the river carries about 2.4 billion kilograms of sediment into the Gulf each year. Deposition of some of this sediment has been building up the current delta, called the Birdfoot Delta, for obvious reasons, for about 700 years. The coastal waters are alive with microscopic organisms called phytoplankton, which contain colorful pigments, including chlorophyll, for harvesting sunlight. Beyond the sediment plume off Louisiana, the waters are very dark, which could indicate that a large amount of chlorophyll is present, absorbing lots of sunlight and causing the water to appear dark. Farther south, the waters appear bright blue, which could be a signature of coccolithophores, which use highly reflective calcium carbonate to build scaly coverings for themselves. The brighter offshore waters could also be caused by a blue-green algae called Trichodesmium, an organism that can not only harness carbon dioxide for photosynthesis, but can also take nitrogen from the air and turn it into a form that can be used by living organisms. Credit: Jacques Descloitres, MODIS Land Rapid Response Team, NASA/GSFC

  13. Mississippi Delta

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The streamers of clouds draped over the Gulf of Mexico in this true-color MODIS image from February 27, 2002, suggest that a cold, dry wind was blowing southward over the United States and began to pick up moisture over the Gulf, causing these strips of clouds. That the clouds didn't pick up until some distance from the coastline allowed MODIS to get a perfect view of the dynamic Gulf Coast environment spanning (left to right) Texas, Louisiana, Mississippi, Alabama, and Florida's Western Panhandle. The Mississippi River runs roughly down the center of the image, and is joined in Louisiana by the Red River coming in from the northwest. Over the past 7000 years, the actual delta, where the main river channel empties into the Gulf, has wandered around what we now think of as the Louisiana coast. Considering all the sediment visible in this image, it's not hard to imagine that the river carries about 2.4 billion kilograms of sediment into the Gulf each year. Deposition of some of this sediment has been building up the current delta, called the Birdfoot Delta, for obvious reasons, for about 700 years. The coastal waters are alive with microscopic organisms called phytoplankton, which contain colorful pigments, including chlorophyll, for harvesting sunlight. Beyond the sediment plume off Louisiana, the waters are very dark, which could indicate that a large amount of chlorophyll is present, absorbing lots of sunlight and causing the water to appear dark. Farther south, the waters appear bright blue, which could be a signature of coccolithophores, which use highly reflective calcium carbonate to build scaly coverings for themselves. The brighter offshore waters could also be caused by a blue-green algae called Trichodesmium, an organism that can not only harness carbon dioxide for photosynthesis, but can also take nitrogen from the air and turn it into a form that can be used by living organisms. Credit: Jacques Descloitres, MODIS Land Rapid Response Team, NASA/GSFC

  14. General effect of endotoxin on glucocorticoid receptors in mammalian tissues

    SciTech Connect

    Stith, R.D.; McCallum, R.E.

    1986-01-01

    Considering the ubiquitous nature of glucocorticoid actions and the fact that endotoxin inhibits glucocorticoid action in the liver, we proposed to examine whether endotoxin affected extrahepatic actions of glucocorticoids. Fasted C57BL/6J mice were injected intraperitoneally with endotoxin (LD50) at 0800 and were killed 6 h later. Control mice were injected with an equal volume of saline. /sup 3/H-dexamethasone binding, measured by a new cytosol exchange assay utilizing molybdate plus dithiothreitol, in liver, kidney, skeletal muscle, spleen, lung, and heart tissue was significantly lower in treated than in control mice. The equilibrium dissociation constants were not significantly different, but the number of available binding sites in each tissue was reduced by endotoxin treatment. Phosphoenolpyruvate carboxykinase activity was significantly reduced in liver but not in kidney. Endotoxin treatment lowered glycogen content in liver but not in skeletal muscle. The reduction observed in the a form of liver glycogen synthase due to endotoxin was not seen in skeletal muscle glycogen synthase a. These data support the proposal that endotoxin or a mediator of its action inhibits systemic glucocorticoid action. The results also emphasize the central role of the liver in the metabolic disturbances of the endotoxin-treated mouse.

  15. Airborne endotoxin in different background environments and seasons.

    PubMed

    Madsen, Anne Mette

    2006-01-01

    Endotoxin is a cell wall component from Gram-negative bacteria, and inhaled endotoxin contributes significantly to the induction of airway inflammation and dysfunction. Background levels of endotoxin have not yet been extensively described. In this study, airborne endotoxin was measured with a standardized protocol in 5 types of background environment (169 samples) in Denmark from October to May. Endotoxin levels in a greenhouse (median = 13.2 EU/m3) were significantly higher than in the other environments. The air from biofuel plants (median = 5.3 EU/m3), the air on congested streets (median = 4.4 EU/m3) and on an agricultural field (median = 2.9 EU/m3) had higher endotoxin contents than the air in industrial areas (median = 1.3 EU/m3) or in towns (median = 0.33 EU/m3). Levels in industrial areas were significantly higher than in towns. A literature study revealed background levels of endotoxin on different continents between 0.063-410 EU/m3, with median or mean values between 0.063-3.6 EU/m3. Endotoxin concentrations in towns and industrial areas were higher in April and May than in autumn and winter, and were higher in October than in winter. These data of exposure in background environments and of seasonal variation are helpful for public health practitioners, epidemiologists and industrial hygienists.

  16. EFFECTS OF LIME (CAO) ON THE ENDOTOXIN LEVELS OF BIOSOLIDS

    EPA Science Inventory

    Lime addition is a common practice for treating biosolids in order to meet EPA 503 requirements for land application. Since this treatment kills the majority of microorganisms, will it increase the level of endotoxins present in biosolids? And, if endotoxin levels are increased, ...

  17. EFFECTS OF LIME (CAO) ON THE ENDOTOXIN LEVELS OF BIOSOLIDS

    EPA Science Inventory

    Lime addition is a common practice for treating biosolids in order to meet EPA 503 requirements for land application. Since this treatment kills the majority of microorganisms, will it increase the level of endotoxins present in biosolids? And, if endotoxin levels are increased, ...

  18. Endotoxin Exposure Is a Risk Factor for Asthma

    PubMed Central

    Thorne, Peter S.; Kulhánková, Katarina; Yin, Ming; Cohn, Richard; Arbes, Samuel J.; Zeldin, Darryl C.

    2005-01-01

    Background: Although research has shown that early life exposure to household endotoxin protects against development of allergies, studies are less clear on the relationship between household endotoxin exposure and prevalence of wheezing and asthma. We as- sayed 2,552 house dust samples in a representative nationwide sam- ple to explore relationships between endotoxin exposures and risk factors for asthma, asthma symptoms, and medication use. Methods: House dust was vacuum-sampled from five locations within homes and assayed for endotoxin. Health, demographic, and housing information was assessed through questionnaire and on-site evaluation of 2,456 residents of 831 homes selected to represent the demographics of the United States. Results: Endotoxin concentration (EU/mg) and load (EU/m2) were highly correlated (r = 0.73–0.79). Geometric mean endotoxin concentrations were as follows (in EU/mg): bedroom floors, 35.3 (5th–95th percentile, 5.0–260); bedding, 18.7 (2.0–142); family room floors, 63.9 (11.5–331); sofas, 44.8 (6.4–240); and kitchen floors, 80.5 (9.8–512). Multivariate analysis demonstrated significant relationships between increasing endotoxin levels and diagnosed asthma, asthma symptoms in the past year, current use of asthma medications, and wheezing among residents of the homes. These relationships were strongest for bedroom floor and bedding dust and were observed in adults only. Modeling the joint effect of bedding and bedroom floor endotoxin on recent asthma symptoms yielded an adjusted odds ratio of 2.83 (95% confidence interval, 1.01–7.87). When stratified by allergy status, allergic subjects with higher endotoxin exposure were no more likely to have diagnosed asthma or asthma symptoms than nonallergic subjects. Conclusion: This study demonstrates that household endotoxin exposure is a significant risk factor for increased asthma prevalence. PMID:16141442

  19. Endotoxin detection in end-stage kidney disease.

    PubMed

    Wong, Jonathan; Jeraj, Hassan; Vilar, Enric; Viljoen, Adie; Farrington, Ken

    2015-01-01

    Endotoxin detection assays are not validated for use in end-stage kidney disease (ESKD). We investigated the accuracy and precision of the kinetic turbidimetric Limulus amoebocyte lysate (LAL) assay to detect endotoxin in plasma from patients with ESKD. Optimisation of endotoxin recovery from plasma using the detergent Tween 80 was also explored. Plasma samples from 7 patients with ESKD and 7 healthy subjects were spiked with different concentrations of endotoxin. Repeated measurements for endotoxin at each level of spike were performed to assess the accuracy and precision of spike recovery. Endotoxin recovery in plasma samples diluted in Tween 80 and water was compared. Mean endotoxin spike recovery was 111.6% and 125.2% in ESKD and healthy subjects, respectively. There was no statistical difference in spike recovery between ESKD and healthy plasma. Precision of the LAL assay in plasma spiked with low (0.05 EU/mL) and high (0.5 EU/mL) concentration of endotoxin spikes was 24.1% and 8.9%, respectively. The use of Tween 80 as a diluent for plasma significantly improved spike recovery in ESKD plasma (100.1% vs 70.4%, p<0.001). The kinetic LAL turbidimetric assay is a valid tool for the detection of blood endotoxin in patients with ESKD, although in blood specimens with low-level endotoxemia (≤0.05 EU/mL) the assay may be less accurate and precise. Tween 80 can be used as a diluent to optimise recovery of endotoxin in ESKD plasma. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  20. Endotoxins in tobacco smoke: shifting tobacco industry positions.

    PubMed

    Barnes, Richard L; Glantz, Stanton A

    2007-10-01

    In the 1980s, the tobacco industry started a campaign to divert attention away from secondhand tobacco smoke (SHS) as a major source of indoor air pollution in workplaces by highlighting the roles of other indoor air pollutants. The industry, working through "third parties," highlighted endotoxins, naturally occurring substances that cause numerous inflammatory reactions in humans, as an alternative explanation to SHS as causing indoor air problems. In 1995, Hasday and colleagues were the first to present findings that cigarette smoke contains significant quantities of endotoxins. This discovery surprised tobacco industry scientists. The 1999 publication of the full Hasday et al. findings received only limited media attention but got the full attention of Philip Morris scientists concerned about a new public health issue and a new basis for regulation of workplace smoking by the U.S. Occupational Safety and Health Administration, which already regulated workplace endotoxin exposures from other sources. Philip Morris undertook an internal endotoxin research project to test the Hasday et al. findings and to determine if endotoxin-free cigarettes were possible. Although experiments were conducted to remove endotoxin from the tobacco, there is no evidence that they were successful. Following confirmation of SHS as an important source of endotoxins, the scientist promoting endotoxins as an important indoor air pollutant for the tobacco industry softened his position on the role of endotoxins as indoor pollutants. The presence of endotoxins in SHS provides an additional mechanism for the adverse effects of SHS that should be researched further, and the risk of exposure should be assessed.

  1. BIOLOGICAL PROPERTIES OF PARENT ENDOTOXINS AND LIPOID FRACTIONS, WITH A KINETIC STUDY OF ACID-HYDROLYZED ENDOTOXIN

    PubMed Central

    Haskins, Willard T.; Landy, Maurice; Milner, Kelsey C.; Ribi, Edgar

    1961-01-01

    The biological potencies of a number of lipid fractions separated from endotoxins by acid hydrolysis, including the material known as lipid A, were determined in parallel with those of their parent endotoxins, employing bio-assays based on the following dose-related host responses: fever, resistance to infection, tumor damage, primary inflammation of skin, and lethality. Without exception, lipid fractions dispersed by detergents exerted less than 1 per cent of the biological activity of the potent endotoxins from which they were derived. A study was made of the rate at which biologic activities diminished in relation to the release of bound lipid during progressive hydrolysis of Salmonella enteritidis endotoxin with dilute acid. Each of the five assays for endotoxin revealed that biological activity had been reduced to negligible proportions prior to any significant liberation from the endotoxin of water-insoluble firmly bound lipid. The major pharmacological activity of endotoxins, therefore, is acid-labile and cannot be accounted for in isolated lipids. This conclusion is also supported by the finding that lipids with activity similar to that of lipid A could be obtained by non-hydrolytic methods without diminishing the potency of the parent endotoxins. PMID:13905235

  2. Collagenase Production by Endotoxin-Activated Macrophages

    PubMed Central

    Wahl, Larry M.; Wahl, Sharon M.; Mergenhagen, Stephan E.; Martin, George R.

    1974-01-01

    Peritoneal exudate macrophages, when exposed to bacterial lipopolysaccharide in culture, were found to produce collagenase (EC 3.4.24.3). This enzyme was not detected in extracts of the macrophages or in media from nonstimulated macrophage cultures. Lipidcontaining fractions of the lipopolysaccharide, including a glycolipid from the rough mutant of Salmonella minnesota (R595) and lipid A, were potent stimulators of collagenase production. The lipid-free polysaccharide fraction had no effect. Cycloheximide prevented the production of collagenase by endotoxin-treated macrophages, suggesting that it was newly synthesized. Images PMID:4372628

  3. Polymorphisms of endotoxin pathway and endotoxin exposure: in vitro IgE synthesis and replication in a birth cohort.

    PubMed

    Sahiner, U M; Semic-Jusufagic, A; Curtin, J A; Birben, E; Belgrave, D; Sackesen, C; Simpson, A; Yavuz, T S; Akdis, C A; Custovic, A; Kalayci, O

    2014-12-01

    Genetic variants in endotoxin signaling pathway are important in modulating the effect of environmental endotoxin on asthma and atopic phenotypes. Our objective was to determine the single nucleotide polymorphisms (SNPs) in the endotoxin signaling pathway that may influence in vitro IgE synthesis and to investigate the relationship between these variants and endotoxin exposure in relation to the development of asthma and atopy in a birth cohort. Peripheral blood mononuclear cells from 45 children with asthma were stimulated with 2 and 200 ng/ml lipopolysaccharide in vitro and IgE was measured in the culture supernatants. Children were genotyped for 121 SNPs from 30 genes in the endotoxin signaling pathway. Variants with a dose-response IgE production in relation to lipopolysaccharide (LPS) were selected for replication in a population-based birth cohort, in which we investigated the interaction between these SNPs and endotoxin exposure in relation to airway hyper-responsiveness, wheeze, and atopic sensitization. Twenty-one SNPs in nine genes (CD14, TLR4, IRF3, TRAF-6, TIRAP, TRIF, IKK-1, ST-2, SOCS1) were found to modulate the effect of endotoxin on in vitro IgE synthesis, with six displaying high linkage disequilibrium. Of the remaining 15 SNPs, for seven we found significant relationships between genotype and endotoxin exposure in the genetic association study in relation to symptomatic airway hyper-responsiveness (CD14-rs2915863 and rs2569191, TRIF-rs4807000), current wheeze (ST-2-rs17639215, IKK-1-rs2230804, and TRIF-rs4807000), and atopy (CD14-rs2915863 and rs2569192, TRAF-6-rs5030411, and IKK-1-rs2230804). Variants in the endotoxin signaling pathway are important determinants of asthma and atopy. The genotype effect is a function of the environmental endotoxin exposure. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Mississippi Delta

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The Mississippi River delta teems with sediment deposited by the river as it flows into the Gulf of Mexico in this true-color image captured by MODIS on October 15, 2001. The sediment, which is marked by brown swirls in the Gulf, provides nutrients for the bloom of phytoplankton visible as blue-green swirls off the coastline. In the high-resolution image the city of Memphis can be seen in the southwest corner of Tennessee, which is just to left of center at the top of the image. The brown coloration that encompasses Memphis and either side of the river, as flows north to south along the left side of the image, is the river's flood plain. Also visible, in the upper-right hand corner of the image is the southern end of the Appalachian Mountains.

  5. Compositions of seed, forage, and processed fractions from insect-protected soybean MON 87701 are equivalent to those of conventional soybean.

    PubMed

    Berman, Kristina H; Harrigan, George G; Riordan, Susan G; Nemeth, Margaret A; Hanson, Christy; Smith, Michelle; Sorbet, Roy; Zhu, Eddie; Ridley, William P

    2009-12-09

    Monsanto Co. has developed biotechnology-derived, insect-protected soybean MON 87701 that produces the Cry1Ac insecticidal crystal (delta-endotoxin) protein derived from Bacillus thuringiensis (Bt) subsp. kurstaki. Cry1Ac provides protection from feeding damage caused by certain targeted lepidopteran pests. The purpose of this work was to assess whether the compositions of seed, forage, and processed fractions (meal, oil, protein isolate, and lecithin) of MON 87701 are comparable to those of conventional soybean. Compositional analyses were conducted on seed and forage tissues harvested from MON 87701 and conventional soybean grown in multiple replicated sites in the United States during the 2007 growing season and in Argentina during the 2007-2008 growing season. Seed, forage, and processed fractions from conventional soybean varieties currently in the marketplace were included in the analyses to establish a range of natural variability for each compositional component; the range of variability was defined by a 99% tolerance interval. Additional seed was collected from soybean grown in a separate U.S. production during the 2007 season. This seed and processed fractions (meal, oil, protein isolate, and crude lecithin) derived from it were also subjected to compositional analyses. Forage samples were analyzed for levels of proximates (ash, fat, moisture, and protein), carbohydrates by calculation, and fiber. Seed samples were analyzed for proximates, carbohydrates by calculation, fiber, amino acids, fatty acids, antinutrients, and vitamin E. Toasted, defatted (TD) meal was analyzed for proximates, fiber, amino acids, and antinutrients. Refined, bleached, and deodorized (RBD) oil was analyzed for fatty acids and vitamin E. Protein isolate was analyzed for amino acids and moisture. Crude lecithin was analyzed for phosphatides. Overall, results demonstrated that the seed, forage, and processed fractions of MON 87701 are compositionally equivalent to those of

  6. Intestinal radiation syndrome: sepsis and endotoxin

    SciTech Connect

    Geraci, J.P.; Jackson, K.L.; Mariano, M.S.

    1985-03-01

    Rats were whole-body irradiated with 8-MeV cyclotron-produced neutrons and /sup 137/Cs ..gamma.. rays to study the role of enteric bacteria and endotoxin in the intestinal radiation syndrome. Decrease in intestinal weight was used as an index of radiation-induced breakdown of the mucosa. Neutron and ..gamma..-ray doses that were sublethal for intestinal death resulted in a dose-dependent decrease in intestinal weight, reaching minimal values 2 to 3 days after exposure, followed by recovery within 5 days after irradiation. Neutron and photon doses that caused intestinal death resulted in greater mucosal breakdown with little or no evidence of mucosal recovery. The presence of fluid in the intestine and diarrhea, but not bacteremia or endotoxemia, were related to mucosal breakdown and recovery. Neither sepsis nor endotoxin could be detected in liver samples taken at autopsy from animals which died a short time earlier from intestinal injury. These results suggest that overt sepsis and endotoxemia do not play a significant role in the intestinal radiation syndrome.

  7. Contamination of nanoparticles by endotoxin: evaluation of different test methods

    PubMed Central

    2012-01-01

    Background Nanomaterials can be contaminated with endotoxin (lipopolysaccharides, LPS) during production or handling. In this study, we searched for a convenient in vitro method to evaluate endotoxin contamination in nanoparticle samples. We assessed the reliability of the commonly used limulus amebocyte lysate (LAL) assay and an alternative method based on toll-like receptor (TLR) 4 reporter cells when applied with particles (TiO2, Ag, CaCO3 and SiO2), or after extraction of the endotoxin as described in the ISO norm 29701. Results Our results indicate that the gel clot LAL assay is easily disturbed in the presence of nanoparticles; and that the endotoxin extraction protocol is not suitable at high particle concentrations. The chromogenic-based LAL endotoxin detection systems (chromogenic LAL assay and Endosafe-PTS), and the TLR4 reporter cells were not significantly perturbed. Conclusion We demonstrated that nanoparticles can interfere with endotoxin detection systems indicating that a convenient test method must be chosen before assessing endotoxin contamination in nanoparticle samples. PMID:23140310

  8. Streptomycetes in house dust: associations with housing characteristics and endotoxin

    PubMed Central

    Johansson, Elisabet; Vesper, Stephen; Levin, Linda; LeMasters, Grace; Grinshpun, Sergey; Reponen, Tiina

    2011-01-01

    In addition to mold, indoor bioaerosols also contain bacterial components that may have implications for human health. Endotoxin is a cell wall component in Gram-negative bacteria present at varying levels indoors that has been found to have respiratory health implications. Streptomyces is a large genus of Gram-positive bacteria, and some species have been shown to produce inflammatory reactions in vitro and in vivo. The aim of this study was to determine predictors of streptomycetes levels in house dust, and to compare the variation in streptomycetes levels with that in endotoxin levels. Dust was collected by floor vacuuming from 178 homes in the Cincinnati metropolitan area. streptomycetes levels were measured by quantitative PCR and endotoxin was assayed by the Limulus Amebocyte Lysate method. Associations between home characteristics and bacterial contaminants, expressed as concentration and load, were investigated through multiple regression analyses. The presence of two or more dogs was a strong predictor of both streptomycetes and endotoxin levels. Season of dust collection and levels of outdoor molds were predictors of streptomycetes but not endotoxin levels. In contrast, number of inhabitants was a significant predictor of endotoxin load only. Neither streptomycetes nor endotoxin levels were associated with metrics of moisture damage. PMID:21204988

  9. Duration of in vivo endotoxin tolerance in horses.

    PubMed

    Holcombe, Susan J; Jacobs, Carrie C; Cook, Vanessa L; Gandy, Jeffery C; Hauptman, Joseph G; Sordillo, Lorraine M

    2016-05-01

    Endotoxemia models are used to study mechanisms and treatments of early sepsis. Repeated endotoxin exposures induce periods of endotoxin tolerance, characterized by diminished proinflammatory responses to lipopolysaccharide (LPS) and modulated production of proinflammatory cytokines. Repeated measure designs using equine endotoxemia models are rarely performed, despite the advantages associated with reduced variability, because the altered responsiveness would confound study results and because the duration of equine endotoxin tolerance is unknown. We determined the interval of endotoxin tolerance, in vivo, in horses based on physical, clinicopathologic, and proinflammatory gene expression responses to repeated endotoxin exposures. Six horses received 30 ng/kg LPS in saline infused over 30 min. Behavior pain scores, physical examination parameters, and blood for complete blood count and proinflammatory gene expression were obtained at predetermined intervals for 24h. Horses received a total of 3 endotoxin exposures. The first exposure was LPS 1, followed 7 days later by LPS 7 or 14-21 days later by LPS 14-21. Lipopolysaccharide exposures were allocated in a randomized, crossover design. Lipopolysaccharide produced clinical and clinicopathologic signs of endotoxemia and increased expression of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and IL-8, P<0.001. Horses exhibited evidence of endotoxin tolerance following LPS 7 but not following LPS 14-21. Horses had significantly lower pain scores, heart rates, respiratory rates and duration of fever, after LPS 7 compared to LPS 1 and LPS 14-21, P<0.001, and expression of TNFα was lower in the whole blood of horses after LPS 7, P=0.05. Clinical parameters and TNFα gene expression were similar or slightly increased in horses following LPS 14-21 compared to measurements made in horses following LPS 1, suggesting that endotoxin tolerance had subsided. A minimum of 3 weeks between experiments is warranted if

  10. Adherent endotoxin on dental implant surfaces: a reappraisal.

    PubMed

    Morra, Marco; Cassinelli, Clara; Bollati, Daniele; Cascardo, Giovanna; Bellanda, Marco

    2015-02-01

    Osteoimmunology is the crosstalk between cells from the immune and skeletal systems, suggesting a role of pro-inflammatory cytokines in the stimulation of osteoclast activity. Endotoxin or bacterial challenges to inflammatory cells are directly relevant to dental implant pathologies involving bone resorption, such as osseointegration failure and peri-implantitis. While the endotoxin amount on implant devices is regulated by standards, it is unknown whether commercially available dental implants elicit different levels of adherent-endotoxin stimulated cytokines. The objective of this work is to develop a model system and evaluate endotoxin-induced expression of pro-inflammatory cytokine genes relevant to osteoclast activation on commercially available dental implants. Murine J774-A1 macrophages were cultured on Ti disks with different level of lipopolysaccharide (LPS) contamination to define the time-course of the inflammatory response to endotoxin, as evaluated by reverse transcription polymerase chain reaction analysis. The developed protocol was then used to measure adherent endotoxin on commercially available packaged and sterile dental implants in the "as-implanted" condition. Results show that tested dental implants induce variable expression of endotoxin-stimulated genes, sometimes above the level expected to promote bone resorption in vivo. Results are unaffected by the specific surface treatment; rather, they likely reflect care in cleaning and packaging protocols. In conclusion, expression of genes that enhance osteoclast activity through endotoxin stimulation of inflammatory cells is widely different on commercially available dental implants. A reappraisal of the clinical impact of adherent endotoxins on dental (and bone) implant devices is required in light of increasing knowledge on crosstalk between cells from the immune and skeletal systems.

  11. Inactivation of Escherichia coli Endotoxin by Soft Hydrothermal Processing▿

    PubMed Central

    Miyamoto, Toru; Okano, Shinya; Kasai, Noriyuki

    2009-01-01

    Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250°C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130°C for 60 min or at 140°C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130°C for 60 min or at 140°C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments. PMID:19502435

  12. Temperature Responses of Mice to Escherichia Coli Endotoxin

    PubMed Central

    Prashker, D.; Wardlaw, A. C.

    1971-01-01

    SJL mice kept in a 23° environment and injected intravenously with Escherichia coli endotoxin developed a marked hypothermia compared with animals given pyrogen-free saline (PFS). In contrast, giving endotoxin to SJL mice which had been “pre-conditioned” for 4 hr at 36° caused relative hyperthermia. Both responses were best observed at 1½ hr after injection. An endotoxin dose of 0·02 μg. was at the threshold of detectability in mice pre-conditioned at 36°, while with 23° animals, the threshold dose was 0·2 μg. Dose-response curves, with an index of precision (λ) of about 0·73, were obtained for mice in both environments, endotoxin doses of about 20 μg. being in the plateau regions of maximum responses. Mouse strains SWR/J and CMRL behaved similarly to SJL, but the temperature responses at both 23° and 36° were smaller. BALB/cJ and AKR/J mice showed a hyperthermic response to endotoxin at 36° but no hypothermia at 23°, while ST/bJ mice showed the converse pattern of hypothermia after endotoxin at 23° but no hyperthermia at 36°. Thus the strain of mouse is an important variable. We suggest that a hypothermia test in SJL mice may provide a simple and convenient bioassay procedure for endotoxin. Although its sensitivity is much less than the rabbit pyrogenicity test, it may be useful for the quantitative measurement of endotoxin activity of such preparations as typhoid, pertussis and cholera vaccines which are rich in endotoxin. PMID:4926536

  13. Some metabolic effects of bacterial endotoxins in salmonid fishes

    USGS Publications Warehouse

    Wedemeyer, G.A.; Ross, A.J.; Smith, L.

    1968-01-01

    Coho salmon (Oncorhynchus kisutch) and rainbow trout (Salmo gairdneri) were highly resistant to endotoxins from both Escherichia coli and Aeromonas salmonicida (a fish pathogen) at 14 and 18 C.This resistance was investigated with liver tryptophan pyrrolase, liver glycogen depletion in vitro, and the arterial blood pressure as indicators. Liver glycogen depletion was accelerated by both endotoxins, but there was no significant cardiovascular response or effect on liver tryptophan pyrrolase activity. Since the cardiovascular effects of histamine were also limited, it was concluded that the metabolic effects of bacterial endotoxins in salmonids are qualitatively different from those of the higher vertebrates.

  14. Comparative personal exposures to organic dusts and endotoxin.

    PubMed

    Simpson, J C; Niven, R M; Pickering, C A; Oldham, L A; Fletcher, A M; Francis, H C

    1999-02-01

    The aims of the study were to provide valid comparative data for personal exposures to dust and endotoxins for different occupations and to calculate comparative data for the contamination of organic dusts with endotoxin. Nine different occupational settings were studied, drawn from the textile, agricultural and animal handling industries. Samples were collected by personal sampling techniques, using the Institute of Occupational Medicine (IOM) sampling head, glass fibre filters and rechargeable sampling pumps. The dust exposures were calculated by gravimetric analysis and using the calculated volume of air sampled were expressed as mg/m3. Endotoxin exposures were measured using a simple water extraction from the collected dusts, followed by a quantitative turbidimetric assay. Results were expressed as ng/m3, using the calculated volume of air sampled. In addition, the levels of the contamination of dusts with endotoxin for individual industries were expressed as ng/mg of collected dust. Two hundred and fifty-nine samples, collected from 9 different industries and across 36 different sites were analysed. This represented a sampling rate of 25% for the total work force. The average sampling time was 4.62 h. For all the dusts collected, a significant correlation between the collected dust and endotoxin was seen (r = 0.7 and p < 0.001). The highest dust exposures occurred during cleaning activities (grain handling: 72.5 mg/m3). The individuals exposed to the highest median level of dust and endotoxin were the animal handlers (poultry handlers, dust: 11.53 mg/m3, endotoxin: 71,995 ng/m3). Weaving and mushroom cultivation had the lowest exposures for dust and endotoxins. The mostly highly contaminated dusts (median values expressed as ng of endotoxin per mg of collected dust) were found in the animal handling (poultry: 1,030 ng/mg, swine: 152 ng/mg) and cotton spinning (522 ng/mg) industries. Processing of cotton and wool fibres was found to reduce the levels of

  15. Bacterial endotoxins and liver haemorrhage in the oestrogenised chicken.

    PubMed

    Curtis, M J; Pearson, A W; Butler, E J

    1980-11-01

    Hepatic steatosis and haemorrhage in oestrogenised chickens were not associated with any increase in the endotoxin content of the plasma. The haemorrhage was not ameliorated by suppressing enteric bacteria with neomycin or exacerbated by the repeated injection of Escherichia coli O111 endotoxin and there were no relevant changes in plasma enzyme activities that are indicators of liver damage. These results therefore do not support the hypothesis that, as in the choline deficient rat, hepatic steatosis impairs the ability of the oestrogenised chicken to dispose of bacterial endotoxins and that these then damage the liver.

  16. Endotoxins in surgical instruments of hip arthroplasty.

    PubMed

    Goveia, Vania Regina; Mendoza, Isabel Yovana Quispe; Guimarães, Gilberto Lima; Ercole, Flavia Falci; Couto, Bráulio Roberto Gonçalves Marinho; Leite, Edna Marilea Meireles; Stoianoff, Maria Aparecida Resende; Ferreira, José Antonio Guimarães

    2016-01-01

    To investigate endotoxins in sterilized surgical instruments used in hip arthroplasties. A descriptive exploratory study conducted in a public teaching hospital. Six types of surgical instruments were selected, namely: acetabulum rasp, femoral rasp, femoral head remover, chisel box, flexible bone reamer and femoral head test. The selection was based on the analysis of the difficulty in removing bone and blood residues during cleaning. The sample was made up of 60 surgical instruments, which were tested for endotoxins in three different stages. The EndosafeTM Gel-Clot LAL (Limulus Amebocyte Lysate method) was used. There was consistent gel formation with positive analysis in eight instruments, corresponding to 13.3%, being four femoral rasps and four bone reamers. Endotoxins in quantity ≥0.125 UE/mL were detected in 13.3% of the instruments tested. Investigar endotoxinas em instrumentais cirúrgicos esterilizados empregados em artroplastias do quadril. Estudo exploratório, descritivo, desenvolvido em um hospital público de ensino. Foram selecionados seis tipos de instrumentais, a saber: raspa acetabular, raspa femural, saca-cabeça de fêmur, formão box, fresa de fêmur e cabeça de prova de fêmur. A seleção foi feita a partir da análise da dificuldade para a remoção de resíduos de sangue e osso durante a limpeza. A amostra foi constituída por 60 instrumentais cirúrgicos, que foram testados para endotoxinas em três momentos distintos. Foi utilizado o método de gel-clot pelo Limulus Amebócito Lisado (LAL) Endosafe(tm). Houve formação de gel consistente com análise positiva em oito instrumentais, o que corresponde a 13,3%, sendo quatro raspas de fêmur e quatro fresas de fêmur. Foram detectadas endotoxinas em quantidade ≥0,125 UE/mL em 13,3% dos instrumentais testados.

  17. Delta III—an evolutionary delta growth

    NASA Astrophysics Data System (ADS)

    Arvesen, R. J.; Simpson, J. S.

    1996-03-01

    In order to remain competitive in the future and expand the McDonnell Douglas Aerospace market share, MDA has developed an expendable launch system strategy that devices cost-effective launch systems from the Delta II with a growth vehicle configuration called Delta III. The Delta III evolves from the Delta II launch system through development of a larger payload fairing (4-meter diameter), new cryogenically propelled upper stage, new first stage fuel tank, and larger strap-on solid rocket motors. We are developing the Delta III using Integrated Product Development Teams that capitalize on the experience base that has led us to a world record breaking mission success of 49 consecutive Delta II missions. The Delta III first-launch capability is currently planned for the spring of 1998 in support of our first spacecraft customer, Hughes Space and Communications International.

  18. Endotoxin-Induced Structural Transformations in Liquid Crystalline Droplets

    NASA Astrophysics Data System (ADS)

    Lin, I.-Hsin; Miller, Daniel S.; Bertics, Paul J.; Murphy, Christopher J.; de Pablo, Juan J.; Abbott, Nicholas L.

    2011-06-01

    The ordering of liquid crystals (LCs) is known to be influenced by surfaces and contaminants. Here, we report that picogram per milliliter concentrations of endotoxin in water trigger ordering transitions in micrometer-size LC droplets. The ordering transitions, which occur at surface concentrations of endotoxin that are less than 10-5 Langmuir, are not due to adsorbate-induced changes in the interfacial energy of the LC. The sensitivity of the LC to endotoxin was measured to change by six orders of magnitude with the geometry of the LC (droplet versus slab), supporting the hypothesis that interactions of endotoxin with topological defects in the LC mediate the response of the droplets. The LC ordering transitions depend strongly on glycophospholipid structure and provide new designs for responsive soft matter.

  19. THE LETHAL EFFECT OF ENDOTOXINS ON THE CHICK EMBRYO

    PubMed Central

    Smith, Richard T.; Thomas, Lewis

    1956-01-01

    Inoculation of the CAM of the 10-day chick embryo with endotoxin preparations derived from the meningococcus and other Gram-negative microorganisms has been shown to result in multiple hemorrhages and death of the embryo within a few hours. Evidence has been presented to indicate that this lethal effect is specific for the general class of endotoxins derived from Gram-negative bacteria. Susceptibility to endotoxin was maximal in 10-day old embryos, and younger or older embryos showed little or no response. The optimal incubation temperature for the effect of endotoxin was 39.5°C., and embryos incubated at 28°C. were completely protected. The lethal effect was prevented by small amounts of cortisone, hydrocortisone, and 9-alpha fluorohydrocortisone, but not by cholesterol, desoxycorticosterone, or 1-dehydrocortisone. PMID:13345967

  20. Induction of hyperreactivity to endotoxin in mice by Coxiella burnetii.

    PubMed Central

    Schramek, S; Kazar, J; Sekeyova, Z; Freudenberg, M A; Galanos, C

    1984-01-01

    Intraperitoneal inoculation of mice with live or killed Coxiella burnetii phase I or phase II cells induced a marked hyperreactivity to the lethal effect of bacterial endotoxin and was accompanied by a marked hepatosplenomegaly. The degree and duration of hyperreactivity depended on the dose of C. burnetii administered and were higher with phase I than with phase II cells. Sensitization to the lethal effects of endotoxin and induction of splenomegaly by phase I C. burnetii cells also proceeded in the endotoxin-resistant C3H/HeJ strain of mice. Preincubation of C. burnetii cells with the corresponding immune serum significantly diminished the ability of phase I but not phase II cells to induce hyperreactivity to endotoxin. PMID:6469358

  1. Methylprednisolone Protection Against Endotoxin in Lead-Sensitized Mice

    PubMed Central

    Marecki, Nelda M.

    1974-01-01

    Lead-sensitized BALB/c male mice are protected against Escherichia coli endotoxin by the intraperitoneal administration of 6α-methylprednisolone. This protection is dependent on time of administration of the methylprednisolone. PMID:4596282

  2. Endotoxin in Size-Separated Metal Working Fluid Aerosol Particles.

    PubMed

    Dahlman-Höglund, Anna; Lindgren, Åsa; Mattsby-Baltzer, Inger

    2016-08-01

    Patients with airway symptoms working in metal working industries are increasing, despite efforts to improve the environmental air surrounding the machines. Our aim was to analyse the amount of endotoxin in size-separated airborne particles of metal working fluid (MWF) aerosol, by using the personal sampler Sioutas cascade impactor, to compare filter types, and to compare the concentration of airborne endotoxin to that of the corresponding MWFs. In a pilot field study, aerosols were collected in two separate machine halls on totally 10 occasions, using glass fibre and polytetrafluoroethylene (PTFE) filters in parallel at each station. Airborne endotoxin was distributed over all size fractions. While a major part was found in the largest size fraction (72%, 2.5-10 µm), up to 8% of the airborne endotoxin was detected in the smallest size fraction (<0.25 µm). Comparing the efficiency of the filter types, a significantly higher median endotoxin level was found with glass fibres filters collecting the largest particle-size fraction (1.2-fold) and with PTFE filters collecting the smallest ones (5-fold). The levels of endotoxin in the size-separated airborne particle fractions correlated to those of the MWFs supporting the aerosol-generating machines. Our study indicates that a significant part of inhalable aerosols of MWFs consists of endotoxin-containing particles below the size of intact bacteria, and thus small enough to readily reach the deepest part of the lung. Combined with other chemical irritants of the MWF, exposure to MWF aerosols containing endotoxin pose a risk to respiratory health problems. © The Author 2016. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.

  3. Ultrasensitive detection of endotoxins using computationally designed nanoMIPs.

    PubMed

    Altintas, Zeynep; Abdin, Mohammed J; Tothill, Alexander M; Karim, Kal; Tothill, Ibtisam E

    2016-09-07

    Novel molecularly imprinted polymer nanoparticles (nanoMIPs) were designed for endotoxin from Escherichia coli 0111:B4, using computational modeling. The screening process based on binding energy between endotoxin and each monomer was performed with 21 commonly used monomers, resulting in the selection of itaconic acid, methacrylic acid and acrylamide as functional monomers due to their strong binding interaction with the endotoxin template. The nanoMIPs were successfully synthesized with functional groups on the outer surface to aid in the immobilization onto sensor surface. The solid phase photopolymerization approach used for the synthesis of nanoMIPs ranging from 200 to 235 nm in diameter. The limit of detection and KD were significantly improved when endotoxin samples were prepared using a novel triethylamine method. This improved the efficiency of gold nanoparticle functionalization by targeting the subunits of the endotoxin. Compared to the vancomycin MIP control, the endotoxin MIPs displayed outstanding affinity and selectivity towards the endotoxin with KD values in the range of 4.4-5.3 × 10(-10) M, with limits of detection of 0.44 ± 0.02 ng mL(-1) as determined by surface plasmon resonance (SPR) sensor when itaconic acid was used as the functional monomer. The MIP surface can be regenerated >30 times without significant loss of binding activity making this approach highly cost effective for expensive analyte templates. The combination of molecular modeling and solid phase synthesis enabled the successful synthesis of nanoMIPs capable of recognition and ultrasensitive detection of endotoxins using the highly sensitive SPR biosensor with triethylamine method.

  4. The Delta 2 launcher

    NASA Astrophysics Data System (ADS)

    Ousley, Gilbert W., Sr.

    1991-12-01

    The utilization of the Delta 2 as the vehicle for launching Aristoteles into its near Sun synchronous orbit is addressed. Delta is NASA's most reliable launch vehicle and is well suited for placing the present Aristoteles spacecraft into a 400 m circular orbit. A summary of some of the Delta 2 flight parameters is presented. Diagrams of a typical Delta 2 two stage separation are included along with statistics on delta reliability and launch plans.

  5. Systemic endotoxin levels in chronic indolent periodontal infections

    PubMed Central

    Ebersole, J. L.; Stevens, J.; Steffen, M. J.; Dawson, D.; Novak, M. J.

    2014-01-01

    Background and Objective: Periodontal disease has been linked with an increased risk of various systemic diseases. A plausible biologic explanation for this link includes the opportunity for oral pathogens to translocate to the circulation as a result of breakdown in integrity of the oral epithelium. This study refined a methodology used to detect endotoxin activity in the serum of subjects with indolent periodontal infections. Material and Methods: The QCL® Kinetic Chromogenic Assay (Cambrex) is a kinetic measure of endotoxin activity. Sera from 211 pregnant women with periodontitis enrolled in the Obstetrics and Periodontal Therapy Trial were used to develop the assay further and to evaluate the detection of endotoxin activity that might accompany a low-level bacteremia in chronic periodontitis. Results: We optimized the system to increase the sensitivity and reproducibility of the assay. The refined system was able to detect endotoxin activity in serum at > 0.0125 EU/mL. At baseline (13–16 wk of gestation), 35.5% of the women were positive for endotoxin activity (1.62 ± 2.21; range: 0.38–15 EU/mL). Conclusion: This report describes a sensitive measure of endotoxin activity in serum. The procedure allowed us to document levels of this microbial virulence factor in serum of individuals with indolent infections such as periodontal disease. PMID:20465752

  6. Endotoxin exposure and respiratory symptoms in the cotton textile industry.

    PubMed

    Latza, Ute; Oldenburg, Marcus; Baur, Xaver

    2004-10-01

    One hundred fourteen male employees of a cotton spinning mill in western Germany participated in a cross-sectional study, the purpose of which was to clarify the dose effect of endotoxin exposure on respiratory symptoms. Airborne endotoxin exposures were classified as low (< or = 100 endotoxin units [EU]/m3), medium (> 100-450 EU/m3), or high (> 450 EU/m3), on the basis of endotoxin activity in the Limulus amoebocyte lysate assay. Age- and smoking-adjusted odds ratios (ORs) and confidence intervals (CIs) were estimated. The dose-response relationship between current endotoxin exposure and prevalence of wheezing (medium: OR = 2.15, 95% CI = 0.48-9.62; high: OR = 5.49, 95% CI = 1.17- 25.81) and cough (medium: OR = 2.11; 95% CI = 0.59-7.56; high: OR = 3.93; 95% CI = 1.02-15.12) was significant (test for linear trend: p values = 0.020 and 0.040, respectively). The association between exposure and wheezing was stronger among atopic workers. The higher prevalence of chest tightness and shortness of breath among workers with medium and high current endotoxin exposure did not reach statistical significance. The results suggested that there was a dose-dependent increase in bronchial symptoms, with significant effects occurring at exposures that exceeded 450 EU/m3.

  7. Endotoxins-the invisible companion in biomaterials research.

    PubMed

    Lieder, Ramona; Petersen, Pétur Henry; Sigurjónsson, Ólafur Eysteinn

    2013-10-01

    Metal implants and polymeric devices for the application in the clinical treatment of orthopedic tissue injuries are increasingly coated with bioactive biomaterials derived from natural substances to induce desirable biological effects. Many metals and polymers used in biomaterials research show high affinity for endotoxins, which are abundant in the environment. Endotoxin contamination is indicated in the pathology of periodontitis and aseptic implant loosening, but may also affect the evaluation of a biomaterial's bioactivity by inducing strong inflammatory reactions. In this review, we discuss the high affinity of three commonly used implant biomaterials for endotoxins and how the contamination can affect the outcome of the orthopedic fixation. The chemical nature of bacterial endotoxins and some of the clinical health implications are described, as this knowledge is critically important to tackle the issues associated with the measurement and removal of endotoxins from medical devices. Commonly used methods for endotoxin testing and removal from natural substances are examined and the lack of standard guidelines for the in vitro evaluation of biomaterials is discussed.

  8. Detection of endotoxin antibody in long-term dialysis patients.

    PubMed

    Yamagami, S; Adachi, T; Sugimura, T; Wada, S; Kishimoto, T; Maekawa, M; Yoshimura, R; Niwa, M; Terano, Y; Shaldon, S

    1990-04-01

    Endotoxins are often seen in dialysate. They are derived from Gram-negative bacteria especially Pseudomonas, E. coli and Serratia. Endotoxins are large-molecular-weight substances with an average molecular weight of 10(8). These large units can be divided into subunits down to a molecular weight of 10,000 which are thought to pass through dialyzer membranes. To investigate this, endotoxin antibody levels were measured in two groups of patients on chronic regular hemodialysis, a low-flux group using cellulosic membrane dialyzers (cuprophan and cuproammonium rayon (CAR) and a high-flux group using synthetic polymer membrane dialyzers (PMMA, EVAL). Using an ELISA based on standard endotoxin antibodies the percentages of patients in the low flux group with endotoxin antibodies were 26.9% with Cuprophan and 25% with CAR, not significantly different from a normal control group. In the PMMA and EVAL groups, it was 53.6% and 68.4% respectively. Back filtration of dialysate into blood is understood as the main reason for the entry of endotoxin in patients treated with high-flux dialyzers.

  9. Endotoxin inactivation by selected drinking water treatment oxidants.

    PubMed

    Anderson, William B; Mayfield, Colin I; Dixon, D George; Huck, Peter M

    2003-11-01

    Exposure to endotoxins in treated drinking water can occur through ingestion, dermal abrasions, inhalation of water vapor, intravenous injection or during dialysis. While the risks associated with endotoxin ingestion and entry through dermal abrasions are not well quantified, adverse effects of intravenous injection and dialysis are well known and some studies indicate that inhalation of moisture-laden air may impact human health. This study quantifies the inactivation of endotoxin derived from Escherichia coli O55:B5 by three substances used either as disinfectants or oxidants in drinking water treatment: chlorine, monochloramine and potassium permanganate. Inactivation rates were found to be 1.4, 1.0 and 0.7 endotoxin units (EU)/mL h, for free chlorine, potassium permanganate and monochloramine, respectively. These rates are relatively slow given that contact times in drinking water distribution systems are typically less than 48 h. While small amounts of endotoxin may be removed by oxidation the observed removals are much less than those provided by physical removal processes. The significance of this finding is important for dialysis considerations but is as yet unclear with regard to inhalation, as the risk of inhaling sufficient quantities of endotoxin-containing aerosolized water droplets to adversely affect human health has not yet been adequately quantified.

  10. Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale.

    PubMed

    Jin, Xueting; Xu, Qing; Champion, Keith; Kruth, Howard S

    2015-05-01

    This technical report addresses the problem of endotoxin contamination of apolipoprotein reagents. Using a bromodeoxyuridine incorporation cell proliferation assay, we observed that human plasma ApoA-I as low as 1 μg/ml resulted in a >90% inhibition in macrophage proliferation. However, not all ApoA-I from different sources showed this effect. We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation>90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus total endotoxin, the latter which included endotoxin that was masked due to binding to protein, suggested that free endotoxin mediated inhibition of macrophage proliferation. Despite the use of an advanced endotoxin removal procedure and agents commonly used to inhibit endotoxin action, the potency of endotoxin precluded successful elimination of endotoxin effect. Our findings show that endotoxin contamination can significantly influence apparent apolipoprotein-mediated cell effects (or effects of any other biological products), especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages.

  11. Endotoxin Contamination of Apolipoprotein A-I: Effect on Macrophage Proliferation – A Cautionary Tale

    PubMed Central

    Jin, Xueting; Xu, Qing; Champion, Keith; Kruth, Howard S.

    2015-01-01

    This technical report addresses the problem of endotoxin contamination of apolipoprotein reagents. Using a bromodeoxyuridine incorporation cell proliferation assay, we observed that human plasma ApoA-I as low as 1 μg/ml resulted in a >90% inhibition in macrophage proliferation. However, not all ApoA-I from different sources showed this effect. We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation >90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus total endotoxin, the latter which included endotoxin that was masked due to binding to protein, suggested that free endotoxin mediated inhibition of macrophage proliferation. Despite the use of an advanced endotoxin removal procedure and agents commonly used to inhibit endotoxin action, the potency of endotoxin precluded successful elimination of endotoxin effect. Our findings show that endotoxin contamination can significantly influence apparent apolipoprotein-mediated cell effects (or effects of any other biological products), especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages. PMID:25778625

  12. Field evaluation of endotoxin air sampling assay methods.

    PubMed

    Thorne, P S; Reynolds, S J; Milton, D K; Bloebaum, P D; Zhang, X; Whitten, P; Burmeister, L F

    1997-11-01

    This study tested the importance of filter media, extraction and assay protocol, and bioaerosol source on the determination of endotoxin under field conditions in swine and poultry confinement buildings. Multiple simultaneous air samples were collected using glass fiber (GF) and polycarbonate (PC) filters, and these were assayed using two methods in two separate laboratories: an endpoint chromogenic Limulus amebocyte lysate (LAL) assay (QCL) performed in water and a kinetic chromogenic LAL assay (KQCL) performed in buffer with resistant-parallel line estimation analysis (KLARE). In addition, two aqueous filter extraction methods were compared in the QCL assay: 120 min extraction at 22 degrees C with vigorous shaking and 30 min extraction at 68 degrees C with gentle rocking. These extraction methods yielded endotoxin activities that were not significantly different and were very highly correlated. Reproducibility of endotoxin determinations from duplicate air sampling filters was very high (Cronbach alpha all > 0.94). When analyzed by the QCL method GF filters yielded significantly higher endotoxin activity than PC filters. QCL and KLARE methods gave similar estimates for endotoxin activity from PC filters; however, GF filters analyzed by the QCL method yielded significantly higher endotoxin activity estimates, suggesting enhancement of the QCL assay or inhibition of the KLARE asay with GF filters. Correlation between QCL-GF and QCL-PC was high (r = 0.98) while that between KLARE-GF and KLARE-PC was moderate (r = 0.68). Analysis of variance demonstrated that assay methodology, filter-type, barn-type, and interactions between assay and filter-type and between assay and barn-type were important factors influencing endotoxin exposure assessment.

  13. Endotoxin increases pulmonary vascular protein permeability in the dog

    SciTech Connect

    Welsh, C.H.; Dauber, I.M.; Weil, J.V.

    1986-10-01

    Endotoxin increases pulmonary vascular permeability consistently in some species but fails to reliably cause injury in the dog. We wondered whether this phenomenon depended on the method of injury assessment, as others have relied on edema measurement; we quantified injury by monitoring the rate of extravascular protein accumulation. /sup 113m/In-labeled protein and /sup 99m/Tc-labeled erythrocytes were injected into anesthetized dogs and monitored by an externally placed lung probe. A protein leak index, the rate of extravascular protein accumulation, was derived from the rate of increase in lung protein counts corrected for changes in intravascular protein activity. After administration of Salmonella enteriditis endotoxin (4 micrograms/kg), the protein leak index was elevated 2.5-fold (41.1 +/- 4.6 X 10(-4) min-1) compared with control (16.0 +/- 2.8 X 10(-4) min-1). In contrast, wet-to-dry weight ratios failed to increase after endotoxin (4.6 +/- 0.8 vs. control values of 4.2 +/- 0.5 g/g dry bloodless lung). However, we observed that endotoxin increased lung dry weight (per unit body weight), which may have attenuated the change in wet-to-dry weight ratios. To determine whether low microvascular pressures following endotoxin attenuated edema formation, we increased pulmonary arterial wedge pressures in five dogs by saline infusion, which caused an increase in wet-to-dry weight ratios following endotoxin but no change in the five controls. We conclude that low dose endotoxin causes pulmonary vascular protein leak in the dog while edema formation is minimal or absent.

  14. The natural immunity to evolutionary atavistic endotoxin for human cancer.

    PubMed

    Moncevičiūtė-Eringienė, Elena; Rotkevič, Kristina; Grikienis, Ruta Grikienyte

    2015-11-01

    We propose a new theory of the immunological control of cancer corresponding to the hypothesis that the specific natural immunity to evolutionary atavistic endotoxin has a potential role in human cancer prevention. The results of our studies have shown that IgMNAE, i.e. endogenous or spontaneous IgM class antibodies to enterobacterial lipopolysaccharide molecules (lipid A), control the immune mechanisms responsible for the internal medium stability not only against the damaging impact of the carcinogenic factors, but also against the malignant transformation of its own degenerated cells. Among people who in 1979 and 1982 had IgMNAE in their blood serum, after 15-30years fell ill with cancer 10%, versus 15% among people who had no IgMNAE (p<0.05). Therefore, it is possible to maintain that the stimulation of IgMNAE synthesis would help in the destruction and elimination of damaged somatic cells or prevent their mechanisms from the formation of invasiveness, metastatic and other properties of their parasitism. In the mechanism of the natural immunity to endotoxin it is possible to see the formation of the respective evolutionary protective reactions which protect the damaged cells from acquiring resistance to damaging factors and thus from becoming an independent new parasitic population. Thereby the presented theory of the immunological control of cancer has a causal connection with our evolutionary resistance theory of the origin of cancer. Collectively, these data suggest that activation of natural immunity to endotoxin and production of vaccines against evolutionary atavistic endotoxin or gram-negative bacterial endotoxin can be helpful when applied in cancer prophylaxis for persons with a low level of natural immunity to endotoxin and perhaps in creating immunotherapeutic methods for stopping the endogenous parasitism of tumour cells by binding IgMNAE to atavistic endotoxin in cancer patients.

  15. Effect of endotoxin on lung fluid balance in unanesthetized sheep.

    PubMed

    Gabel, J C; Hansen, T N; Drake, R E

    1984-02-01

    We used a gravimetric technique to test for increased pulmonary capillary permeability after Escherichia coli endotoxin infusion in unanesthetized sheep. The sheep were chronically prepared with cannulas placed into the left atrium and pulmonary artery 1-2 wk before the experiments. We estimated pulmonary capillary pressure (Pc) as the average of pulmonary arterial and left atrial pressures, and used the modified method of Pierce to estimate the ratio of extravascular fluid weight (EVF) to blood-free dry weight. In 15 sheep we inflated a left atrial balloon to raise Pc to -10.7, 5, 10, or 15 mmHg above plasma oncotic pressure (IIc) for 3 h, then measured EVF. EVF averaged 4.0 +/- 0.2 (base line), 4.3 +/- 0.1, 4.5 +/- 0.1, and 5.1 +/- 0.5 (SD), respectively, for the four levels of Pc - IIc. We gave seven additional sheep 1 microgram/kg of E. coli endotoxin (0127:B8) and measured EVF after 3 h of stable Pc. Endotoxin increased Pc in each sheep. EVF was higher than control for the endotoxin sheep with Pc - IIc greater than -1. This finding is consistent with an increase in pulmonary capillary permeability caused by endotoxin. However, EVF was not elevated in the endotoxin sheep with Pc - IIc less than 1 mmHg. This shows that the increased permeability was insufficient to cause edema unless Pc was elevated. Thus endotoxin may cause edema by two mechanisms, 1) an increase in capillary permeability, and 2) an increase in Pc.

  16. Stress-Derived Corticotropin Releasing Factor Breaches Epithelial Endotoxin Tolerance

    PubMed Central

    Yu, Yong; Geng, Xiao-Rui; Yang, Gui; Liu, Zhi-Gang; Zheng, Peng-Yuan; Yang, Ping-Chang

    2013-01-01

    Background and aims Loss of the endotoxin tolerance of intestinal epithelium contributes to a number of intestinal diseases. The etiology is not clear. Psychological stress is proposed to compromise the intestinal barrier function. The present study aims to elucidate the role of the stress-derived corticotropin releasing factor (CRF) in breaching the established intestinal epithelial endotoxin tolerance. Methods Epithelial cells of HT-29, T84 and MDCK were exposed to lipopolysaccharide to induce the endotoxin tolerance; the cells were then stimulated with CRF. The epithelial barrier function was determined using as indicators of the endotoxin tolerant status. A water-avoid stress mouse model was employed to test the role of CRF in breaching the established endotoxin tolerance in the intestine. Results The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP). The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells. Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP. After treating mice with the 10-day chronic stress, the intestinal epithelial barrier function was markedly compromised, which could be prevented by blocking either CRF, or TLR4, or Cldn2. Conclusions Psychological stress-derived CRF can breach the established endotoxin tolerance in the intestinal mucosa. PMID:23840363

  17. LOW-DOSE AIRBORNE ENDOTOXIN EXPOSURE ENHANCES BRONCHIAL RESPONSIVENESS TO INHALED ALLERGEN IN ATOPIC ASTHMATICS

    EPA Science Inventory

    Endotoxin exposure has been associated with both protection against development of TH2-immune responses during childhood and exacerbation of asthma in persons who already have allergic airway inflammation.1 Occupational and experimental inhalation exposures to endotoxin have been...

  18. LOW-DOSE AIRBORNE ENDOTOXIN EXPOSURE ENHANCES BRONCHIAL RESPONSIVENESS TO INHALED ALLERGEN IN ATOPIC ASTHMATICS

    EPA Science Inventory

    Endotoxin exposure has been associated with both protection against development of TH2-immune responses during childhood and exacerbation of asthma in persons who already have allergic airway inflammation.1 Occupational and experimental inhalation exposures to endotoxin have been...

  19. ALLERGEN PROVOCATION AUGMENTS ENDOTOXIN-INDUCED NASAL INFLAMMATION IN ATOPIC ASTHMATICS

    EPA Science Inventory

    Background: Recent epidemiologic and in vivo studies have suggested that inhaled endotoxin plays an important role in asthma pathogenesis.
    Objective: The present study examines the effect of nasal allergen provocation on subsequent endotoxin challenges in subjects with atopi...

  20. Histological analysis of the association between formocresol and endotoxin in the subcutaneous tissue of mice.

    PubMed

    Sant'anna, Ana Teresa; Spolidório, Luis Carlos; Ramalho, Lizeti Toledo Oliveira

    2008-01-01

    This study performed a histological analysis of the effect of formocresol associated to endotoxin (LPS) in the subcutaneous connective tissue of mice. Ninety mice were randomly assigned to 3 groups (n=30). Each animal received one plastic tube implant containing endotoxin solution (10 mg/mL), formocresol (original formula) or a mixture of endotoxin and formocresol. The endotoxin and formocresol groups served as controls. The periods of analysis were 7, 15 and 30 days. At each experimental period, tissue samples were collected and submitted to routine processing for histological analysis. Endotoxin and formocresol produced necrosis and chronic inflammation at 7 and 15 days. At 30 days, the endotoxin group showed no necrosis, while in the formocresol group necrosis persisted. The formocresol-endotoxin association produced necrosis and chronic inflammation in the same way as observed with formocresol at all experimental periods. In conclusion, formocresol seems not to be able to inactive the toxic effects of endotoxin in connective tissues.

  1. delta-Hexachlorocyclohexane (delta-HCH)

    Integrated Risk Information System (IRIS)

    delta - Hexachlorocyclohexane ( delta - HCH ) ; CASRN 319 - 86 - 8 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Ass

  2. Puerarin ameliorates experimental alcoholic liver injury by inhibition of endotoxin gut leakage, Kupffer cell activation, and endotoxin receptors expression.

    PubMed

    Peng, Jing-Hua; Cui, Tuan; Huang, Fu; Chen, Liang; Zhao, Yu; Xu, Lin; Xu, Li-Li; Feng, Qin; Hu, Yi-Yang

    2013-03-01

    Puerarin, an isoflavone component extracted from Kudzu (Pueraria lobata), has been demonstrated to alleviate alcohol-related disorders. Our study examined whether puerarin ameliorates chronic alcoholic liver injury through inhibition of endotoxin gut leakage, the subsequent Kupffer cell activation, and endotoxin receptors expression. Rats were provided with the Liber-DeCarli liquid diet for 8 weeks. Puerarin (90 mg/kg or 180 mg/kg daily) was orally administered from the beginning of the third week until the end of the experiment. Chronic alcohol intake caused increased serum alanine aminotransferase, aspartate aminotransferase, hepatic gamma-glutamyl transpeptidase, and triglyceride levels as well as fatty liver and neutrophil infiltration in hepatic lobules as determined by biochemical and histologic assays. A significant increase of liver tumor necrosis factor α was detected by enzyme-linked immunosorbent assay. These pathologic effects correlated with increased endotoxin level in portal vein and upregulated protein expression of hepatic CD68, lipopolysaccharide-binding protein, CD14, Toll-like receptor 2, and Toll-like receptor 4. Meanwhile, the intestinal microvilli were observed to be sparse, shortened, and irregularity in distribution under the transmission electron microscope in conjunction with the downregulated intestinal zonula occludens-1 protein expression. These hepatic pathologic changes were significantly inhibited in puerarin-treated animals as were the endotoxin levels and hepatic CD68 and endotoxin receptors. Moreover, the pathologic changes in intestinal microvillus and the decreased intestinal zonula occludens-1 were also ameliorated with puerarin treatment. These results thus demonstrate that puerarin inhibition of endotoxin gut leakage, Kupffer cell activation, and endotoxin receptors expression is involved in the alleviation of chronic alcoholic liver injury in rats.

  3. Toxicologic interactions between ozone and bacterial endotoxin

    SciTech Connect

    Peavy, D.L.; Fairchild, E.J. II

    1987-02-01

    The effects of acute exposure of mice to bacterial lipopolysaccharide (LPS), the endotoxin of gram negative microorganisms, and ozone (O3) have been investigated. Intraperitoneal (ip) administration of 5 mg/kg LPS to CD-1 mice followed by exposure to 15 ppm O3 for 1.5 hr produced synergistic effects as measured by pulmonary edemagenesis and lethality assays. In contrast, ip administration of 0.1-1.6 mg/kg LPS to CD-1 mice over 5 consecutive days, a dose regimen resulting in LPS tolerance, protected against a lethal challenge of 20 ppm O3 for 3 hr. A statistically significant increase in catalase and glutathione peroxidase activity was measured in homogenates of lungs obtained from CD-1 mice receiving a tolerance-inducing regimen of LPS. These results demonstrate that two, distinct toxicologic interactions can occur between O3 and bacterial LPS. Synergism between these agents could explain, in part, the increased susceptibility of O3-exposed animals to respiratory infection with gram negative microorganisms. Protection resulting from LPS-induced increases in pulmonary antioxidant activity provides additional evidence that O3 and, possibly, LPS mediate their toxicity through oxidative mechanisms.

  4. Exposure to airborne endotoxins among sewer workers: an exploratory study.

    PubMed

    Duquenne, Philippe; Ambroise, Denis; Görner, Pierre; Clerc, Frédéric; Greff-Mirguet, Guylaine

    2014-04-01

    Exploratory bioaerosol sampling was performed in order to assess exposure to airborne endotoxins during sewer work. Personal samples were collected in underground sewer pipes using 37-mm closed-face cassettes containing fibreglass filters (CFC-FG method) or polycarbonate filters (CFC-PC method). Endotoxins were quantified using the limulus amoebocyte lysate assay. Concentrations of airborne endotoxins at sewer workplaces (16-420 EU m(-3)) were higher than those measured outside the sewer network (0.6-122 EU m(-3)). Sewer worker exposure to airborne endotoxins depended on the workplace and on the tasks. Exposure levels were the highest for tasks involving agitation of water and matter, especially for 'chamber cleanup' and 'pipes cleanup' with a high-pressure water jet. Airborne endotoxin levels at the workplace tended to be higher when CFC-FG was used as the sampling method rather than CFC-PC. The adjusted mean of the measured concentrations for CFC-PC represents 57% of the mean observed with CFC-FG. The number of samples collected in the descriptive study was too low for drawing definitive conclusions and further exposure investigations are needed. Therefore, our exploratory study provides new exposure data for the insufficiently documented sewer working environment and it would be useful for designing larger exposures studies.

  5. Evolution of endotoxin contamination during production of a therapeutic serum.

    PubMed

    Massaldi, Hugo; Morais, Victor

    2007-01-01

    A comparative bench-scale study of endotoxin contamination is presented for two common processes of immunoglobulin purification from equine plasma: ammonium sulphate fractionation of F(ab')2 fragments and caprylic acid precipitation of non-IgG proteins. To this end, both processes were carried out under normal sterile conditions, using sanitized material and equipment and optimal water quality in a clean but open environment. Stream samples, taken at different stages from each process, were analyzed for endotoxin content by the Limulus Amebocyte Lysate (LAL) test. It was found that exogenous contamination preferentially came from endotoxins already present in reagents and/or raw materials, whereas contamination from the environment was minimal. Endogenous endotoxin accumulation, concomitant with the concentration of proteins during processing, was found to be an important factor. With classic technology, blood extraction and sterilizing filtration are critical points for both processes. It is concluded that sterility is not a sufficient condition to obtain an endotoxin-free product. Only with proper sanitization of material, and by applying the caprylic acid purification process with a starting plasma below 4-5 EU/mL, would it be possible to achieve a final product within the norm.

  6. Bacterial endotoxin adhesion to different types of orthodontic adhesives

    PubMed Central

    ROMUALDO, Priscilla Coutinho; GUERRA, Thaís Rodrigues; ROMANO, Fábio Lourenço; da SILVA, Raquel Assed Bezerra; BRANDÃO, Izaíra Tincani; SILVA, Célio Lopes; da SILVA, Lea Assed Bezerra; NELSON-FILHO, Paulo

    2017-01-01

    Abstract Bacterial endotoxin (LPS) adhesion to orthodontic brackets is a known contributing factor to inflammation of the adjacent gingival tissues. Objective The aim of this study was to assess whether LPS adheres to orthodontic adhesive systems, comparing two commercial brands. Material and Methods Forty specimens were fabricated from Transbond XT and Light Bond composite and bonding agent components (n=10/component), then contaminated by immersion in a bacterial endotoxin solution. Contaminated and non-contaminated acrylic resin samples were used as positive and negative control groups, respectively. LPS quantification was performed by the Limulus Amebocyte Lysate QCL-1000™ test. Data obtained were scored and subjected to the Chi-square test using a significance level of 5%. Results There was endotoxin adhesion to all materials (p<0.05). No statistically significant difference was found between composites/bonding agents and acrylic resin (p>0.05). There was no significant difference (p>0.05) among commercial brands. Affinity of endotoxin was significantly greater for the bonding agents (p=0.0025). Conclusions LPS adhered to both orthodontic adhesive systems. Regardless of the brand, the endotoxin had higher affinity for the bonding agents than for the composites. There is no previous study assessing the affinity of LPS for orthodontic adhesive systems. This study revealed that LPS adheres to orthodontic adhesive systems. Therefore, additional care is recommended to orthodontic applications of these materials. PMID:28877283

  7. House dust endotoxin and allergic sensitization in children.

    PubMed

    Gehring, Ulrike; Bischof, Wolfgang; Fahlbusch, Bärbel; Wichmann, Heinz-Erich; Heinrich, Joachim

    2002-10-01

    A higher exposure to endotoxin was hypothesized to contribute to lower prevalence of allergic sensitization and hay fever in children growing up on a farm. We studied the association between house dust endotoxin and allergic sensitization. We randomly selected 740 children, aged between 5 and 10 years, from a group of children who participated in two cross-sectional surveys performed in Saxony-Anhalt, Germany, from 1992 to 1993 and from 1995 to 1996, such that 50% of the children were atopic or had a diagnosis of asthma. From 1996 to 1998, we collected living-room floor dust in the homes of 454 of these children (61%). The content of endotoxin in house dust was quantified using a chromogenic kinetic limulus amoebocyte lysate test and was related with health outcomes measured in the preceding cross-sectional surveys. Multiple logistic regression analyses adjusted for place of residence, sex, age, parental education, parental atopy, and pet ownership showed a negative association between exposure to endotoxin and sensitization to one or more allergens (aOR [95% CI] 0.95 [0.83; 1.10]) and two or more allergens (aOR [95% CI] 0.80 [0.67; 0.97]) using 0.35 kU/L as the cutoff value for sensitization. The protective effect was strengthened with increasing degree of sensitization. In conclusion, exposure to higher levels of house dust endotoxin is associated with lower prevalence of allergic sensitization in children.

  8. Endotoxin Neutralization as a Biomonitor for Inflammatory Bowel Disease

    PubMed Central

    Champion, Keith; Chiu, Laura; Ferbas, John; Pepe, Michael

    2013-01-01

    Gram-negative bacterial endotoxin is a potent immunostimulant implicated in the development and/or progression of a variety of diseases. The mammalian immune system has both innate and adaptive immune responses to neutralize endotoxin. In this study, a system was developed to monitor bacterial exposure by measuring the extent and nature of endotoxin neutralization in plasma. In control patients, females had higher levels of endotoxin neutralization than males, mirroring clinical outcomes from bacterial infection and sepsis. In addition to the total amount of neutralization, we used inactivation techniques to elucidate the nature of this activity and develop a system to compare early and late immune responses. Using this method to monitor patients with inflammatory bowel disease, we found a more robust total response that relies more on long-term, adaptive components of the immune system and less on early, innate components. Our results indicate that endotoxin neutralization is a valuable method to discern inflammatory bowel disease patients from a control population. Additionally, the nature of neutralization may be valuable in monitoring disease severity and/or the role of medication. PMID:23826338

  9. The effect of bacterial endotoxin of phagocytosis of Tetrahymena and serotonin induced imprinting.

    PubMed

    Kovács, G; Nagy, S U; Csaba, G

    1986-01-01

    Endotoxin inhibited the phagocytosis of Tetrahymena pyriformis after a short exposure and, to a lesser degree, after repeated treatments during one week (about 35 generations). Endotoxin also prevented the development of serotonin imprinting. Detoxified endotoxin (Tolerin) affected the phagocytosis of Tetrahymena much less, indicating that the lipid-A part of the molecule may account for the membrane-toxic effect.

  10. Granulocyte activation by endotoxin. I. Correlation between adherence and other granulocyte functions, and role of endotoxin structure on biologic activity.

    PubMed

    Dahinden, C; Galanos, C; Fehr, J

    1983-02-01

    Reminiscent of high concentrations of formylated chemotactic peptides, another group of bacteria-derived products, the lipopolysaccharides and lipid A, stimulate PMN adherence to petri dishes. Attachment and spreading of PMN is accompanied by intense release of secondary granule constituents and marked stimulation of the hexose monophosphate shunt activity. Dose-response studies with endotoxin preparations of diverse activity show that induction of PMN adherence, enzyme release, and respiratory burst activation are highly correlated, suggesting that this functional triad is mediated by a common mechanism. Hyperadhesiveness inducing concentrations of the chemotactically inert endotoxin lead to marked inhibition of PMN migration without affecting the direction-finding mechanism of the cell toward formylated peptides and C-derived chemotaxin(s). Endotoxin preparations at a lower grade of aggregation are more active, and the polysaccharide chains of the molecule are not essential with respect to PMN stimulation. Under our experimental conditions, endotoxin-induced stimulation of PMN is not inhibited by indomethacin, suggesting independence of cyclooxygenase-derived products. This type of PMN activation may play an important role in endotoxin-mediated tissue damage in vivo. Furthermore, hyperadhesion-induced inhibition of PMN migration to inflammatory sites during endotoxemia might hamper host resistance.

  11. Determination of endotoxins in sugar with the Limulus test.

    PubMed

    Haskå, G; Nystrand, R

    1979-12-01

    The Limulus amebocyte lysate test has been used for determination of pyrogens in sugar of different qualities. All the samples of domestic white sugar and beet raw sugar produced in Sweden during 1976 had a very low content of endotoxins, less than 10 ng/g of sugar. Imported cane raw sugar was, however, highly contaminated. The highest value obtained corresponds to about 100 mg of Escherichia coli endotoxin per g of raw sugar. Such crude sugar cannot, even after refining, be used for medical purposes. Instead, Swedish beet sugar is used as the raw material for production of invert sugar solutions for parenteral administration. The amount of endotoxin in this sugar is less than 1 ng/g.

  12. Personal endotoxin exposure in a panel study of school children with asthma

    PubMed Central

    2011-01-01

    Background Endotoxin exposure has been associated with asthma exacerbations and increased asthma prevalence. However, there is little data regarding personal exposure to endotoxin in children at risk, or the relation of personal endotoxin exposure to residential or ambient airborne endotoxin. The relation between personal endotoxin and personal air pollution exposures is also unknown. Methods We characterized personal endotoxin exposures in 45 school children with asthma ages 9-18 years using 376 repeated measurements from a PM2.5 active personal exposure monitor. We also assayed endotoxin in PM2.5 samples collected from ambient regional sites (N = 97 days) and from a subset of 12 indoor and outdoor subject home sites (N = 109 and 111 days, respectively) in Riverside and Whittier, California. Endotoxin was measured using the Limulus Amoebocyte Lysate kinetic chromogenic assay. At the same time, we measured personal, home and ambient exposure to PM2.5 mass, elemental carbon (EC), and organic carbon (OC). To assess exposure relations we used both rank correlations and mixed linear regression models, adjusted for personal temperature and relative humidity. Results We found small positive correlations of personal endotoxin with personal PM2.5 EC and OC, but not personal PM2.5 mass or stationary site air pollutant measurements. Outdoor home, indoor home and ambient endotoxin were moderately to strongly correlated with each other. However, in mixed models, personal endotoxin was not associated with indoor home or outdoor home endotoxin, but was associated with ambient endotoxin. Dog and cat ownership were significantly associated with increased personal but not indoor endotoxin. Conclusions Daily fixed site measurements of endotoxin in the home environment may not predict daily personal exposure, although a larger sample size may be needed to assess this. This conclusion is relevant to short-term exposures involved in the acute exacerbation of asthma. PMID:21810249

  13. Coarse particulate matter and airborne endotoxin within wood stove homes

    PubMed Central

    McNamara, M.; Thornburg, J.; Semmens, E.; Ward, T.; Noonan, C.

    2015-01-01

    Emissions from indoor biomass burning are a major public health concern in developing areas of the world. Less is known about indoor air quality, particularly airborne endotoxin, in homes burning biomass fuel in residential wood stoves in higher income countries. A filter-based sampler was used to evaluate wintertime indoor coarse particulate matter (PM 10–2.5) and airborne endotoxin (EU/m3, EU/mg) concentrations in 50 homes using wood stoves as their primary source of heat in western Montana. We investigated number of residents, number of pets, dampness (humidity), and frequency of wood stove usage as potential predictors of indoor airborne endotoxin concentrations. Two 48-h sampling events per home revealed a mean winter PM10–2.5 concentration (± s.d.) of 12.9 (± 8.6) μg/m3, while PM2.5 concentrations averaged 32.3 (± 32.6) μg/m3. Endotoxin concentrations measured from PM10–2.5 filter samples were 9.2 (± 12.4) EU/m3 and 1010 (± 1524) EU/mg. PM10–2.5 and PM2.5 were significantly correlated in wood stove homes (r = 0.36, P < 0.05). The presence of pets in the homes was associated with PM10–2.5 but not with endotoxin concentrations. Importantly, none of the other measured home characteristics was a strong predictor of airborne endotoxin, including frequency of residential wood stove usage. PMID:23551341

  14. Antibiotic-induced release of endotoxin in chronically bacteriuric patients.

    PubMed Central

    Hurley, J C; Louis, W J; Tosolini, F A; Carlin, J B

    1991-01-01

    A novel in vivo model for the study of antibiotic-induced release of endotoxin from gram-negative bacteria is described. The model uses the chronically colonized urinary tracts of patients whose spinal cords have been injured. At baseline, the organisms were present in the range of 1 x 10(3) to 2 x 10(7) CFU/ml, and the concentration of endotoxin ranged from 2 x 10(-1) to 1 x 10(3) ng/ml in 44 studies. In 10 control studies, the concentration of endotoxin and the numbers of viable gram-negative bacteria over time changed by an average of less than 0.15 log10 units from the baseline values. At 2 h after antibiotic administration, the average decrease in CFU was 0.93 log10 units, and because antibiotics cause the release of endotoxin, an average increase in endotoxin concentration of 0.59 log10 units was noted in 21 studies with susceptible bacteria. Similar changes in response to antibiotic exposure were seen in studies with susceptible Pseudomonas bacteria in comparison with those seen in studies with susceptible members of the family Enterobacteriaceae. These results provide evidence that this novel model may be useful for comparing the effects of antibiotics with different modes of action, both as single agents and in combination, on the concentration of endotoxin in relation to changes in the numbers of bacteria, under conditions of bacterial replication and antibiotic exposure more closely resembling those found in vivo than is possible in other models. PMID:1804012

  15. Evaluation of quantification methods of occupational endotoxin exposure.

    PubMed

    Liebers, V; Raulf-Heimsoth, M; Linsel, G; Goldscheid, N; Düser, M; Stubel, H; Brüning, Th

    2007-11-01

    Endotoxin has been identified as important component of organic-dust exposure and is suspected as main cause of work-related adverse health effects in dusty areas. Although the determination of endotoxin levels by using the Limulus amoebocyte lysate (LAL) assay is internationally accepted, reliability and variation of values measured with this test remain a point of discussion. Therefore, the purpose of the study was to determine the influence of different parameters on endotoxin activity measured in airborne samples. This study thus analyzed: (a) dust filter extraction procedures, (b) storage of samples, (c) usage of different commercially available LAL assays, and (d) results of the whole blood assay (WBA) compared to the LAL test. Using a parallel sampler, 120 filters were loaded with dust at 4 different occupational settings and extracted in 2 labs using a standardized protocol. Parameters like Tween in the extraction medium, extraction volume, centrifugation speed, and material of tubes used for extraction were tested. The LAL test and the WBA were able to determine the differences in dust load of filters obtained from the settings investigated. In addition, results varied significantly with modifications in extraction procedures. Using Tween for filter extraction mainly influenced the resulting endotoxin activity. In addition, LAL test differences according to manufacturer of LAL test, extraction volume, and whether the samples are freshly processed or frozen also resulted in significant variations in the endotoxin levels. In conclusion, a reliable assessment of exposure to endotoxin activity is only possible if standard operation procedures (SOPs) for sampling and determination are established.

  16. Imaging Phenotype of Occupational Endotoxin-Related Lung Function Decline.

    PubMed

    Lai, Peggy S; Hang, Jing-Qing; Zhang, Feng-Ying; Sun, J; Zheng, Bu-Yong; Su, Li; Washko, George R; Christiani, David C

    2016-09-01

    Although occupational exposures contribute to a significant proportion of obstructive lung disease, the phenotype of obstructive lung disease associated with work-related organic dust exposure independent of smoking remains poorly defined. We identified the relative contributions of smoking and occupational endotoxin exposure to parenchymal and airway remodeling as defined by quantitative computed tomography (CT). The Shanghai Textile Worker Study is a longitudinal study of endotoxin-exposed cotton workers and endotoxin-unexposed silk workers that was initiated in 1981. Spirometry, occupational endotoxin exposure, and smoking habits were assessed at 5-year intervals. High-resolution computed tomography (CT) was performed in 464 retired workers in 2011, along with quantitative lung densitometric and airway analysis. Significant differences in all CT measures were noted across exposure groups. Occupational endotoxin exposure was associated with a decrease (-1.3%) in percent emphysema (LAAI-950), a 3.3-Hounsfield unit increase in 15th percentile density, an 18.1-g increase in lung mass, and a 2.3% increase in wall area percent. Current but not former smoking was associated with a similar CT phenotype. Changes in LAAI-950 were highly correlated with 15th percentile density (correlation -1.0). Lung mass was the only measure associated with forced expiratory volume in 1 sec (FEV1) decline, with each 10-g increase in lung mass associated with an additional loss (-6.1 mL) of FEV1 (p = 0.001) between 1981 and 2011. There are many similarities between the effects of occupational endotoxin exposure and those of tobacco smoke exposure on lung parenchyma and airway remodeling. The effects of occupational endotoxin exposure appear to persist even after the cessation of exposure. LAAI-950 may not be a reliable indicator of emphysema in subjects without spirometric impairment. Lung mass is a CT-based biomarker of accelerated lung function decline. Lai PS, Hang J, Zhang F, Sun J

  17. Occurrence of endotoxin in dialysis fluid from 39 dialysis units.

    PubMed

    Kulander, L; Nisbeth, U; Danielsson, B G; Eriksson, O

    1993-05-01

    Endotoxin exposure during haemodialysis may cause acute and chronic adverse reactions. In order to estimate the risk to the patient, samples of dialysis fluid from 39 of the 45 dialysis units in Sweden were analysed by the chromogenic Limulus amoebocyte lysate assay. Higher levels were obtained after the usual weekend shutdowns. The length of the tubing delivering the reverse osmosis water seemed to influence the extent of contamination. Fifty-nine percent of the units showed low mean endotoxin levels (i.e. mean concentration below the recommended limit in Sweden: < 25 ng l-1), while 18% of units had high levels (mean concentration > 100 ng l-1).

  18. Endotoxin has acute and chronic effects on the cerebral circulation of fetal sheep.

    PubMed

    Feng, Susan Y S; Phillips, David J; Stockx, Elaine M; Yu, Victor Y H; Walker, Adrian M

    2009-03-01

    We studied the impact of endotoxemia on cerebral blood flow (CBF), cerebral vascular resistance (CVR), and cerebral oxygen transport (O(2) transport) in fetal sheep. We hypothesized that endotoxemia impairs CBF regulation and O(2) transport, exposing the brain to hypoxic-ischemic injury. Responses to lipopolysaccharide (LPS; 1 microg/kg iv on 3 consecutive days, n = 9) or normal saline (n = 5) were studied. Of LPS-treated fetuses, five survived and four died; in surviving fetuses, transient cerebral vasoconstriction at 0.5 h (DeltaCVR approximately +50%) was followed by vasodilatation maximal at 5-6 h (DeltaCVR approximately -50%) when CBF had increased (approximately +60%) despite reduced ABP (approximately -20%). Decreased CVR and increased CBF persisted 24 h post-LPS and the two subsequent LPS infusions. Cerebral O(2) transport was sustained, although arterial O(2) saturation was reduced (P < 0.05). Histological evidence of neuronal injury was found in all surviving LPS-treated fetuses; one experienced grade IV intracranial hemorrhage. Bradykinin-induced cerebral vasodilatation (DeltaCVR approximately -20%, P < 0.05) was abolished after LPS. Fetuses that died post-LPS (n = 4) differed from survivors in three respects: CVR did not fall, CBF did not rise, and O(2) transport fell progressively. In conclusion, endotoxin disrupts the cerebral circulation in two phases: 1) acute vasoconstriction (1 h) and 2) prolonged vasodilatation despite impaired endothelial dilatation (24 h). In surviving fetuses, LPS causes brain injury despite cerebral O(2) transport being maintained by elevated cerebral perfusion; thus sustained O(2) transport does not prevent brain injury in endotoxemia. In contrast, cerebral hypoperfusion and reduced O(2) transport occur in fetuses destined to die, emphasizing the importance of sustaining O(2) transport for survival.

  19. Effect of endotoxin and radio-detoxified endotoxin on the serum T4 level of rats and response of their thyroid gland to exogenous TSH

    SciTech Connect

    Bertok, L.; Nagy, S.U.

    1984-12-01

    Experiments were performed to demonstrate that, while the shock-inducing dose of parent (toxic) endotoxin significantly decreases the serum T4 level of rats and inhibits the T4 response given to exogenous thyroid stimulating hormone (TSH), the radio-detoxified (/sup 60/Co-gamma, 150 kGy) endotoxin preparation does not inhibit the response to exogenous TSH. It also decreases serum T4 level to a lesser extent than untreated endotoxin.

  20. Endotoxin levels in settled airborne dust in European schools: the HITEA school study.

    PubMed

    Jacobs, J H; Krop, E J M; Borras-Santos, A; Zock, J-P; Taubel, M; Hyvarinnen, A; Pekkanen, J; Doekes, G; Heederik, D J J

    2014-04-01

    Indoor exposure to microbial agents is known to influence respiratory health. Besides home exposure, exposure in schools can affect respiratory health. In this study, we measured endotoxin in settled dust in primary schools in three European countries from three different geographical regions with different climates. Our aim was to characterize endotoxin levels in primary schools and evaluate associations with potential determinants. Endotoxin levels were repeatedly assessed in 23 schools in Spain (n = 7), the Netherlands (n = 10), and Finland (n = 6) using electrostatic dustfall collectors. In total, 645 measurements were taken in 237 classrooms. Endotoxin levels differed significantly between countries; Dutch schools had the highest levels, while Finnish schools showed the lowest levels. In each country, differences in endotoxin levels were observed between schools and over the sampling periods. Estimates improved after adjustment for sampling period. Factors affecting endotoxin levels in a school differed per country. In general, endotoxin levels were higher in lower grades and in classrooms with higher occupancy. School endotoxin levels may contribute significantly to total endotoxin exposure in children and teachers. As the correlation between the repeated measurements is reasonable, single endotoxin measurements form a reasonable basis for estimating annual endotoxin levels in schools.

  1. Airborne endotoxin concentrations in indoor and outdoor particulate matter and their predictors in an urban city.

    PubMed

    Yoda, Y; Tamura, K; Shima, M

    2017-02-04

    Endotoxins are an important biological component of particulate matter and have been associated with adverse effects on human health. There have been some recent studies on airborne endotoxin concentrations. We collected fine (PM2.5 ) and coarse (PM10-2.5 ) particulate matter twice on weekdays and weekends each for 48 hour, inside and outside 55 homes in an urban city in Japan. Endotoxin concentrations in both fractions were measured using the kinetic Limulus Amebocyte Lysate assay. The relationships between endotoxin concentrations and household characteristics were evaluated for each fraction. Both indoor and outdoor endotoxin concentrations were higher in PM2.5 than in PM10-2.5 . In both PM2.5 and PM10-2.5 , indoor endotoxin concentrations were higher than outdoor concentrations, and the indoor endotoxin concentrations significantly correlated with outdoor concentrations in each fraction (R(2) =0.458 and 0.198, respectively). Indoor endotoxin concentrations in PM2.5 were significantly higher in homes with tatami or carpet flooring and in homes with pets, and lower in homes that used air purifiers. Indoor endotoxin concentrations in PM10-2.5 were significantly higher in homes with two or more children and homes with tatami or carpet flooring. These results showed that the indoor endotoxin concentrations were associated with the household characteristics in addition to outdoor endotoxin concentrations.

  2. Distribution of radiolabeled endotoxin with particular reference to the eye: concise communication

    SciTech Connect

    Rosenbaum, J.T.; Hendricks, P.A.; Shively, J.E.; McDougall, I.R.

    1983-01-01

    A single systemic injection of endotoxin (lipopolysaccharide or LPS) reproducibly induces a cellular infiltrate in the uveal tract of the rat eye within 24 hr. Other organs are not comparably sensitive to systemic endotoxin. One hypothesis to explain this unique sensitivity is that endotoxin is preferentially bound by ocular tissue. Researchers tested this hypothesis by studying the distribution in the rat of intravenously injected endotoxin that had been radiolabeled with /sup 99m/Tc or /sup 32/P. With either radionuclide the concentration of endotoxin per gram of tissue at a variety of times after injection ranging from 5 min to 3 hr and 45 min, was markedly less in the eye than in liver, kidney, or spleen. A study with radiolabeled albumin indicated that these differences could not be ascribed solely to the organ's blood volume. They demonstrate, therefore, that the eye does not preferentially bind endotoxin, and they are compatible with the hypothesis that endotoxin's ocular effects are indirectly mediated.

  3. Distribution of radiolabeled endotoxin with particular reference to the eye: concise communication.

    PubMed

    Rosenbaum, J T; Hendricks, P A; Shively, J E; McDougall, I R

    1983-01-01

    A single systemic injection of endotoxin (lipopolysaccharide or LPS) reproducibly induces a cellular infiltrate in the uveal tract of the rat eye within 24 hr. Other organs are not comparably sensitive to systemic endotoxin. One hypothesis to explain this unique sensitivity is that endotoxin is preferentially bound by ocular tissue. We tested this hypothesis by studying the distribution in the rat of intravenously injected endotoxin that had been radiolabeled with Tc-99m or P-32. With either radionuclide the concentration of endotoxin per gram of tissue at a variety of times after injection ranging from 5 min to 3 hr and 45 min, was markedly less in the eye than in liver, kidney, or spleen. A study with radiolabeled albumin indicated that these differences could not be ascribed solely to the organ's blood volume. They demonstrate, therefore, that the eye does not preferentially bind endotoxin, and they are compatible with the hypothesis that endotoxin's ocular effects are indirectly mediated.

  4. A Cytophaga species endotoxin as a putative agent of occupation-related lung disease.

    PubMed Central

    Flaherty, D K; Deck, F H; Hood, M A; Liebert, C; Singleton, F; Winzenburger, P; Bishop, K; Smith, L R; Bynum, L M; Witmer, W B

    1984-01-01

    A previous study suggested that a biologically active bacterial endotoxin was a putative agent of lung disease in a textile-producing facility. The endotoxin was isolated from the biomass growing in a chilled-water spray air humidification system. The bacterial flora of the air humidification system were isolated and taxonomically identified to the genus level. By using indirect immunofluorescence assays, a serologically reactive Cytophaga species was identified. A serologically reactive, biologically active (Limulus assay) endotoxin was purified from phenol extracts of the Cytophaga species. The endotoxin contained sugars, hexosamines, and lipids identical to those found in the humidifier biomass endotoxin. All subjects with biopsy-proven and suspected lung disease had antibodies directed toward the purified Cytophaga endotoxin. The data suggest that the Cytophaga endotoxin is the putative agent of lung disease in the textile facility. PMID:6360896

  5. [Review on characteristics and detecting assay of bacterial endotoxin contamination in water environment].

    PubMed

    Zhang, Can; Liu, Wen-Jun; Zhang, Ming-Lu; Tian, Fang; Yang, Yi; An, Dai-Zhi

    2014-04-01

    Endotoxins, also known as lipopolysaccharide complexes, are anchored in the outer membrane cell wall of most Gram-negative bacteria and some cyanobacteria. They are continuously released to environment during cell decay. Being common pyrogens and highly immunogenic molecules, endotoxins are related to many human diseases. Due to the tolerances and thermo-stability of endotoxin molecules, they were hard to be removed by common methods. The health risk caused by the endotoxin contamination in drinking water and water environment by various exposure pathways have attracted more and more attention in recent years. In this paper, the physical and chemical properties, biological activities and detection assay of the endotoxin contamination were reviewed, and interfere factors of the main assay, the LAL/TAL (Limulus amebocyte lysate/Tachypleus amebocyte lysate) assay, for detecting endotoxin in water sample were investigated, and the development tendency of the endotoxin detection assay was analyzed.

  6. Binding of /sup 125/I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

    SciTech Connect

    Meyers, K.M.; Boehme, M.; Inbar, O.

    1982-10-01

    Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using /sup 125/I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.

  7. Endotoxin contamination and control in surface water sources and a drinking water treatment plant in Beijing, China.

    PubMed

    Can, Zhang; Wenjun, Liu; Wen, Sun; Minglu, Zhang; Lingjia, Qian; Cuiping, Li; Fang, Tian

    2013-07-01

    In this paper, endotoxin contamination was determined in treated water following each unit of a drinking water treatment plant (WTP) in Beijing, China and its source water (SW) from a long water diversion channel (Shijiazhuang-Beijing) originating from four reservoirs in Hebei province, China. The total-endotoxin activities in SW ranged from 21 to 41 EU/ml at five selected cross sections of the diversion channel. The total-endotoxin in raw water of the WTP ranged from 11 to 16 EU/ml due to dilution and pretreatment during water transportation from Tuancheng Lake to the WTP, and finished water of the WTP ranged from 4 to 10 EU/ml, showing a 49% decrease following the full-scale treatment process at the WTP. Compared with the 31% removal of free-endotoxin, the WTP removed up to 71% of bound-endotoxin in raw water. The traditional treatment processes (coagulation, sedimentation and filtration) in the WTP removed substantial amounts of total-endotoxin (up to 63%), while endotoxin activities increased after granular activated carbon (GAC) adsorption and chlorination. The total-endotoxin in the actual water was composed of free-endotoxin and bound-endotoxin (endotoxin aggregates, bacteria-bound endotoxins and particle-attached endotoxins). The endotoxin aggregates, bacteria-bound endotoxins and particle-attached endotoxins co-exist as suspended particles in water, and only the bacteria-bound endotoxins were correlated with bacterial cells suspended in water. The particle distribution of endotoxin aggregates in ultrapure water was also tested and the results showed that the majority (64-89%) of endotoxin aggregates had diameters <2 μm. The endotoxin contamination and control in treated water following each unit of the WTP processes and its SW from reservoirs are discussed and compared with regard to bacterial cell counts and particle characteristics, which were dependent, to a certain extent, on different flow rates and turbulence of the water environments.

  8. Integrating murine gene expression studies to understand obstructive lung disease due to chronic inhaled endotoxin.

    PubMed

    Lai, Peggy S; Hofmann, Oliver; Baron, Rebecca M; Cernadas, Manuela; Meng, Quanxin Ryan; Bresler, Herbert S; Brass, David M; Yang, Ivana V; Schwartz, David A; Christiani, David C; Hide, Winston

    2013-01-01

    Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD). These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease. We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke. A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature. Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene expression were observed. The endotoxin component of tobacco

  9. Integrating Murine Gene Expression Studies to Understand Obstructive Lung Disease Due to Chronic Inhaled Endotoxin

    PubMed Central

    Lai, Peggy S.; Hofmann, Oliver; Baron, Rebecca M.; Cernadas, Manuela; Meng, Quanxin Ryan; Bresler, Herbert S.; Brass, David M.; Yang, Ivana V.; Schwartz, David A.; Christiani, David C.; Hide, Winston

    2013-01-01

    Rationale Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD). These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease. Methods We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke. Results A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature. Conclusions Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene expression were observed

  10. A selective sorbent for removing bacterial endotoxins from blood

    NASA Astrophysics Data System (ADS)

    Morozov, A. S.; Kopitsyna, M. N.; Bessonov, I. V.; Karelina, N. V.; Nuzhdina, A. V.; Sarkisov, I. Yu.; Pavlova, L. A.; Tsyurupa, M. P.; Blinnikova, Z. K.; Davankov, V. A.

    2016-12-01

    Synthetic ligands carrying a positive charge and capable of selective binding of bacterial endotoxins are covalently immobilized on surfaces of domestic hemosorbent Styrosorb-514 based on hypercrosslinked polystyrene. It is shown that the resulting sorbent aimed at treating sepsis exceeds imported specific hemosorbent in Toraymyxin™ columns in removing lipopolysaccharides, and can be used in domestically-produced Desepta columns.

  11. Streptomycetes in house dust: associations with housing characteristics and endotoxin

    EPA Science Inventory

    In addition to mold, indoor bioaerosols also contain bacterial components that may have implications for human health. Endotoxin is a cell wall component in Gram-negative bacteria present at varying levels indoors that has been found to have respiratory health implications. Stre...

  12. Mouse C-Reactive Protein and Endotoxin-Induced Resistance

    PubMed Central

    Patterson, L. T.; Higginbotham, R. D.

    1965-01-01

    Patterson, L. T. (University of Texas, Galveston), and R. D. Higginbotham. Mouse C-reactive protein and endotoxin-induced resistance. J. Bacteriol. 90:1520–1524. 1965.—The relationship between the level of C-reactive protein (CRP) in the sera of mice and resistance to Staphylococcus aureus infection after the injection of Escherichia coli endotoxin was studied. The CRP level was essentially unchanged at 6 hr after endotoxin, and resistance was slightly decreased. At 24 hr after endotoxin, both CRP levels and resistance were increased. Since the increase in the CRP level and resistance appeared to be associated, it was of interest that, when mouse CRP was tested for in vitro reactions with several strains of bacteria, cells of all species of gram-positive bacteria tested (including S. aureus) were agglutinated by CRP. E. coli was not agglutinated under the conditions of the test. It is proposed that mouse CRP is an opsonin, and possibly a lysin, and is involved in nonspecific resistance to infection with S. aureus. PMID:5322718

  13. Resistance of MMP9 and TIMP1 to endotoxin tolerance.

    PubMed

    Muthukuru, Manoj; Cutler, Christopher W

    2015-07-01

    Inflammatory cytokines activate tissue collagenases such as matrix metalloproteinases (MMPs). MMPs are antagonized by tissue inhibitors of metalloproteinases (TIMPs) that attempt to regulate excessive collagenase activity during inflammatory conditions. During chronic inflammatory conditions, induction of endotoxin tolerance negatively regulates the cytokine response in an attempt to curtail excessive host tissue damage. However, little is known about how downregulation of inflammatory cytokines during endotoxin tolerance regulates MMP activities. In this study, human monocyte-derived macrophages were either sensitized or further challenged to induce tolerance with lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) or Escherichia coli (EcLPS). Inflammatory cytokines, such as TNF-α and IL-1β, and levels of MMP9 and TIMP1 were analyzed by a combination of cytometric bead array, western blot/gelatin zymography and real-time RT-PCR. Functional blocking with anti-TLR4 but not with anti-TLR2 significantly downregulated TNF-α and IL-1β. However, MMP9 levels were not inhibited by toll-like receptor (TLR) blocking. Interestingly, endotoxin tolerance significantly upregulated TIMP1 relative to MMP9 and downmodulated MMP9 secretion and its enzymatic activity. These results suggest that regulatory mechanisms such as induction of endotoxin tolerance could inhibit MMP activities and could facilitate restoring host tissue homeostasis.

  14. Endotoxin-induced mortality in rats is reduced by nitrones

    SciTech Connect

    Hamburger, S.A.; McCay, P.B. )

    1989-12-01

    The goal of these investigations was to determine if nitrone spin-trapping agents can alter mortality associated with endotoxemia in the rat. Reactive free radicals attack nitrone spin-trapping agents forming relatively reactive, persistent free radical spin adducts. We administered 85 mM (10 ml/kg) of alpha-phenyl N-tert-butyl nitrone (PBN), alpha-4-pyridyl-N-oxide N-tert-butyl nitrone (4-POBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), or vehicle (saline i.p.) 30 min before endotoxin (25 mg/kg i.p.) or vehicle to Sprague-Dawley (SD) or Holtzman virus-free (HVF) rats (n = 10-17/group). All vehicle-treated rats receiving endotoxin were dead by 1 day. At 7 days, 83% of PBN-treated SD, 42% of PBN- or POBN-treated HVF, and 25% of DMPO-treated HVF rats were alive. The difference in survival of PBN-treated animals between strains may reflect the higher susceptibility of HVF rats to endotoxin. The observed reduction in mortality may be related to the well-established capacity of spin-trapping agents to capture reactive free radicals that may be generated in target tissues in response to endotoxin, and that would otherwise react with cell components and produce tissue injury.

  15. Bacterial endotoxin adhesion to different types of orthodontic adhesives.

    PubMed

    Romualdo, Priscilla Coutinho; Guerra, Thaís Rodrigues; Romano, Fábio Lourenço; Silva, Raquel Assed Bezerra da; Brandão, Izaíra Tincani; Silva, Célio Lopes; Silva, Lea Assed Bezerra da; Nelson-Filho, Paulo

    2017-01-01

    The aim of this study was to assess whether LPS adheres to orthodontic adhesive systems, comparing two commercial brands. Forty specimens were fabricated from Transbond XT and Light Bond composite and bonding agent components (n=10/component), then contaminated by immersion in a bacterial endotoxin solution. Contaminated and non-contaminated acrylic resin samples were used as positive and negative control groups, respectively. LPS quantification was performed by the Limulus Amebocyte Lysate QCL-1000™ test. Data obtained were scored and subjected to the Chi-square test using a significance level of 5%. There was endotoxin adhesion to all materials (p<0.05). No statistically significant difference was found between composites/bonding agents and acrylic resin (p>0.05). There was no significant difference (p>0.05) among commercial brands. Affinity of endotoxin was significantly greater for the bonding agents (p=0.0025). LPS adhered to both orthodontic adhesive systems. Regardless of the brand, the endotoxin had higher affinity for the bonding agents than for the composites. There is no previous study assessing the affinity of LPS for orthodontic adhesive systems. This study revealed that LPS adheres to orthodontic adhesive systems. Therefore, additional care is recommended to orthodontic applications of these materials.

  16. [The importance of endotoxin producing bacterias for practical purposes

    PubMed

    Schimmel, Dietrich

    1994-01-01

    Lipopolysaccharides (endotoxin) cause according to resorption out of the intestinal tract or aerogenic inhalation or by a septic infection clinical signs. The clinical reactions are praeshock symptoms, acute forms of shock and death. The experimental intratracheally administration of lipopolysaccharides into calves caused pneumonic lesions without bacterial experimental infection.

  17. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin

    NASA Astrophysics Data System (ADS)

    Borovikova, Lyudmila V.; Ivanova, Svetlana; Zhang, Minghuang; Yang, Huan; Botchkina, Galina I.; Watkins, Linda R.; Wang, Haichao; Abumrad, Naji; Eaton, John W.; Tracey, Kevin J.

    2000-05-01

    Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of proinflammatory and anti-inflammatory pathways. Endotoxin (lipopolysaccharide), produced by all gram-negative bacteria, activates macrophages to release cytokines that are potentially lethal. The central nervous system regulates systemic inflammatory responses to endotoxin through humoral mechanisms. Activation of afferent vagus nerve fibres by endotoxin or cytokines stimulates hypothalamic-pituitary-adrenal anti-inflammatory responses. However, comparatively little is known about the role of efferent vagus nerve signalling in modulating inflammation. Here, we describe a previously unrecognized, parasympathetic anti-inflammatory pathway by which the brain modulates systemic inflammatory responses to endotoxin. Acetylcholine, the principle vagal neurotransmitter, significantly attenuated the release of cytokines (tumour necrosis factor (TNF), interleukin (IL)-1β, IL-6 and IL-18), but not the anti-inflammatory cytokine IL-10, in lipopolysaccharide-stimulated human macrophage cultures. Direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxaemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF amounts, and prevented the development of shock.

  18. Fighting the stranger-antioxidant protection against endotoxin toxicity.

    PubMed

    Cadenas, Susana; Cadenas, Ana M

    2002-10-30

    Septic shock is a serious problem in critically ill and surgical patients throughout the world. It is a systemic inflammatory response caused by excessive secretion of proinflammatory mediators, such as tumor necrosis factor-alpha, mainly induced by endotoxin, a major component of the Gram-negative bacterial outer membrane. Experimental evidence suggests that reactive oxygen species (ROS) may be important mediators of cellular injury during endotoxemia, either as a result of macromolecular damage or by interfering with extracellular and intracellular regulatory processes. In addition, nitric oxide is thought to play a key role in the pathogenesis of sepsis. This review begins with a brief overview of the toxic effects of endotoxin at organism level, paying particular attention to cardiovascular damage. It continues by analysing the mechanism by which endotoxin is recognized by specific cells of the immune system, which then respond to bacterial infection and the pathway leading to nuclear factor-kappaB activation and proinflammatory gene transcription. With regard to this process, the review focuses on the involvement of reactive oxygen and nitrogen species. Lastly, the protective role of antioxidants against endotoxin toxicity and their potential clinical use is discussed.

  19. Streptomycetes in house dust: associations with housing characteristics and endotoxin

    EPA Science Inventory

    In addition to mold, indoor bioaerosols also contain bacterial components that may have implications for human health. Endotoxin is a cell wall component in Gram-negative bacteria present at varying levels indoors that has been found to have respiratory health implications. Stre...

  20. Endotoxins in indoor air and settled dust in primary schools in a subtropical climate.

    PubMed

    Salonen, Heidi; Duchaine, Caroline; Létourneau, Valérie; Mazaheri, Mandana; Clifford, Sam; Morawska, Lidia

    2013-09-03

    Endotoxins can significantly affect the air quality in school environments. However, there is currently no reliable method for the measurement of endotoxins, and there is a lack of reference values for endotoxin concentrations to aid in the interpretation of measurement results in school settings. We benchmarked the "baseline" range of endotoxin concentration in indoor air, together with endotoxin load in floor dust, and evaluated the correlation between endotoxin levels in indoor air and settled dust, as well as the effects of temperature and humidity on these levels in subtropical school settings. Bayesian hierarchical modeling indicated that the concentration in indoor air and the load in floor dust were generally (<95th percentile) <13 EU/m(3) and <24,570 EU/m(2), respectively. Exceeding these levels would indicate abnormal sources of endotoxins in the school environment and the need for further investigation. Metaregression indicated no relationship between endotoxin concentration and load, which points to the necessity for measuring endotoxin levels in both the air and settled dust. Temperature increases were associated with lower concentrations in indoor air and higher loads in floor dust. Higher levels of humidity may be associated with lower airborne endotoxin concentrations.

  1. Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

    PubMed

    Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

    2014-12-01

    The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.

  2. Endotoxin concentrations within the breathing zone of horses are higher in stables than on pasture.

    PubMed

    Berndt, Annerose; Derksen, Frederik J; Edward Robinson, N

    2010-01-01

    Inflammatory airway disease is common in stabled horses, with a prevalence of 17.3% in Michigan pleasure horses. Stable dust is rich in endotoxin, which may induce neutrophilic airway inflammation. Climatological conditions (ambient temperature and relative humidity) may influence endotoxin concentrations in pastures. The aim of this project was to determine if endotoxin levels in the breathing zone of horses in stables were higher than of horses on pasture, and if the endotoxin on pasture was associated with climatological conditions. Endotoxin exposure of six horses that were stabled or on pasture was determined by a Limulus amebocyte lysate assay. Climatological data were obtained from the US National Climatic Data Center. Endotoxin exposure was significantly higher (about 8-fold) in stables than on pasture. On pasture, endotoxin varied widely, despite constant climatological conditions. It was concluded that stabled horses are exposed to higher endotoxin concentrations than horses on pastures. Local endotoxin concentrations may be more important than ambient climatological conditions in determining endotoxin exposure of individual horses. Copyright 2008 Elsevier Ltd. All rights reserved.

  3. Airborne endotoxin concentrations at a large open-lot dairy in southern idaho.

    PubMed

    Dungan, Robert S; Leytem, April B

    2009-01-01

    Endotoxins are derived from gram-negative bacteria and are a potential respiratory health risk for animals and humans. To determine the potential for endotoxin transport from a large open-lot dairy, total airborne endotoxin concentrations were determined at an upwind location (background) and five downwind locations on three separate days. The downwind locations were situated at of the edge of the lot, 200 and 1390 m downwind from the lot, and downwind from a manure composting area and wastewater holding pond. When the wind was predominantly from the west, the average endotoxin concentration at the upwind location was 24 endotoxin units (EU) m(-3), whereas at the edge of the lot on the downwind side it was 259 EU m(-3). At 200 and 1390 m downwind from the edge of the lot, the average endotoxin concentrations were 168 and 49 EU m(-3), respectively. Average airborne endotoxin concentrations downwind from the composting site (36 EU m(-3)) and wastewater holding pond (89 EU m(-3)) and 1390 m from the edge of the lot were not significantly different from the upwind location. There were no significant correlations between ambient weather data collected and endotoxin concentrations over the experimental period. The downwind data show that the airborne endotoxin concentrations decreased exponentially with distance from the lot edge. Decreasing an individual's proximity to the dairy should lower their risk of airborne endotoxin exposure and associated health effects.

  4. Restriction of gut-derived endotoxin impairs DNA synthesis for liver regeneration.

    PubMed

    Cornell, R P

    1985-11-01

    The influence of restricting gut-derived endotoxin availability on liver regeneration after partial hepatectomy was evaluated. Partial hepatectomy was performed by 67% liver resection of ether-anesthetized rats. Liver regeneration was quantified after partial hepatectomy by [3H]thymidine incorporation into hepatic DNA; endotoxemia due to absorption of endogenous endotoxin from the gut into the portal circulation was determined by qualitative lysate assay of perchloric acid-extracted plasma samples, and plasma levels of the hepatotrophic factors insulin and glucagon were measured by radioimmunoassay. Treatments to restrict gut-derived endotoxin included chronic gavage with neomycin and cefazolin for gut sterilization, chronic gavage with cholestyramine to bind endotoxin within the gut, subcutaneous administration of polymyxin B to neutralize the lipid A portion of circulating endotoxin, intraperitoneal induction of endotoxin tolerance by progressively higher doses of endotoxin, and experimentation with isolator-reared defined flora Fisher rats that were Gram-negative bacteria deficient and therefore endotoxin deficient. All treatments to restrict endogenous endotoxin impaired DNA synthesis in regenerating livers particularly 21 h posthepatectomy when replication was increasing most rapidly in normal rats. We hypothesize that impairment of DNA synthesis after partial hepatectomy in endotoxin-restricted animals was due to the observed lack of normal systemic endotoxemic as well as hyperinsulinemic and hyperglucagonemic responses to 67% liver resection.

  5. Rapid detection of bacterial endotoxins in drinking water and renovated wastewater.

    PubMed Central

    Jorgensen, J H; Lee, J C; Pahren, H R

    1976-01-01

    A pilot study was conducted to determine the feasibility of using the Limulus endotoxin assay to detect endotoxins in potable waters and from reclaimed advanced waste treatment (AWT) plant effluents. Water samples were tested using both Limulus lysates prepared in our laboratory and a commercial product, Difco Pyrotest. The Limulus assay procedure was easily adapted to the testing of water samples for endotoxin. Measured endotoxin concentrations varied from 0.78 ng/ml to 1,250 ng/ml. Levels of endotoxin were not predictable based on whether the water was drinking water or AWT water, i.e., some AWT water samples had less endotoxin activity than some samples of drinking water, and some AWT waters had greater endotoxin activity than drinking water. Only three of the water samples tested were free of any detectable endotoxin. Breakpoint chlorination procedures seemed to reduce measurable endotoxin content, whereas passage through activated carbon columns was associated with greater final endotoxin concentrations in test waters. PMID:791115

  6. Polymyxin B-immobilized fiber column hemoperfusion removes endotoxin throughout a 24-hour treatment period.

    PubMed

    Mitaka, Chieko; Fujiwara, Naoto; Yamamoto, Mamoru; Toyofuku, Takahiro; Haraguchi, Go; Tomita, Makoto

    2014-10-01

    The purpose of this study was to evaluate the extent of endotoxin adsorption by polymyxin B-immobilized fiber column hemoperfusion (PMX) performed for a 24-hour treatment period in patients with septic shock. Nineteen patients with septic shock were retrospectively studied. The plasma endotoxin concentrations of blood drawn from the radial artery and from the outlet circuit of the PMX column were measured by kinetic turbidimetric limulus assay using an MT-358 Toxinometer (Wako Pure Chemical Industries, Ltd, Osaka, Japan) after 24 hours of PMX treatment. The endotoxin removal rate was defined by the following equation: ([radial artery endotoxin concentration - outlet circuit of PMX column endotoxin concentration]/radial artery endotoxin concentration) × 100%. The patients had a median Acute Physiology and Chronic Health Evaluation II score of 29 at intensive care unit admission and a 28-day mortality of 47%. Before the start of the PMX treatment, the median radial arterial plasma endotoxin concentration was 16.48 pg/mL. After 24 hours of PMX treatment, the median radial plasma endotoxin concentration had decreased to 1.857 pg/mL, and the concentration at the outlet circuit of the PMX column was further decreased to 0.779 pg/mL. The median endotoxin removal rate was 74.4%. These findings suggest that 24-hour PMX treatment was effective in removing endotoxin continuously throughout the entire treatment period. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Lactulose inhibits endotoxin induced tumour necrosis factor production by monocytes. An in vitro study.

    PubMed Central

    Greve, J W; Gouma, D J; von Leeuwen, P A; Buurman, W A

    1990-01-01

    Preoperative oral treatment with lactulose is used to prevent complications after surgery in patients with obstructive jaundice. The effect is perhaps the result of an inactivation of gut derived endotoxins but the exact mechanism of action is, however, unknown. Tumour necrosis factor is an important mediator of endotoxin toxicity. The cytokine tumour necrosis factor is mainly produced by mononuclear phagocytes. In this study, the effect of lactulose on the endotoxin induced tumour necrosis factor release by monocytes was investigated. The direct effect of lactulose on endotoxin was tested in a chromogenic limulus amoebocyte lysate assay. Polymyxin B a known inactivator of endotoxin was used as control in both experiments. Lactulose has a limited capacity to inactivate endotoxin as measured in the endotoxin assay. In contrast lactulose significantly reduced endotoxin induced tumour necrosis factor production by monocytes. In conclusion lactulose inhibits tumour necrosis factor production by a direct inhibitory effect on monocytes, rather than by inactivation of endotoxin. Because tumour necrosis factor is an important mediator of endotoxin toxicity, this inhibitory effect could explain the beneficial effect of lactulose in obstructive jaundice. PMID:2311978

  8. Endotoxin inactivation via steam-heat treatment in dilute simethicone emulsions used in biopharmaceutical processes.

    PubMed

    Britt, Keith A; Galvin, Jeffrey; Gammell, Patrick; Nti-Gyabaah, Joseph; Boras, George; Kolwyck, David; Ramirez, José G; Presente, Esther; Naugle, Gregory

    2014-01-01

    Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam-heat treatment was fit to a four-parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature-related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration-related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre-exponential factor was >10(12) s(-1) suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam-heat treatment decreased endotoxin levels by 1-2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam-heat treatment. The results from this study show that steam-heat treatment is a viable endotoxin control strategy that can be implemented to support large-scale biopharmaceutical manufacturing.

  9. Endotoxins associated with cyanobacteria and their removal during drinking water treatment.

    PubMed

    Rapala, Jarkko; Lahti, Kirsti; Räsänen, Leena A; Esala, Anna-Liisa; Niemelä, Seppo I; Sivonen, Kaarina

    2002-05-01

    The aim of this study was to investigate endotoxin concentrations in cyanobacterial water blooms and strains, and to assess the removal of endotoxins during drinking water treatment. Endotoxin concentrations were measured from 151 hepatotoxic, neurotoxic and non-toxic cyanobacterial water blooms by using Limulus amebocyte lysate (LAL) assay, and the results were compared to bacterial data. Endotoxin activities ranged from 20 to 3.8 x 10(4) endotoxin units (EU) per ml. Endotoxicity of the samples correlated with phycobiliprotein concentration that was used to assess cyanobacterial abundance, heterotrophic plate count, and Aeromonas spp. but it did not correlate with the number of coliforms or streptococci. The high endotoxin concentrations occasionally detected in the water bloom samples were probably due to Gram negative bacteria that existed together with cyanobacteria since the 26 axenic cyanobacterial strains from different genera that were studied showed very low endotoxin activity. No differences in endotoxin activity were detected between hepatotoxic, neurotoxic and non-toxic strains. Removal of endotoxins during drinking water treatment was studied at nine waterworks that previously had been associated with high numbers of cyanobacteria and that used different processes for water purification. Endotoxin concentration in raw waters ranged from 18 to 356 EU ml(-1). The treatment processes reduced 59-97% of the endotoxin activity; in the treated water the concentration ranged from 3 to 15 EU ml(-1). The most significant reduction occurred at the early stages of water treatment, during coagulation, settling and sand filtration. Activated carbon filtration either increased or had no effect on endotoxin concentration. Ozonation and chlorination had little effect on the endotoxin concentrations.

  10. In vivo quantitation of the rat liver's ability to eliminate endotoxin from portal vein blood

    SciTech Connect

    Yamaguchi, Y.; Yamaguchi, K.; Babb, J.L.; Gans, H.

    1982-12-01

    The in vivo uptake of endotoxin by the liver from portal vein blood was assessed during a single passage through the liver. /sup 51/Cr labeled and unlabeled endotoxin were infused in different amounts into the femoral vein of three groups of lead-sensitized rats: a nonoperated, a sham-operated, and a surgically created reversed Eck fistula (REF) group. Whereas in the former two the infused endotoxin encounters the lung as the first filter organ, the liver performs this function in the latter experimental model. The mortality rates observed in control and sham-operated, lead-sensitized rats were found to correlate closely and reproducibly to the degree of endotoxemia. This assay was then applied to determine the amount of endotoxin eliminated by the liver by establishing, in the REF rat, the amounts of endotoxin that escaped hepatic clearance. The capacity of the liver to eliminate endotoxin from portal vein blood during a single passage increases as the portal vein endotoxin level rises; it approaches a maximum, suggesting that endotoxin's interaction with the Kupffer cells conforms to classical saturation kinetics. A Lineweaver-Burk plot prepared from these data indicates that the maximal in vivo capacity of the liver to remove endotoxin from portal vein blood approximates 1.5 micrograms/gm liver/hr. Data obtained with the use of radiolabeled endotoxin corroborate the information obtained with the bioassay technique. Endotoxin eliminated by the Kupffer cells in these quantities is slowly disintegrated; 4 hr after termination of the endotoxin infusion, less than 4% of the radiolabel is found in the urine and none in the bile. These observations indicate that the Kupffer cell's functional capacity to sequester and detoxify endotoxin is extensive and far exceeds the requirements imposed by physiological and most pathological conditions.

  11. [Effects of heat and cool-producing needling manipulations on rectal temperature and serum endotoxin content in endotoxin-induced heat syndrome rabbits].

    PubMed

    Zhou, Hai-Yan; Yang, Jie; Feng, Yue; Yang, Shen-Qiao

    2012-08-01

    To observe the effect of traditional manipulations of "Shaoshanhuo" (heat-producing needling) and "Toutianliang"(cool-producing needling) on body temperature and serum endotoxin level in heat syndrome rabbits. Twenty-four Japanese rabbits were randomly divided into control, model, Shaoshanhuo and Toutianliang groups. Heat-syndrome model was established by subcutaneous injection of bacterium coli endotoxin solution (40 microg/mL, 2 mL/kg). Heat-producing and cool-producing needling was applied to bilateral "Quchi" (LI 11) for 5 min, respectively. Rectal temperature was detected by using a thermometer, and serum endotoxin content assayed by using Limulus Ameboyte Lysate kit (luminescence measuring). In comparison with the control group, both rectal temperature and serum endotoxin levels were increased significantly in the model group (P < 0.01). While compared to the model group, the rectal temperature and serum endotoxin levels were down-regulated considerably in both Shaoshanhuo and Toutianliang groups (P < 0.05, P < 0.01). The effect of the Toutianliang group was obviously superior to that of the Shaoshanhuo group in reducing serum endotoxin content (P < 0.01). Both heat-producing needling and cool-producing needling can lower rectal temperature and serum endotoxin levels in heat-syndrome rabbits, and the effect of cool-producing needling is relatively better in reducing endotoxin content.

  12. Connectivity in river deltas

    NASA Astrophysics Data System (ADS)

    Passalacqua, P.; Hiatt, M. R.; Sendrowski, A.

    2016-12-01

    Deltas host approximately half a billion people and are rich in ecosystem diversity and economic resources. However, human-induced activities and climatic shifts are significantly impacting deltas around the world; anthropogenic disturbance, natural subsidence, and eustatic sea-level rise are major causes of threat to deltas and in many cases have compromised their safety and sustainability, putting at risk the people that live on them. In this presentation, I will introduce a framework called Delta Connectome for studying connectivity in river deltas based on different representations of a delta as a network. Here connectivity indicates both physical connectivity (how different portions of the system interact with each other) as well as conceptual (pathways of process coupling). I will explore several network representations and show how quantifying connectivity can advance our understanding of system functioning and can be used to inform coastal management and restoration. From connectivity considerations, the delta emerges as a leaky network that evolves over time and is characterized by continuous exchanges of fluxes of matter, energy, and information. I will discuss the implications of connectivity on delta functioning, land growth, and potential for nutrient removal.

  13. Pen Branch delta expansion

    SciTech Connect

    Nelson, E.A.; Christensen, E.J.; Mackey, H.E.; Sharitz, R.R.; Jensen, J.R.; Hodgson, M.E.

    1984-02-01

    Since 1954, cooling water discharges from K Reactor ({anti X} = 370 cfs {at} 59 C) to Pen Branch have altered vegetation and deposited sediment in the Savannah River Swamp forming the Pen Branch delta. Currently, the delta covers over 300 acres and continues to expand at a rate of about 16 acres/yr. Examination of delta expansion can provide important information on environmental impacts to wetlands exposed to elevated temperature and flow conditions. To assess the current status and predict future expansion of the Pen Branch delta, historic aerial photographs were analyzed using both basic photo interpretation and computer techniques to provide the following information: (1) past and current expansion rates; (2) location and changes of impacted areas; (3) total acreage presently affected. Delta acreage changes were then compared to historic reactor discharge temperature and flow data to see if expansion rate variations could be related to reactor operations.

  14. Enhanced susceptibility of mice to combinations of delta 9-tetrahydrocannabinol and live or killed gram-negative bacteria.

    PubMed Central

    Bradley, S G; Munson, A E; Dewey, W L; Harris, L S

    1977-01-01

    Combinations of delta 9-tetrahydrocannabinol (delta 9-THC) and bacterial endotoxin were shown to be hyperadditively toxic for mice. A variety of purified lipopolysaccharide (LPS) preparations elicted enhanced mortality in combination with delta 9-THC. Escherichia coli O26:B6 LPS (Boivin preparation) at an essentially nonlethal dose of 2.5 mg/kg reduced the dose of delta 9-THC required to kill 50% of the treated mice from ca. 350 to 150 mg/kg. Inbred BALB, DBA, and C3H/HeCr mice, noninbred ICR mice, and hybrid CDF1 and BDF1 mice were hyperreactive to combinations of delta 9-THC and LPS. Moreover, a variety of heat-killed intestinal and gram-negative bacteria, live E. coli, and complexes of lipid A with a variety of proteins substituted for LPS in the synergistic toxicity of LPS and delta 9-THC. Extracts of marijuana also elicited hyperreactivity to LPS. The hyperadditive lethality of combinations of delta 9-THC and LPS was markedly less in mice rendered refractory to LPS or delta 9-THC by repeated administration of LPS or delta 9-THC, respectively. PMID:330405

  15. Passive Immunization against Cachectin/Tumor Necrosis Factor Protects Mice from Lethal Effect of Endotoxin

    NASA Astrophysics Data System (ADS)

    Beutler, B.; Milsark, I. W.; Cerami, A. C.

    1985-08-01

    A highly specific polyclonal rabbit antiserum directed against murine cachectin/tumor necrosis factor (TNF) was prepared. When BALB/c mice were passively immunized with the antiserum or with purified immune globulin, they were protected against the lethal effect of the endotoxin lipopolysaccharide produced by Escherichia coli. The prophylactic effect was dose-dependent and was most effective when the antiserum was administered prior to the injection of the endotoxin. Antiserum to cachectin/TNF did not mitigate the febrile response of endotoxin-treated animals, and very high doses of endotoxin could overcome the protective effect. The median lethal dose of endotoxin in mice pretreated with 50 microliters of the specific antiserum was approximately 2.5 times greater the median lethal dose for controls given nonimmune serum. The data suggest that cachectin/TNF is one of the principal mediators of the lethal effect of endotoxin.

  16. Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay.

    PubMed

    Brawner, Andrew; Hinrichs, Steven H; Larson, Marilynn A; Lockridge, Oksana

    2016-04-05

    The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min(-1) and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 °C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations.

  17. Endotoxin contamination: a key element in the interpretation of nanosafety studies.

    PubMed

    Li, Yang; Boraschi, Diana

    2016-02-01

    The study of toxicity and potential risks of engineered nanoparticles is of particular importance in nanomedicine. Endotoxin, a common contaminant of bacterial origin, has biological effects that can mask the true biological effects of nanoparticles, if its presence is overlooked. In this review, we report the features of nanoparticle contamination by endotoxin, and the different biological effects of endotoxin-contaminated nanoparticles. We will describe different methods for endotoxin detection applied to nanoparticles, and discuss their pros and cons. Eventually, we describe various methods for eliminating endotoxin contamination in nanoparticles. Although there is no universal technique for efficiently removing endotoxin from nanoparticles, specific solutions can be found case by case, which can allow us to perform nanosafety studies in biologically relevant conditions.

  18. Concentration, physical state, and purity of bacterial endotoxin affect its detoxification by ionizing radiation

    SciTech Connect

    Csako, G.; Tsai, C.M.; Hochstein, H.D.; Elin, R.J.

    1986-11-01

    Increasing concentrations of a highly purified bacterial lipopolysaccharide preparation, the U.S. Reference Standard Endotoxin, were exposed to increasing doses of ionizing radiation from a 60Co source. At identical radiation doses both the structural change and Limulus amebocyte lysate (LAL) reactivity were progressively smaller with increasing concentrations of the lipopolysaccharide in an aqueous medium. Under the experimental conditions used, there was a linear relationship between the endotoxin concentration and radiation dose for the structural changes. In contrast to endotoxin in aqueous medium, endotoxin irradiated in its dry state showed no decrease in LAL reactivity and rabbit pyrogenicity. Endotoxin exposed to radiation in water in the presence of albumin showed a much smaller decrease in LAL and pyrogenic activities than expected. The results show that the concentration, physical state, and purity of endotoxin influence its structural and functional alteration by ionizing radiation.

  19. The relationship between faecal endotoxin and faecal microflora of the C57BL mouse.

    PubMed Central

    Rogers, M. J.; Moore, R.; Cohen, J.

    1985-01-01

    We studied the effect of oral selective antibiotic decontamination (SD) on the faecal endotoxin content and microflora in individual C57BL mice. Suppression of the coliform count was associated with an initial rise in faecal endotoxin concentration from 0.1 to 3.1 mg/g wet faeces during the first week of SD, which fell to 0.04 mg/g during the second week of treatment. Cessation of SD resulted in an immediate sharp increase in coliform count followed by its decline and gradual recovery to pre-treatment counts. Faecal endotoxin levels followed a parallel course. SD did not effect significantly the counts of lactobacilli, bacteroides and enterococci. It appears that the coliform population is responsible for the overall level of faecal endotoxin, and that during the initial period of SD endotoxin levels are elevated, an effect which may be mediated by antibiotic-enhanced release of endotoxin. PMID:4067295

  20. Relationships between metabolic changes and clinical signs in pregnant sheep given endotoxin.

    PubMed Central

    Naylor, J M; Kronfeld, D S

    1986-01-01

    Groups of four pregnant ewes were allocated to the following feeding and intravenous endotoxin treatments: fed, Escherichia coli endotoxin (50 micrograms/kg X 75), fed, saline, fasted, E. coli endotoxin (50 micrograms/kg X 75) and fasted, saline. Endotoxin administration resulted in depression, fever, hypoglycemia, hypocalcemia and a reduction in nonesterified fatty acid and ketone body concentrations. Depression correlated best with body temperature (r = 0.76), fasted sheep showed smaller increases in body temperature and were less depressed following endotoxin. Three of eight endotoxin treated sheep died, mortality was not related to rectal temperature but was associated with lactic acidosis. Hypoglycemia was not associated with either death or depression. Fed sheep that were unable to stand had lower serum calcium concentrations than standing sheep. PMID:3527393

  1. Delta hepatitis in Malaysia.

    PubMed

    Sinniah, M; Dimitrakakis, M; Tan, D S

    1986-06-01

    Sera from one hundred and fifty nine Malaysian individuals were screened for the prevalence of delta markers. These included 15 HBsAg positive homosexuals, 16 acute hepatitis B cases, 9 chronic hepatitis B patients, 13 healthy HBsAg carriers and 106 intravenous (i.v.) drug abusers, of whom 27 were positive for HBsAg only and the rest were anti-HBc IgG positive but HBsAg negative. The prevalence of delta markers in the homosexuals was found to be 6.7%, in the HBsAg positive drug abusers 17.8%, in acute hepatitis B cases 12.5%. No evidence of delta infection was detected in healthy HBsAg carriers, chronic hepatitis B cases and HBsAg negative i.v. drug abusers. With reference to i.v. drug abusers, the prevalence of delta markers was higher in Malays (23%) than in Chinese (7%) although the latter had a higher HBsAg carrier rate. Although the HBsAg carrier rate in the homosexuals was high, their delta prevalence rate was low as compared to drug abusers. In Malaysia, as in other non-endemic regions, hepatitis delta virus transmission appeared to occur mainly via the parenteral and sexual routes. This is the first time in Malaysia that a reservoir of delta infection has been demonstrated in certain groups of the population at high risk for hepatitis B.

  2. Endotoxins in baled cottons and airborne dusts in textile mills in the People's Republic of China.

    PubMed Central

    Olenchock, S A; Christiani, D C; Mull, J C; Ye, T T; Lu, P L

    1983-01-01

    Bulk cotton samples and airborne vertical elutriated cotton dusts were obtained from textile mills in Shanghai, People's Republic of China. Analysis of endotoxin contents revealed that baled cottons which were grown in different countries varied in endotoxin contamination. The two textile mills, which operated at similar overall airborne dust levels, differed markedly in the levels of airborne endotoxins. The data suggest that the biological activity or "toxicity" of airborne cotton dusts may not be correlated directly with gravimetric dust levels. PMID:6639029

  3. Treatment Characteristics of Polysaccharides and Endotoxin Using Oxygen Plasma Produced by RF Discharge

    NASA Astrophysics Data System (ADS)

    Kitazaki, Satoshi; Hayashi, Nobuya; Goto, Masaaki

    2010-10-01

    Treatment of polysaccharides and endotoxin were attempted using oxygen plasma produced by RF discharge. Oxygen radicals observed by optical light emission spectra are factors of decomposition of polysaccharides and endotoxin. Fourier transform infrared spectra indicate that most of chemical bonds in the polysaccharides are dissociated after irradiation of the oxygen plasma. Also, the decomposition rate of endotoxin was approximately 90% after irradiation of the oxygen plasma for 180 min.

  4. Treatment Characteristics of Polysaccharides and Endotoxin Using Oxygen Plasma Produced by RF Discharge

    SciTech Connect

    Kitazaki, Satoshi; Hayashi, Nobuya; Goto, Masaaki

    2010-10-13

    Treatment of polysaccharides and endotoxin were attempted using oxygen plasma produced by RF discharge. Oxygen radicals observed by optical light emission spectra are factors of decomposition of polysaccharides and endotoxin. Fourier transform infrared spectra indicate that most of chemical bonds in the polysaccharides are dissociated after irradiation of the oxygen plasma. Also, the decomposition rate of endotoxin was approximately 90% after irradiation of the oxygen plasma for 180 min.

  5. CARDIORESPIRATORY AND METABOLIC RESPONSES TO LIVE E. COLI AND ENDOTOXIN IN THE MONKEY

    DTIC Science & Technology

    of live organisms (Escherichia coli) were compared with endotoxin and saline in rhesus monkeys. Six animals were given E . coli , six endotoxin, and...five served as controls. Studies were conducted for 2-4 hours. The mean cardiac output decreased 62% within 60-90 minutes in the E . coli group and 41...Pco2 decreased to 24 mm Hg in the E . coli group and 26 mm Hg in the endotoxin group. Arterial hypoxemia developed in four animals and high

  6. School Endotoxin Exposure and Asthma Morbidity in Inner-city Children.

    PubMed

    Lai, Peggy S; Sheehan, William J; Gaffin, Jonathan M; Petty, Carter R; Coull, Brent A; Gold, Diane R; Phipatanakul, Wanda

    2015-11-01

    Endotoxin exposure is associated with airway inflammation. Children spend 6 to 8 h/d in school, yet the effect of school-specific endotoxin exposure on asthma morbidity is not well understood. In this longitudinal cohort study, 248 students with asthma, from 38 inner-city schools, underwent baseline phenotyping and follow-up. Clinical outcomes were evaluated throughout the academic school year and linked to classroom-specific dust and air endotoxin levels as well as home dust endotoxin levels. The primary outcome was maximum asthma symptom-days per 2-week period. Classrooms had higher settled dust endotoxin levels compared with homes (14.3 endotoxin unit/mg vs 11.3 endotoxin unit/mg; P = .02). Airborne endotoxin levels exceeding recommended occupational exposure limits for adults were recorded in 22.0% of classrooms. Classroom air endotoxin levels were independently associated with increased maximum symptom-days in children with nonatopic asthma, but not in those with atopic asthma (interaction P = .03). Adjusting for home exposures, classroom endotoxin exposure was independently associated with a dose-dependent increase in asthma symptom-days for children with nonatopic asthma (adjusted incidence rate ratio, 1.16 [95% CI, 1.03-1.31]; P = .02). In these subjects, maximum symptom-days increased by 1.3 days for each 14-day period when comparing students in classrooms with the lowest endotoxin levels compared with average measured levels. Inner-city children with asthma are exposed to high levels of airborne endotoxin at school, resulting in increased asthma symptoms in children with nonatopic asthma. Mitigation of school-related exposures may represent a strategy to decrease asthma morbidity in this population. ClinicalTrials.gov; No.: NCT01756391; URL: www.clinicaltrials.gov.

  7. Indoor dust and air concentrations of endotoxin in urban and rural environments.

    PubMed

    Barnig, C; Reboux, G; Roussel, S; Casset, A; Sohy, C; Dalphin, J-C; de Blay, F

    2013-03-01

    Rural dairy farming is associated with high exposure to indoor endotoxins as compared to rural nonfarming houses and urban houses. The time spent on the mattress (7 h for an adult) and of the proximity of the contaminated source should be taken into account with the other causes of exposure. Studies in European children from a farming background have shown that these children have a reduced risk of asthma and atopic sensitization compared to their urban counterparts. It has been suggested that this might be due to exposure to high levels of endotoxin in the farming environment. The aim of this study was to compare indoor endotoxin concentrations in air and dust samples from randomly selected urban and rural dwellings. In the rural area, endotoxins were analysed in farmhouses and nonfarmhouses as well as housing characteristics, lifestyle factors and agricultural practices likely to influence air and dust endotoxin levels. Endotoxin levels were significantly higher in floor (6600 ± 6100 vs 3600 ± 5600 and 3800 ± 17,000 ng g⁻¹; P < 0·001) and mattress dust (2900 ± 4100 vs 1100 ± 2400 and 800 ± 2600 ng g⁻¹; P < 0·001) from farmhouses compared to other rural and urban homes. However, no difference was observed between endotoxin concentrations in the air of urban and rural houses, and airborne endotoxin levels did not correlate to dust levels. Lack of ventilation and direct entry into the house were correlated with an increase in dust endotoxin levels. These results confirm that dairy farming is associated with high exposure to endotoxins in indoor dust samples. No difference was observed between indoor airborne concentrations between urban and rural houses. These results suggest that measuring endotoxin in dust is the most relevant method to assess endotoxin exposure. © 2012 The Society for Applied Microbiology.

  8. Endotoxin suppresses surfactant synthesis in cultured rat lung cells

    SciTech Connect

    Li, J.J.; Sanders, R.L.; McAdam, K.P.; Gelfand, J.A.; Burke, J.F.

    1989-02-01

    Pulmonary complications secondary to postburn sepsis are a major cause of death in burned patients. Using an in vitro organotypic culture system, we examined the effect of E. coli endotoxin (LPS) on lung cell surfactant synthesis. Our results showed that E. coli endotoxin (1.0, 2.5, 10 micrograms LPS/ml) was capable of suppressing the incorporation of /sup 3/H-choline into de novo synthesized surfactant, lamellar bodies (LB), and common myelin figures (CMF) at 50%, 68%, and 64%, respectively. In a similar study, we were able to show that LPS also inhibited /sup 3/H-palmitate incorporation by cultured lung cells. LPS-induced suppression of surfactant synthesis was reversed by hydrocortisone. Our results suggest that LPS may play a significant role in reducing surfactant synthesis by rat lung cells, and thus contribute to the pathogenesis of sepsis-related respiratory distress syndrome (RDS) in burn injury.

  9. Endotoxin-like activity associated with Lyme disease Borrelia.

    PubMed

    Fumarola, D; Munno, I; Marcuccio, C; Miragliotta, G

    1986-12-01

    The newly recognized spirochete, Borrelia burgdorferi, the causative agent of Lyme Disease, has been examined for endotoxin-like activities as measured by the standard Farmacopea Ufficiale della Republica Italiana rabbit fever test and the Limulus amoebocyte lysate assay. The suspension of heat-killed microorganism caused a febrile response at a dose of 1 X 10(8) bacteria pro kilo. Similar results were obtained in the Limulus assay where the heat-killed spirochetes stimulated formation of solid clot until the concentration of 1 X 10(5) per ml. Both in pyrogen test and in Limulus assay heat-killed Escherichia coli exhibited a higher degree of potency. These results show that LD-Borrelia possess endotoxin-like activities which could help in understanding the pathogenesis of the clinical symptomatology of the disease.

  10. [Endotoxin adsortion as adjuvant therapy in gram negative severe sepsis].

    PubMed

    Candel, F J; Martínez-Sagasti, F; Borges, M; Maseda, E; Herrera-Gutiérrez, M; Garnacho-Montero, J; Maynar, F J; Zaragoza, R; Mensa, J; Azanza, J R

    2010-09-01

    The mortality rate of severe sepsis and septic shock remains still high. Within the last years a better knowledge of its physiopathology and the implementation of a group of measures addressed to a fast identification and early treatment of the septic patients have proved to reduce mortality rate. Likewise, it continues being investigated in modulating the inflammatory response and limiting the harmful action of the bacterial products on the immune system. As a result of this research some endotoxin adsorber devices have been designed to control one of the most important targets that start the inflammatory cascade when gram negative microorganisms are involved.The usefulness that these endotoxin removal devices might have as adjuvant treatment in the Septic Syndrome and its applicability are reviewed in this paper. Likewise a profile of patient that might be to the benefit of this therapy is suggested according to the current knowledge.

  11. Enhanced endotoxin effects in plasma fibronectin-deficient rats.

    PubMed Central

    Yoder, M C; Kilpatrick-Smith, L; Arbittier, D; Douglas, S D; Polin, R A

    1987-01-01

    Immunoreactive plasma fibronectin depletion has been associated with the presence of collagen-fibronectin complexes in patients after trauma and in animal models of traumatic and burn injuries. However, the role of plasma fibronectin in the development of sepsis after traumatic and burn injuries in patients is unknown. Treatment of patients and animals with purified human plasma fibronectin ameliorates some of the clinical and metabolic effects of systemic endotoxemia. We report that the induction of immunoreactive plasma fibronectin deficiency by gelatin infusion is associated with enhanced effects of intraperitoneal Escherichia coli endotoxin injection. We have observed a significant increase in the concentrations of ammonia in plasma of treated rats compared with those in control rats administered the same dose of endotoxin. PMID:3298066

  12. Endotoxin removal by radio frequency gas plasma (glow discharge)

    NASA Astrophysics Data System (ADS)

    Poon, Angela

    2011-12-01

    Contaminants remaining on implantable medical devices, even following sterilization, include dangerous fever-causing residues of the outer lipopolysaccharide-rich membranes of Gram-negative bacteria such as the common gut microorganism E. coli. The conventional method for endotoxin removal is by Food & Drug Administration (FDA)-recommended dry-heat depyrogenation at 250°C for at least 45 minutes, an excessively time-consuming high-temperature technique not suitable for low-melting or heat-distortable biomaterials. This investigation evaluated the mechanism by which E. coli endotoxin contamination can be eliminated from surfaces during ambient temperature single 3-minute to cumulative 15-minute exposures to radio-frequency glow discharge (RFGD)-generated residual room air plasmas activated at 0.1-0.2 torr in a 35MHz electrodeless chamber. The main analytical technique for retained pyrogenic bio-activity was the Kinetic Chromogenic Limulus Amebocyte Lysate (LAL) Assay, sufficiently sensitive to document compliance with FDA-required Endotoxin Unit (EU) titers less than 20 EU per medical device by optical detection of enzymatic color development corresponding to < 0.5 EU/ml in sterile water extracts of each device. The main analytical technique for identification of chemical compositions, amounts, and changes during sequential reference Endotoxin additions and subsequent RFGD-treatment removals from infrared (IR)-transparent germanium (Ge) prisms was Multiple Attenuated Internal Reflection (MAIR) infrared spectroscopy sensitive to even monolayer amounts of retained bio-contaminant. KimaxRTM 60 mm x 15 mm and 50mm x 15mm laboratory glass dishes and germanium internal reflection prisms were inoculated with E. coli bacterial endotoxin water suspensions at increments of 0.005, 0.05, 0.5, and 5 EU, and characterized by MAIR-IR spectroscopy of the dried residues on the Ge prisms and LAL Assay of sterile water extracts from both glass and Ge specimens. The Ge prism MAIR

  13. Peripherally administered orexin improves survival of mice with endotoxin shock

    PubMed Central

    Ogawa, Yasuhiro; Irukayama-Tomobe, Yoko; Murakoshi, Nobuyuki; Kiyama, Maiko; Ishikawa, Yui; Hosokawa, Naoto; Tominaga, Hiromu; Uchida, Shuntaro; Kimura, Saki; Kanuka, Mika; Morita, Miho; Hamada, Michito; Takahashi, Satoru; Hayashi, Yu; Yanagisawa, Masashi

    2016-01-01

    Sepsis is a systemic inflammatory response to infection, accounting for the most common cause of death in intensive care units. Here, we report that peripheral administration of the hypothalamic neuropeptide orexin improves the survival of mice with lipopolysaccharide (LPS) induced endotoxin shock, a well-studied septic shock model. The effect is accompanied by a suppression of excessive cytokine production and an increase of catecholamines and corticosterone. We found that peripherally administered orexin penetrates the blood-brain barrier under endotoxin shock, and that central administration of orexin also suppresses the cytokine production and improves the survival, indicating orexin’s direct action in the central nervous system (CNS). Orexin helps restore body temperature and potentiates cardiovascular function in LPS-injected mice. Pleiotropic modulation of inflammatory response by orexin through the CNS may constitute a novel therapeutic approach for septic shock. DOI: http://dx.doi.org/10.7554/eLife.21055.001 PMID:28035899

  14. Delta Scuti stars: Theory

    NASA Technical Reports Server (NTRS)

    Guzik, J. A.

    1998-01-01

    The purpose of asteroseismology is not only to derive the internal structure of individual stars from their observed oscillation frequencies, but also to test and extend one's understanding of the physics of matter under the extremes of temperature, density, and pressure found in stellar interiors. In this review, the author hopes to point out what one can learn about the Sun by studying (delta) Scuti stars, as well as what one can learn about stars more massive or evolved than the Sun. He discusses some of the difficulties in theoretical approaches to asteroseismology for (delta) Scuti stars, using FG Vir, (delta) Scuti, and CD-24(degree) 7599 as examples.

  15. Delta Scuti stars: Theory

    SciTech Connect

    Guzik, J.A.

    1998-03-01

    The purpose of asteroseismology is not only to derive the internal structure of individual stars from their observed oscillation frequencies, but also to test and extend one`s understanding of the physics of matter under the extremes of temperature, density, and pressure found in stellar interiors. In this review, the author hopes to point out what one can learn about the Sun by studying {delta} Scuti stars, as well as what one can learn about stars more massive or evolved than the Sun. He discusses some of the difficulties in theoretical approaches to asteroseismology for {delta} Scuti stars, using FG Vir, {delta} Scuti, and CD-24{degree} 7599 as examples.

  16. Nile River Delta, Egypt

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The Nile River Delta of Egypt (30.0N, 31.0E) irrigated by the Nile River and its many distributaries, is some of the richest farm land in the world and home to some 45 million people, over half of Egypt's population. The capital city of Cairo is at the apex of the delta. Just across the river from Cairo can be seen the ancient three big pyramids and sphinx at Giza and the Suez Canal is just to the right of the delta.

  17. Nile Delta, Egypt

    NASA Technical Reports Server (NTRS)

    1982-01-01

    The Nile Delta of Egypt (30.0N, 31.0E) irrigated by the Nile River and its many distributaries, is some of the richest farm land in the world and home to some 45 million people, over half of Egypt's population of 57 million. The capital city of Cairo is at the apex of the delta in the middle of the scene. Across the river from Cairo can be seen the three big pyramids and sphinx at Giza and the Suez Canal is just to the right of the delta.

  18. Nile River Delta, Egypt

    NASA Technical Reports Server (NTRS)

    1984-01-01

    The Nile River Delta of Egypt (30.0N, 31.0E) irrigated by the Nile River and its many distributaries, is some of the richest farm land in the world and home to some 45 million people, over half of Egypt's population. The capital city of Cairo is at the apex of the delta. Just across the river from Cairo can be seen the ancient three big pyramids and sphinx at Giza and the Suez Canal is just to the right of the delta.

  19. Delta Scuti stars: Theory

    NASA Technical Reports Server (NTRS)

    Guzik, J. A.

    1998-01-01

    The purpose of asteroseismology is not only to derive the internal structure of individual stars from their observed oscillation frequencies, but also to test and extend one's understanding of the physics of matter under the extremes of temperature, density, and pressure found in stellar interiors. In this review, the author hopes to point out what one can learn about the Sun by studying (delta) Scuti stars, as well as what one can learn about stars more massive or evolved than the Sun. He discusses some of the difficulties in theoretical approaches to asteroseismology for (delta) Scuti stars, using FG Vir, (delta) Scuti, and CD-24(degree) 7599 as examples.

  20. Modeling river delta formation

    PubMed Central

    Seybold, Hansjörg; Andrade, José S.; Herrmann, Hans J.

    2007-01-01

    A model to simulate the time evolution of river delta formation process is presented. It is based on the continuity equation for water and sediment flow and a phenomenological sedimentation/erosion law. Different delta types are reproduced by using different parameters and erosion rules. The structures of the calculated patterns are analyzed in space and time and compared with real data patterns. Furthermore, our model is capable of simulating the rich dynamics related to the switching of the mouth of the river delta. The simulation results are then compared with geological records for the Mississippi River. PMID:17940031

  1. Nile River Delta, Egypt

    NASA Image and Video Library

    1984-10-13

    The Nile River Delta of Egypt (30.0N, 31.0E) irrigated by the Nile River and its many distributaries, is some of the richest farm land in the world and home to some 45 million people, over half of Egypt's population. The capital city of Cairo is at the apex of the delta. Just across the river from Cairo can be seen the ancient three big pyramids and sphinx at Giza and the Suez Canal is just to the right of the delta.

  2. Comparison of endotoxin levels in cow's milk samples derived from farms and shops.

    PubMed

    Sipka, Sándor; Béres, Andrea; Bertók, Lóránd; Varga, Tamara; Bruckner, Geza

    2015-07-01

    The observations on the protective effect of bacterial endotoxin in farm-derived cow's milk on childhood asthma and allergy are contradictory. The aim of this study was to determine the endotoxin levels in 'farm-derived whole raw' and 'processed shop' sources of cow's milk, and to test how the temperature and storing conditions might alter their endotoxin concentrations. Milk was collected from farms and shops. The level of endotoxin was measured by micro (gel-clot) Limulus amebocyte lysate test expressed as EU/ml. The concentration ranges of endotoxin were much higher and more widely scattered in the samples of whole raw farm milk than in the processed shop milk. Cold storage or heating increased the endotoxin concentrations in all samples of farm milk, but not in the processed shop milk. These results show that elevated levels of endotoxin in raw farm milk samples can occur from the cowshed or be formed during storage. In processed shop milk, storage does not cause any changes in the amount of endotoxin. Therefore, it is consistent that the handling and storage of raw milk alters the endotoxin concentrations, which may explain previous contradictory findings regarding the beneficial modulating effects on innate immunity toward allergy prevention in early childhood.

  3. Protection against hyperoxia by serum from endotoxin treated rats: absence of superoxide dismutase induction

    SciTech Connect

    Berg, J.T.; Smith, R.M.

    1988-01-01

    Endotoxin greatly reduces lung injury and pleural effusions in adult rats exposed to normobaric hyperoxia (> 98% oxygen for 60 hours). This study reports that serum from endotoxin treated donor rats protects serum recipients against hyperoxic lung injury without altering lung superoxide dismutase (SOD) activity. Rats pretreated with endotoxin alone were protected and exhibited an increase in lung SOD activity as previously reported by others. Protection by serum was not due to the transfer of residual endotoxin or SOD. These results show, that protection from oxygen toxicity can occur in rats without an increase in lung SOD and suggest that a serum factor may be involved.

  4. Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane

    SciTech Connect

    Sawada, Y.; Fujii, R.; Igami, I.; Kawai, A.; Kamiki, T.; Niwa, M.

    1986-04-01

    The microporous polyethylene hollow-fiber membrane has a unique microfibrile structure throughout its depth and has been found to possess the functions of filtration and adsorption of endotoxin in water. The membrane has a maximum pore diameter of approximately 0.04 micron, a diameter which is within the range of microfiltration. Approximately 10 and 20% of the endotoxin in tap water and subterranean water, respectively, was smaller than 0.025 micron. Endotoxin in these water sources was efficiently removed by the microporous polyethylene hollow-fiber membrane. Escherichia coli O113 culture broth contained 26.4% of endotoxin smaller than 0.025 micron which was also removed. Endotoxin was leaked into the filtrate only when endotoxin samples were successively passed through the membrane. These results indicate that endotoxin smaller than the pore size of the membrane was adsorbed and then leaked into the filtrate because of a reduction in binding sites. Dissociation of /sup 3/H-labeled endotoxin from the membrane was performed, resulting in the removal of endotoxin associated with the membrane by alcoholic alkali at 78% efficiency.

  5. Human very low density lipoproteins and chylomicrons can protect against endotoxin-induced death in mice.

    PubMed Central

    Harris, H W; Grunfeld, C; Feingold, K R; Rapp, J H

    1990-01-01

    Endotoxemia stimulates many physiologic responses including disturbances in lipid metabolism. We hypothesized that this lipemia may be part of a defensive mechanism by which the body combats the toxic effects of circulating endotoxin. We tested the effects of mixtures of endotoxin, lipoproteins, and lipoprotein-free plasma and determined the ability of varying concentrations of human very low density lipoproteins (VLDL) and chylomicrons, as well as low density lipoproteins (LDL) and high density lipoproteins (HDL), and of the synthetic lipid emulsion SOYACAL to prevent endotoxin-induced death in mice. This study demonstrates that the triglyceride-rich VLDL and chylomicrons, as well as cholesterol-rich LDL and HDL, and cholesterol-free SOYACAL can protect against endotoxin-induced death. Protection required small amounts of lipoprotein-free plasma, and depended on the incubation time and the concentration of lipoprotein lipid. Despite stringent techniques to prevent exogenous endotoxin contamination eight of ten duplicate VLDL preparations contained endotoxin (5,755 +/- 3,514 ng endotoxin/mg triglyceride, mean +/- SEM) making the isolation of endotoxin-free VLDL difficult. In contrast, simultaneous preparations of LDL and HDL were relatively free of endotoxin contamination (3 +/- 3 and 320 +/- 319 ng/mg total cholesterol, respectively), suggesting that the contamination of VLDL occurs in vivo and not during the isolation procedure. These observations suggest a possible role for increased triglyceride-rich lipoproteins in the host's defense against endotoxemia and infection. Images PMID:2394827

  6. Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane.

    PubMed

    Sawada, Y; Fujii, R; Igami, I; Kawai, A; Kamiki, T; Niwa, M

    1986-04-01

    The microporous polyethylene hollow-fiber membrane has a unique microfibrile structure throughout its depth and has been found to possess the functions of filtration and adsorption of endotoxin in water. The membrane has a maximum pore diameter of approximately 0.04 micron, a diameter which is within the range of microfiltration. Approximately 10 and 20% of the endotoxin in tap water and subterranean water, respectively, was smaller than 0.025 micron. Endotoxin in these water sources was efficiently removed by the microporous polyethylene hollow-fiber membrane. Escherichia coli O113 culture broth contained 26.4% of endotoxin smaller than 0.025 micron which was also removed. Endotoxin was leaked into the filtrate only when endotoxin samples were successively passed through the membrane. These results indicate that endotoxin smaller than the pore size of the membrane was adsorbed and then leaked into the filtrate because of a reduction in binding sites. Dissociation of 3H-labeled endotoxin from the membrane was performed, resulting in the removal of endotoxin associated with the membrane by alcoholic alkali at 78% efficiency.

  7. Toxic shock syndrome toxin 1 enhances synthesis of endotoxin-induced tumor necrosis factor in mice.

    PubMed Central

    Henne, E; Campbell, W H; Carlson, E

    1991-01-01

    Toxic shock syndrome toxin 1 (TSST-1) was tested for its ability to enhance the production of endotoxin-induced tumor necrosis factor (TNF) in C3H/HeN mice. The TNF level in serum was quantified by a sandwich enzyme-linked immunosorbent assay (ELISA). It was found that when mice were injected with 20 micrograms of TSST-1 12 h before exposure to 1 micrograms of endotoxin, the serum endotoxin-induced TNF was 20 times as high as that found in mice exposed to endotoxin alone. Although 20 micrograms of TSST-1 did induce a maximum level of near 1 ng of TNF per ml of serum 1.5 h after exposure, the TNF concentration was greatly diminished after 5 to 6 h and was no longer detectable after 12 h. Pretreatment of mice with 20 micrograms of TSST-1 or 1 micrograms of endotoxin did not influence TNF induction by TSST-1 12 h later. Also, pretreatment of mice with 1 micrograms of endotoxin did not enhance TNF induction by endotoxin 12 h later. Enhancement was achieved only when mice were exposed to TSST-1 more than 4 h and less than 24 h before injection of endotoxin. Despite the relatively high serum TNF levels (30 to 50 ng/ml), no mortality was observed in the mice treated with both TSST and endotoxin. PMID:1879919

  8. Detection of bacterial endotoxin in drinking tap and bottled water in Kuwait.

    PubMed

    Abdulraheem, Abdulkareem; Mustafa, Seham; Al-Saffar, Nabeel; Shahjahan, Muhammed

    2012-12-01

    This study was carried out to measure and compare the concentration of bacterial endotoxin in a variety of samples from drinking tap and bottled water available in Kuwait by using the Limulus Amoebocyte lysate test. A total of 29 samples were tested. Samples were collected from a variety of locations throughout the six governorates of Kuwait and 23 brands of local and imported bottled water samples were collected from the local market. The concentration of bacterial endotoxin was measured by using the standard Limulus Amoebocyte lysate test, gel clot method. This study showed that measured endotoxin concentrations in tap drinking water varied from 2.4 to 33.8 EU/ml with the average endotoxin concentration of 14.2 EU/ml. While the results of endotoxin concentrations in the bottled water were <0.03 to 20.1 EU/ml with an average of 1.96 EU/ml. The average concentration of endotoxin in bottled water is 13.5 % of the average concentration of endotoxin in tap drinking water. This experimental investigation has proved that drinking bottled water has less endotoxin as compared to tap water in Kuwait. It is also demonstrated that the endotoxin concentration did not exceed the acceptable level in drinking tap water.

  9. Schistosomiasis Japonica During Pregnancy Is Associated With Elevated Endotoxin Levels in Maternal and Placental Compartments

    PubMed Central

    McDonald, Emily A.; Pond-Tor, Sunthorn; Jarilla, Blanca; Sagliba, Marianne J.; Gonzal, Annaliza; Amoylen, Amabelle J.; Olveda, Remigio; Acosta, Luz; Gundogan, Fusun; Ganley-Leal, Lisa M.; Kurtis, Jonathan D.; Friedman, Jennifer F.

    2014-01-01

    Schistosomiasis affects approximately 40 million women of reproductive age and has been linked to elevated levels of circulating endotoxin in nonpregnant individuals. We have evaluated endotoxin levels in maternal, placental, and newborn blood collected from women residing in Leyte, Philippines. Endotoxin levels in both maternal and placental compartments in pregnant women with schistosomiasis were 1.3- and 2.4-fold higher, respectively, than in uninfected women. In addition, higher concentrations of endotoxin in placental blood were associated with premature birth, acute chorioamnionitis, and elevated proinflammatory cytokines. By promoting endotoxemia, schistosomiasis may exert additional, maladaptive influences on pregnancy outcomes. PMID:23964108

  10. Influence of endotoxin treatment on dexamethasone induction of hepatic phosphoenolpyruvate carboxykinase.

    PubMed Central

    McCallum, R E; Seale, T W; Stith, R D

    1983-01-01

    Decreased glucocorticoid binding has been observed at a time after endotoxin (3 to 6 h) when imparied liver enzyme induction is known to occur. This study was undertaken to characterize the early time course of hypoglycemia and decreased liver phosphoenolpyruvate carboxykinase (PEPCK) activity in intact and adrenalectomized mice given endotoxin. In addition, altered steroid induction of hepatic PEPCK was examined in adrenalectomized mice given dexamethasone at intervals before and after a median lethal dose of endotoxin. Intact mice demonstrated a dramatic hyperglycemia at 1 h after endotoxin treatment, a response absent in adrenalectomized mice. Plasma glucose levels were significantly reduced from control values at 3 and 6 h posttreatment, with the most pronounced endotoxin-induced hypoglycemia seen in adrenalectomized mice. Hepatic PEPCK activity in intact mice given endotoxin was decreased at 3 and 6 h after treatment, although no change from basal, noninduced levels was seen in poisoned adrenalectomized mice. The increased increment in hepatic PEPCK activity due to fasting of intact control mice was reproduced in adrenalectomized control mice by the administration of dexamethasone. Furthermore, the induction of hepatic PEPCK by dexamethasone was inhibited by 1 h after endotoxin treatment, with enzyme activity falling to basal, noninduced levels by 6 h posttreatment. At these same time intervals after endotoxin treatment, no evidence of histopathology in the liver or adrenal glands was seen. These results coincide with changes in steroid binding seen previously and indicate that endotoxin treatment produces significant alterations in glucocorticoid action at the subcellular or molecular level. PMID:6822414

  11. Neuropharmacological agents modifying endotoxin-induced changes in mice1

    PubMed Central

    Wright, David J M; Weller, Malcolm P I

    1980-01-01

    A variety of neuropharmacological agents were tested to elucidate how chlorpromazine influenced an endotoxin-induced reaction. The results obtained, particularly with beta-adrenergic blocking agents, reserpine and fusaric acid, suggested that the primary locus of chlorpromazine's action was mediated by peripheral beta-adrenergic receptor blockade. Such a locus is compatible with the low doses of propranolol which suppress the reaction, and with successful treatment of shock with dopamine. PMID:6112273

  12. Complex links between dietary lipids, endogenous endotoxins and metabolic inflammation.

    PubMed

    Laugerette, Fabienne; Vors, Cécile; Peretti, Noël; Michalski, Marie-Caroline

    2011-01-01

    Metabolic diseases such as obesity are characterized by a subclinical inflammatory state that contributes to the development of insulin resistance and atherosclerosis. Recent reports also indicate that (i) there are alterations of the intestinal microbiota in metabolic diseases and (ii) absorption of endogenous endotoxins (namely lipopolysaccharides, LPS) can occur, particularly during the digestion of lipids. The aim of the present review is to highlight recently gained knowledge regarding the links between high fat diets, lipid digestion, intestinal microbiota and metabolic endotoxemia & inflammation.

  13. Cardiotrophin-1 attenuates endotoxin-induced acute lung injury.

    PubMed

    Pulido, E J; Shames, B D; Pennica, D; O'leary, R M; Bensard, D D; Cain, B S; McIntyre, R C

    1999-06-15

    Cardiotrophin-1 (CT-1) is a recently discovered member of the gp130 cytokine family, which includes IL-6, IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Recent evidence suggests that, like other members of this family, CT-1 may possess anti-inflammatory properties. We hypothesized that in vivo CT-1 administration would attenuate endotoxin (ETX)-induced acute lung injury. We studied the effects of CT-1 (100 microgram/kg ip, 10 min prior to ETX) in a rat model of ETX-induced acute lung injury (Salmonella typhimurium lipopolysaccharide, 20 mg/kg ip). Six hours after ETX, lungs were harvested for determination of neutrophil accumulation (myeloperoxidase, MPO, assay) and lung edema (wet-to-dry weight ratio). Mechanisms of pulmonary vasorelaxation were examined in isolated pulmonary artery rings at 6 h by interrogating endothelium-dependent (response to acetylcholine) and endothelium-independent (response to sodium nitroprusside) relaxation following alpha-adrenergic (phenylephrine)-stimulated preconstriction. CT-1 abrogated the endotoxin-induced lung neutrophil accumulation: 2.3 +/- 0.2 units MPO/g wet lung (gwl) vs 6. 3 +/- 0.3 units MPO/gwl in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Similarly, CT-1 prevented ETX-induced lung edema: wet-to-dry-weight ratio, 4.473 +/- 0.039 vs 4.747 +/- 0.039 in the ETX group (P < 0.05 vs ETX, P > 0.05 vs control). Endotoxin caused significant impairment of both endothelium-dependent and -independent pulmonary vasorelaxation, and CT-1 attenuated this injury. Thus, cardiotrophin-1 possesses significant anti-inflammatory properties in a model of endotoxin-induced acute lung injury. Copyright 1999 Academic Press.

  14. Septic Participation in Cardiogenic Shock: Exposure to Bacterial Endotoxin.

    PubMed

    Ramirez, Paula; Villarreal, Esther; Gordon, Monica; Gómez, María Dolores; de Hevia, Luis; Vacacela, Karla; Gisbert, Teresa; Quinzá, Adrian; Ruiz, Jesús; Alonso, Ricardo; Bonastre, Juan; Vila, Jordi

    2017-05-01

    In cardiogenic shock (CS), presence of fever, leukocytosis, relatively low systemic vascular resistances, and high serum procalcitonin levels are quite frequent and recurrently involve the search for an infectious complication. We hypothesized that endotoxin exposure in CS could explain this sepsis-like syndrome. Prospective observational study of consecutive CS patients admitted to our intensive care unit (ICU). Patients were followed during the first 3 days after CS onset. All clinical, hemodynamic, and microbiological data were collected. Inflammatory biomarkers and anti-endotoxin antibodies (IgM EndoCAb) were daily measured. We included 37 consecutive CS patients. Twenty-two patients (60%) had body temperature >38.3°C or <35°C; and 23 patients (62%) had a leucocyte count >14,000/mm or <4,000/mm. Microbiological study was performed in 30 patients (81%). No infection was diagnosed in the studied patients. All the patients had serum inflammatory biomarkers levels above normal values including procalcitonin (19 patients [51%] had serum procalcitonin above 2 ng/mL). All the patients had IgM EndoCAb below the normal median value; 22 patients (59.5%) had IgM anti-endotoxin value below 10th percentile range for healthy people. Hemodynamic and respiratory stabilization was achieved in 23 patients (62%) and the ICU mortality rate was 38%, only procalcitonin and interleuquin-6 were associated with higher mortality rate. We have detected extremely low titers of IgM EndoCAb in CS suggesting endotoxin exposure. However, only inflammatory biomarkers were related to ICU mortality.

  15. Assessment of endotoxin activity in wastewater treatment plants.

    PubMed

    Guizani, Mokhtar; Dhahbi, Mahmoud; Funamizu, Naoyuki

    2009-07-01

    Endotoxic material, commonly associated to biological reactions, is thought to be one of the most important constituents in water. This has become a very important topic because of the common interest in microbial products governed by the possible shift to water reuse for drinking purposes. In this light, this study was conducted to provide an assessment of endotoxic activity in reclaimed wastewater. A bacterial endotoxin test (LAL test) was applied to water samples from several wastewater treatment plants (WWTP) in Sapporo, Japan keeping in view the seasonal variation. Samples were taken from several points in WWTP (influent, effluent, return sludge, advanced treatment effluent). The findings of this study indicated that wastewater shows high endotoxin activity. The value of Endotoxin (Endo) to COD ratio in the effluent is usually higher than that of the influent. Moreover, it is found that wastewater contains initially endotoxic active material. Some of those chemicals are biodegradable and but most of them are non-biodegradable. Batch scale activated sludge studies were undertaken to understand the origin of endotoxic active material in the effluent. This study showed that those chemicals are mainly produced during biological reactions, more precisely during decay process. Moreover, raw wastewater (RWW) contains high amounts of organic matter having endotoxicity which remains in the effluent.

  16. Changes in regional plasma extravasation in rats following endotoxin infusion

    SciTech Connect

    van Lambalgen, A.A.; van den Bos, G.C.; Thijs, L.G.

    1987-07-01

    Regional differences in plasma extravasation during endotoxin shock in rats and a possible relationship with changes in regional blood flow were studied with radioactive isotopes (/sup 125/I-HSA, 51Cr-labeled red blood cells, microspheres) in anesthetized rats (pentobarbital). Shock was induced by intravenous infusion of endotoxin (Eschericia coli; 10 mg X kg-1) for 60 min (starting at t = 0); at t = 120 min, the experiments were terminated. These rats (n = 8) were compared with time-matched control rats (n = 8). A third group (rats killed 7.5 min after injection of /sup 125/I-HSA, i.e., no extravasation; n = 8) served as baseline. The amount of plasma extravasated in 2 hr of endotoxin shock was significantly increased over control values in skin (by 67%), colon (88%), skeletal muscle (105%), stomach (230%), pancreas (300%), and diaphragm (1300%). Losses of /sup 125/I-HSA into intestinal lumen and peritoneal cavity had also increased over control values by 146 and 380%, respectively. Blood flow was compromised in most organs except heart and diaphragm. Extravasation when normalized for total plasma supply was correlated with total blood supply; the more the blood supply decreased, the higher the normalized extravasation. In the diaphragm, however, blood supply and plasma leakage increased together. Decreased blood supply and plasma extravasation may be related but they could also be simultaneously occurring independent phenomena with a common origin.

  17. Thoracic epidural anesthesia decreases endotoxin-induced endothelial injury

    PubMed Central

    2014-01-01

    Background The sympathetic nervous system is considered to modulate the endotoxin-induced activation of immune cells. Here we investigate whether thoracic epidural anesthesia with its regional symapathetic blocking effect alters endotoxin-induced leukocyte-endothelium activation and interaction with subsequent endothelial injury. Methods Sprague Dawley rats were anesthetized, cannulated and hemodynamically monitored. E. coli lipopolysaccharide (Serotype 0127:B8, 1.5 mg x kg-1 x h-1) or isotonic saline (controls) was infused for 300 minutes. An epidural catheter was inserted for continuous application of lidocaine or normal saline in endotoxemic animals and saline in controls. After 300 minutes we measured catecholamine and cytokine plasma concentrations, adhesion molecule expression, leukocyte adhesion, and intestinal tissue edema. Results In endotoxemic animals with epidural saline, LPS significantly increased the interleukin-1β plasma concentration (48%), the expression of endothelial adhesion molecules E-selectin (34%) and ICAM-1 (42%), and the number of adherent leukocytes (40%) with an increase in intestinal myeloperoxidase activity (26%) and tissue edema (75%) when compared to healthy controls. In endotoxemic animals with epidural infusion of lidocaine the values were similar to those in control animals, while epinephrine plasma concentration was 32% lower compared to endotoxemic animals with epidural saline. Conclusions Thoracic epidural anesthesia attenuated the endotoxin-induced increase of IL-1β concentration, adhesion molecule expression and leukocyte-adhesion with subsequent endothelial injury. A potential mechanism is the reduction in the plasma concentration of epinephrine. PMID:24708631

  18. Delta agent (Hepatitis D)

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/000216.htm Hepatitis D (Delta agent) To use the sharing features on this page, please enable JavaScript. Hepatitis D is a viral infection caused by the ...

  19. Delta in Terra Cimmeria

    NASA Image and Video Library

    2011-02-18

    This unnamed crater in northern Terra Cimmeria has a small channel that created a delta feature. Such features are important indicators of liquid water in Mars past as shown in this image from NASA Mars Odyssey.

  20. Federal Funding in the Delta.

    ERIC Educational Resources Information Center

    Reeder, Richard J.; Calhoun, Samuel D.

    2002-01-01

    The Lower Mississippi Delta region, especially the rural Delta, faces many economic challenges. The rural Delta has received much federal aid in basic income support and funding for human resource development, but less for community resource programs, which are important for economic development. Federal aid to the Delta is analyzed in terms of…

  1. Quantification of endotoxins in infected root canals and acute apical abscess exudates: monitoring the effectiveness of root canal procedures in the reduction of endotoxins.

    PubMed

    Sousa, Ezilmara L R; Martinho, Frederico C; Nascimento, Gustavo G; Leite, Fabio R M; Gomes, Brenda P F A

    2014-02-01

    This clinical study was condu